KR102760488B1 - Pathogen-resistant animals having modified cd163 genes - Google Patents

Abstract

Non-human animals and offspring thereof are provided which comprise at least one altered chromosomal sequence in a gene encoding a CD163 protein. Animal cells containing such altered chromosomal sequences are also provided. The animals and cells have increased resistance to pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV). The animals and offspring have chromosomal alterations in the CD163 gene. The invention further relates to breeding methods for producing pathogen-resistant animals and to populations of animals produced using such methods.

Description

PATHOGEN-RESISTANT ANIMALS HAVING MODIFIED CD163 GENES

The present invention relates to non-human animals and offspring thereof comprising at least one altered chromosomal sequence in a gene encoding the CD163 protein. The present invention also relates to animal cells containing such altered chromosomal sequences. The animals and cells have increased resistance to pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV). The animals and offspring have chromosomal alterations in the CD163 gene such that entry and replication of PRRSV is inhibited and the animals are thus resistant to diseases and syndromes caused by the virus. The present invention also relates to breeding methods for producing pathogen-resistant animals and to populations of animals produced using such methods. The present invention also relates to methods for gene editing of CD163 involving direct injection of embryos and development of animals, founder animals, and strains that are resistant to pathogens such as PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the group of mammalian arteriviruses, which also includes murine lactate dehydrogenase-enhanced virus, simian hemorrhagic fever virus, and equine arteritis virus. Arteriviruses share important characteristics associated with viral pathogenesis, including tropism for macrophages and the ability to cause severe disease and persistent infection. The clinical disease syndrome caused by infection with porcine reproductive and respiratory syndrome virus (PRRSV) was first reported in the United States in 1987 (Keffaber, 1989) and later in Europe in 1990 (Wensvoort et al., 1991). Infection with PRRSV results in respiratory disease, including cough and fever, reproductive failure during late pregnancy, and reduced growth rate. The virus is also involved in a variety of polymicrobial disease syndrome interactions, maintaining lifelong asymptomatic infections (Rowland et al., 2012).

Since its introduction, PRRS has been the most important disease of commercial pigs in North America, Europe, and Asia, with only Australia and Antarctica remaining free of the disease. In North America alone, PRRSV-related losses are estimated to cost producers $664 million annually (Holtkamp et al., 2013). In 2006, a more severe form of the disease, known as highly pathogenic PRRS (HP-PRRS), decimated pig populations throughout China. Genetic diversity has limited the development of vaccines needed to effectively control and eliminate the disease. Genetic selection for natural resistance may be an option, but results have been limited to date (Boddicker et al., 2014).

A previous model describing PRRSV infection of alveolar macrophages identified SIGLEC1 (CD169) as the primary virus receptor on the surface of macrophages; however, previous studies using SIGLEC1 ^-/- pigs showed no differences in virus replication compared to wild-type pigs.

Control efforts are hampered by a lack of understanding of both PRRSV pathogenesis (particularly at the molecular level) and epizootiology. Currently, producers often vaccinate pigs against PRRSV with modified-attenuated live strains or killed virus vaccines, but current vaccines often fail to provide satisfactory protection. This is due to both strain variation and inadequate stimulation of the immune system. In addition to concerns about the efficacy of available PRRSV vaccines, there is strong evidence that currently used modified-live vaccines can persist in individual pigs and pig herds and increase mutation, as demonstrated by virulent field isolates following experimental infection of pigs (Rowland et al., Virology, 259:262-266 (1999)) (Mengeling et al., Am. J. Vet. Res, 60(3): 334-340 (1999)). In addition, vaccine virus has been shown to be shed in the semen of vaccinated boars (Christopher-Hennings et al., Am. J. Vet. Res, 58(1): 40-45 (1997)). As an alternative to vaccination, some experts have advocated a "test and removal" strategy in breeding herds (Dee and Molitor, Vet. Rec., 143:474-476 (1998)). Successful use of this strategy depends on the removal of all pigs acutely or persistently infected with PRRSV followed by strict controls to prevent reintroduction of the virus. The difficulties and high costs associated with this strategy are due to the fact that little is known about the incidence of persistent PRRSV infection and therefore no reliable techniques are available to identify persistently infected pigs.

As can be seen, there is a need in the art to develop strategies to induce PRRSV resistance in animals.

Non-human animals, offspring thereof, and animal cells comprising at least one altered chromosomal sequence in a gene encoding CD163 protein are provided.

Also provided are methods of breeding animals or lineages having reduced susceptibility to infection by a pathogen. The methods include genetically modifying an oocyte or sperm cell to introduce a modified chromosomal sequence in a gene encoding CD163 protein into at least one of an oocyte and a sperm cell, and fertilizing the oocyte with a sperm cell to produce a fertilized egg containing the modified chromosomal sequence in a gene encoding CD163 protein. Alternatively, the methods include genetically modifying a fertilized egg to introduce a modified chromosomal sequence in a gene encoding CD163 protein into the fertilized egg. The methods further include transferring the fertilized egg into a surrogate female animal, wherein pregnancy and term delivery produce progeny animals, screening the progeny animals for susceptibility to the pathogen, and selecting the progeny animals having reduced susceptibility to the pathogen compared to animals that do not contain the modified chromosomal sequence in a gene encoding CD163 protein.

A population of animals produced by the above breeding method is also provided.

Also provided is a method of increasing the resistance of a livestock animal to infection with a pathogen. The method comprises gene editing at least one chromosomal sequence from a gene encoding a CD163 protein such that production or activity of the CD163 protein is reduced compared to production or activity of the CD63 protein in a livestock animal that does not contain the edited chromosomal sequence in the gene encoding the CD163 protein.

An alteration to the chromosomal sequence in the gene encoding the CD163 protein provided herein reduces the susceptibility of an animal, offspring, cell, or population (e.g., a porcine animal, offspring, cell, or population) to a pathogen (e.g., a virus such as porcine reproductive and respiratory syndrome virus (PRRSV)).

In any of the porcine animals, offspring, cells, and methods provided herein, the alteration in the chromosomal sequence in the gene encoding the CD163 protein is selected from the group consisting of an 11 base pair deletion from nucleotides 3,137 to 3,147 relative to reference sequence SEQ ID NO: 47; a 2 base pair insertion between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47 and a 377 base pair deletion from nucleotides 2,573 to 2,949 relative to reference sequence SEQ ID NO: 47 on the same allele; a 124 base pair deletion from nucleotides 3,024 to 3,147 relative to reference sequence SEQ ID NO: 47; a 123 base pair deletion from nucleotides 3,024 to 3,147 relative to reference sequence SEQ ID NO: 47; A 1 base pair insertion between nucleotides 3147 and 3148 relative to reference sequence SEQ ID NO: 47; a 130 base pair deletion from nucleotides 3030 to 3159 relative to reference sequence SEQ ID NO: 47; a 132 base pair deletion from nucleotides 3030 to 3161 relative to reference sequence SEQ ID NO: 47; a 1506 base pair deletion from nucleotides 1525 to nucleotides 3030 relative to reference sequence SEQ ID NO: 47; a 7 base pair insertion between nucleotides 3148 and 3149 relative to reference sequence SEQ ID NO: 47; a 1280 base pair deletion from nucleotides 2818 to 4097 relative to reference sequence SEQ ID NO: 47; A 1373 base pair deletion from nucleotides 2724 to 4096 as compared to reference sequence SEQ ID NO: 47; a 1467 base pair deletion from nucleotides 2431 to 3897 as compared to reference sequence SEQ ID NO: 47; a 1930 base pair deletion from nucleotides 488 to nucleotides 2417 as compared to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insertion beginning at nucleotide 488 and there is an additional 129 base pair deletion in exon 7 from nucleotides 3044 to 3172 as compared to reference sequence SEQ ID NO: 47; a 28 base pair deletion from nucleotides 3145 to 3172 as compared to reference sequence SEQ ID NO: 47; A 1387 base pair deletion from nucleotides 3145 to 4531 relative to reference sequence SEQ ID NO: 47; A 1382 base pair deletion from nucleotides 3113 to 4494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3113; A 1720 base pair deletion from nucleotides 2440 to 4160 relative to reference sequence SEQ ID NO: 47; or a combination thereof.

An isolated nucleic acid is also provided. The isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence comprising SEQ ID NO: 47; (b) a nucleotide sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 47, wherein the nucleotide sequence contains at least one substitution, insertion, or deletion relative to SEQ ID NO: 47; and (c) a cDNA sequence of (a) or (b).

Additional isolated nucleic acids are also provided. The isolated nucleic acids comprise SEQ ID NO: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, or 114.

Other purposes and features will become somewhat self-evident and will be somewhat mentioned below.

Figure 1. CRISPR and targeting vectors used to modify CD163. Panel A depicts wild-type exons 7, 8, and 9 of the CD163 gene targeted for modification using CRISPR. Panel B depicts a targeting vector designed to replace porcine exon 7 (the porcine domain SRCR5 of CD163) with DNA encoding human SRCR8 of CD163L. This targeting vector is used for drug selection with G418 and transfection. PCR primers for long range, left arm, and right arm assays are indicated by arrows for 1230, 3752, 8791, 7765, and 7775. Panel C depicts the same targeting vector as shown in panel B, but with the Neo cassette removed. This targeting vector was used to target CD163 in cells that were already neomycin resistant. Primers used in the small deletion assay are illustrated by arrows and labeled GCD163F and GCD163R. Panel D highlights the exons targeted by CRISPR. The positions of CRISPR 10, 131, 256, and 282 are indicated by downward arrows on exon 7. CRISPR numbers represent the number of base pairs from the intron-exon junction of intron 6 and exon 7.
Figure 2. CRISPR and targeting vectors used to modify CD1D. Panel A depicts wild-type exons 3, 4, 5, 6, and 7 of the CD1D gene targeted for modification by CRISPR. Panel B shows a targeting vector designed to replace exon 3 with the selectable marker Neo . This targeting vector was used to modify CD1D with CRISPR. PCR primers for the far, left-arm, and right-arm assays are indicated by arrows for 3991, 4363, 7373, and 12806. Panel C depicts the exons targeted by CRISPR. The positions of CRISPR 4800, 5350, 5620, and 5626 are indicated by downward arrows on exon 3. Primers used in the small deletion assay are illustrated by arrows and are labeled GCD1DF and GCD1DR. CRISPR counts represent the number of base pairs from the intron-exon junction of intron 6 and exon 7.
Figure 3. Reproductive processes of CD163 and CD1D knockout pigs by CRISPR/Cas9 and SCNT. A) Targeted deletion of CD163 in somatic cells following transfection with CRISPR/Cas9 and donor DNA. The wild-type (WT) genotype results in a 6545 base pair (bp) band. Lanes 1-6 represent six different colonies from a single transfection with donor DNA containing CRISPR 10 and Neo with Cas9. Lanes 1, 4, and 5 show large homozygous deletions of 1500-2000 bp. Lane 2 shows a smaller homozygous deletion. Lanes 3 and 6 represent biallelic alterations of the WT allele and the small deletion or allele. The exact alteration of each colony was determined by sequence analysis for only the colony used for SCNT. A faint WT band in some of the lanes may indicate cross-contamination of fetal fibroblasts from adjacent WT colonies. NTC = no template control. B) Targeted deletion of CD1D in somatic cells following transfection with CRISPR/Cas9 and donor DNA. The WT genotype gives rise to a 8729 bp band. Lanes 1-4 represent colonies with 500-2000 bp deletions of CD1D. Lane 4 is likely a WT colony. NTC ¼ no template control. C) Image of CD163 knockout pigs produced by SCNT during the study. These male piglets contain a homozygous 1506 bp deletion of CD163. D) Image of CD1D pigs produced during the study. These piglets contain a 1653 bp deletion of CD1D. E) Genotypes of two SCNT litters containing a 1506 bp deletion in CD163. Lanes 1-3 (litter 63) and lanes 1-4 (litter 64) represent genotypes for individual piglets from each litter. Sow represents the recipient female of the SCNT embryo, and WT represents the WT control. NTC = no template control. F) Genotypes of two SCNT litters containing a 1653 bp deletion in CD1D. Lanes 1-7 (litter 158) and lanes 1-4 (litter 159) represent genotypes for individual piglets.
Figure 4. Effect of CRISPR/Cas9 system in pig embryos. A) Frequency of blastocyst formation after injection of different concentrations of CRISPR/Cas9 system into zygotes. The toxicity of CRISPR/Cas9 system was lowest at 10 ng/㎕. B) CRISPR/Cas9 system can successfully disrupt the expression of eGFP in blastocysts when introduced into zygotes. Original magnification X4. C) Types of mutations for eGFP generated using CRISPR/Cas9 system: WT genotype (SEQ ID NO: 16), #1 (SEQ ID NO: 17), #2 (SEQ ID NO: 18), and #3 (SEQ ID NO: 19).
Figure 5. Effectiveness of the CRISPR/Cas9 system in targeting CD163 in pig embryos. A) Examples of mutations generated on CD163 by the CRISPR/Cas9 system: WT genotype (SEQ ID NO: 20), #1-1 (SEQ ID NO: 21), #1-4 (SEQ ID NO: 22), and #2-2 (SEQ ID NO: 23). All embryos tested by DNA sequencing showed mutations on CD163 (18/18). CRISPR 131 is highlighted in bold. B) Sequence analysis readout of homozygous deletions induced by the CRISPR/Cas9 system. The image shows #1-4 from panel A, which has a 2 bp deletion of CD163.
Figure 6. Effectiveness of the CRISPR/Cas9 system when introduced with two types of CRISPR. A) PCR amplification of CD163 in blastocysts injected with CRISPR/Cas9 as zygotes. Lanes 1, 3, 6, and 12 show directed deletions between two different CRISPRs. B) PCR amplification of CD1D in blastocysts injected with CRISPR/Cas9 as zygotes. CD1D had a lower frequency of deletions as measured by gel electrophoresis compared to CD163 (3/23); lanes 1, 8, and 15 show clear deletions in CD1D. C) The CRISPR/Cas9 system successfully targeted two genes when two CRISPRs targeting CD163 and eGFP were provided in the system. Variants of CD163 and eGFP are shown: CD163 WT (SEQ ID NO: 24), CD163 #1 (SEQ ID NO: 25), CD163 #2 (SEQ ID NO: 26), CD163 #3 (SEQ ID NO: 27), eGFP WT (SEQ ID NO: 28), eGFP #1-1 (SEQ ID NO: 29), eGFP #1-2 (SEQ ID NO: 30), eGFP #2 (SEQ ID NO: 31), and eGFP #3 (SEQ ID NO: 32).
Figure 7. CD163 knockout pigs generated by the CRISPR/Cas9 system injected into the zygote. A) PCR amplification of CD163 from the knockout pigs; clear signs of deletion were detected in littermates 67-2 and 67-4. B) Images of CD163 knockout pigs with surrogate mothers. All animals are healthy and show no abnormalities. C) Genotypes of CD163 knockout pigs. The wild-type (WT) sequence is shown as SEQ ID NO: 33. Two animals (from littermates 67-1 (SEQ ID NO: 34) and 67-3 (SEQ ID NO: 37)) carried homozygous deletions or insertions in CD163. Two other animals (from litters 67-2 and 67-4) carried biallelic alterations in CD163: #67-2 A1 (SEQ ID NO: 35), #67-2 A2 (SEQ ID NO: 36), #67-4 A1 (SEQ ID NO: 38), and #67-4 a2 (SEQ ID NO: 39). The deletions were induced by introducing two different CRISPR systems with Cas9. None of the animals from the zygotic injections for CD163 displayed a mosaic genotype.
Figure 8. CD1D knockout pigs generated by CRISPR/Cas9 system injected into zygotes. A) PCR amplification of CD1D from knockout pigs; 166-1 shows a mosaic genotype for CD1D. 166-2, 166-3, and 166-4 showed no size changes for the amplicons, but sequencing of the amplicons showed alterations. WT FF = wild-type fetal fibroblasts. B) PCR amplification of the long-range assay showed clear deletion of one allele in piglets 166-1 and 166-2. C) Image of CD1D knockout pigs with surrogate mothers. D) Sequence data of CD1D knockout pigs; WT (SEQ ID NO: 40), #166-1.1 (SEQ ID NO: 41), #166-1.2 (SEQ ID NO: 42), #166-2 (SEQ ID NO: 43), #166-3.1 (SEQ ID NO: 44), #166-3.2 (SEQ ID NO: 45), and #166-4 (SEQ ID NO: 46). The atg initiation codon in exon 3 is shown in lowercase bold.
Figure 9. Clinical signs during acute PRRSV infection. Results of daily assessment for respiratory signs and presence of fever for CD163 +/+ (n=6) and CD163 -/- (n=3).
Figure 10. Lung histopathology during acute PRRSV infection. Representative photomicrographs of H and E stained tissues from wild-type and knockout pigs. Left panels show mononuclear cell infiltration and edema. Right panels from knockout pigs show lung architecture of normal lung.
Figure 11. Viremia in different genotypes. Note that CD163-/- piglet data are placed along the X-axis.
Figure 12. Antibody production in unresponsive, wild-type, and uncharacterized allele pigs.
Figure 13. Cell surface expression of CD163 in individual pigs. Lines seen towards the right in the uncharacterized A, uncharacterized B, and CD163 +/+ panels represent CD163 antibodies, whereas lines seen towards the left of these panels show no antibody control (background). Note that CD163 staining in the CD163-/- animals overlaps with the background control, and CD163 staining in the uncharacterized allele is roughly intermediate between WT levels and background (note also that this is on a log scale, and thus ~10% less).
Figure 14. Levels of CD169 on alveolar macrophages from three representative pigs and no-antibody controls (FITC-labeled anti-CD169).
Figure 15. Viremia in different genotypes. Note that Δ43 amino acid piglet data are placed along the X-axis.
Fig. 16. Genomic sequence of wild-type CD163 exons 7-10 used as a reference sequence (SEQ ID NO: 47). The sequence includes 3000 bp upstream of exon 7 to the last base of exon 10. The underlined regions indicate the locations of exons 7, 8, 9, and 10, respectively.

Provided herein are methods for producing animals and gene-edited animals having a CD163 gene modification that is resistant to PRRSV and other related respiratory virus infections. The animals have a chromosomal modification (insertion or deletion) that inactivates or otherwise modulates CD163 gene activity. CD163 is required for PRRSV entry into cells and viral replication. Thus, refractory CD163 animals are resistant to PRRSV infection when challenged. Such animals can be produced using any of a number of protocols utilizing gene editing.

Also provided herein is a method of producing a porcine animal, comprising introducing into a porcine animal cell or porcine embryo an agent that specifically binds to a chromosomal surface site of the cell and causes double-stranded DNA cleavage or otherwise inactivates or reduces the activity of a CD163 gene or protein therein, using a gene editing method such as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system, Transcription Activator-Like Effector nuclease (TALEN), ZFN (zinc finger nuclease), recombinase fusion protein, or meganuclease.

Also described herein are uses of one or more specific CD163 loci in combination with polypeptides capable of effecting cleavage and/or integration of specific nucleic acid sequences within the CD163 locus. Examples of uses of a CD163 gene in combination with polypeptides or RNAs capable of effecting cleavage and/or integration of the CD163 locus include polypeptides selected from the group consisting of zinc finger proteins, meganucleases, TAL domains, TALENs, RNA-guided CRISPR/Cas recombinases, leucine zippers, and others known in the art. Specific examples include chimeric (“fusion”) proteins comprising a site-specific DNA binding domain polypeptide and a cleavage domain polypeptide (e.g., a nuclease), e.g., a ZFN protein comprising a zinc-finger polypeptide and a FokI nuclease polypeptide. Described herein are polypeptides comprising a DNA-binding domain that specifically binds to a CD163 gene. Such polypeptides may also comprise a nuclease (cleavage) domain or half-domain (e.g., a homing endonuclease, including a homing endonuclease having a modified DNA-binding domain), and/or a ligase domain, such that the polypeptide can induce a targeted double-stranded cleavage and/or promote recombination of a nucleic acid of interest at the cleavage site. The DNA-binding domain targeting the CD163 locus can be a DNA-cleaving functional domain. The polypeptide can be used to introduce an exogenous nucleic acid into the genome of a host organism (e.g., an animal species) at one or more CD163 loci. The DNA-binding domain can comprise a zinc finger protein having one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers) that is engineered (non-naturally occurring) to bind to any sequence within the CD163 gene. Any of the zinc finger proteins described herein can bind to a target site (e.g., a promoter or other expression element) within the coding sequence of a target gene or within an adjacent sequence. The zinc finger protein can bind to a target site in the CD163 gene.

definition

Units, prefixes, and symbols may be indicated in their SI approved forms. Unless otherwise indicated, nucleic acids are written left to right in the 5' to 3' direction; amino acid sequences are written left to right in the amino to carboxy direction. Numerical ranges cited in the specification are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids may be represented herein by their commonly known three-letter symbols or by their single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be represented herein by their commonly accepted single-letter codes. Unless otherwise provided, software, electrical, and electronics terms used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5th edition, 1993). Terms defined below are more fully defined by reference to the specification as a whole.

As will be understood by those skilled in the art, for any and all purposes, particularly in the context of providing a written description, all ranges recited herein also include any and all possible sub-ranges and combinations of sub-ranges thereof, as well as individual values making up the range, particularly integer values. The recited range includes each particular value, integer, decimal, or identity within the range. It will be readily recognized that any recited range sufficiently describes the same range and can be subdivided into at least equivalent 1/2, 1/3, 1/4, 1/5, or 1/10. As a non-limiting example, each range discussed herein can be readily subdivided into 1/3 below, 1/3 in the middle, and 1/3 above.

When introducing elements of the present invention or preferred embodiments(s) thereof, the articles "a", "an", "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

The term "and/or" means either one of the items, any combination of the items, or all of the items associated with that term. The phrase "one or more" is readily understood by those skilled in the art, especially when read in the context of its usage.

A "binding protein" is a protein that can bind to another molecule. A binding protein can, for example, bind to a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein), and/or a protein molecule (a protein-binding protein). In the case of a protein-binding protein, it can bind to itself (bind to form a homodimer, homotrimer, etc.) and/or bind to one or more molecules of a different protein or proteins. A binding protein can have more than one type of binding activity. For example, a zinc finger protein has DNA-binding, RNA-binding and protein-binding activities.

The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, a "conservatively modified variant" refers to a nucleic acid encoding an identical or conservatively modified variant of the amino acid sequence. Because of the degeneracy of the genetic code, a plurality of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at any position where an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid modifications are "silent variations" and represent a type of conservatively modified change. All nucleic acid sequences herein that encode a polypeptide also describe all possible silent variations of the nucleic acid, with reference to the genetic code.

One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is commonly the only codon for methionine; and UGG, which is commonly the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each asymptomatic variation of a nucleic acid encoding a polypeptide of the invention is encompassed by each disclosed polypeptide sequence and is within the scope of the invention.

With respect to amino acid sequences, the skilled person will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequence which alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants", wherein the alteration replaces an amino acid with a chemically similar amino acid. Thus, any number of amino acid residues selected from the integer group consisting of 1 to 15 may be so altered. Thus, for example, 1, 2, 3, 4, 5, 7 or 10 alterations may be made.

Conservatively modified variants typically provide biological activity similar to the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzymatic activity, or ligand/receptor binding is typically at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the native protein for its native substrate. Conservative substitution tables providing functionally similar amino acids are well known in the art.

Each of the following six groups contains amino acids that are conservative substitutions for each other: [1] alanine (A), serine (S), threonine (T); [2] aspartic acid (D), glutamic acid (E); [3] asparagine (N), glutamine (Q); [4] arginine (R), ligine (K); [5] isoleucine (I), leucine (L), methionine (M), valine (V); and [6] phenylalanine (F), trypsin (Y), tryptophan (W). See also Creighton (1984) Proteins W. H. Freeman and Company.

The term "CRISPR" stands for "clustered regularly interspaced short palindromic repeats". The term "Cas9" refers to "CRISPR associated protein 9". The term "CRISPR/Cas9" or "CRISPR/Cas9 system" refers to a programmable nuclease system for genetic engineering comprising the Cas9 protein, or a derivative thereof, and one or more non-coding RNAs capable of providing the functions of a CRISPR RNA (crRNA) and a transcription-activating RNA (tracrRNA) to Cas9. The crRNA and tracrRNA can be combined or used individually to produce a "guide RNA" (gRNA). The crRNA or gRNA provides a sequence complementary to a genomic target. The CRISPR/Cas9 system is further described below.

Reference herein to a deletion of a nucleotide sequence from nucleotide x to nucleotide y means that all of the nucleotides within and including x and y are deleted. Thus, for example, the phrase "an 11 base pair deletion from nucleotide 3,137 to nucleotide 3,147 relative to SEQ ID NO: 47" means that each of nucleotides 3,317 to 3,147 are deleted, inclusive of nucleotides 3,317 and 3,147.

"Disease resistance" is a characteristic of an animal, wherein the animal avoids disease symptoms that are a result of an animal-pathogen interaction, such as the interaction between a swine animal and PRRSV, i.e., preventing the pathogen from causing the animal disease and associated disease symptoms, or alternatively, reducing the incidence and/or severity of clinical signs or reducing clinical symptoms. Those skilled in the art will recognize that the compositions and methods disclosed herein can be used in conjunction with other compositions and methods available in the art to protect animals from pathogen challenge.

In relation to a particular nucleic acid, "encoding" or "encoded" means that it contains information for translation into a particular protein. A nucleic acid encoding a protein may contain intervening sequences (e.g., introns) within the translated region of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA). The information that encodes the protein is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code. When the nucleic acid is made synthetically or modified, the known codon preferences of the intended host in which the nucleic acid is to be expressed can be utilized.

As used herein, the terms "gene editing", "gene edited", "genetically edited" and "gene editing effectors" refer to the use of homing technology using naturally occurring or artificially engineered nucleases, also called "molecular scissors", "homing endonucleases", or "targeting endonucleases". The nucleases create a specific double-stranded chromosome break (DSB) at a desired location in the genome, which in some cases utilizes the cell's endogenous mechanisms to repair the break induced by the natural processes of homologous recombination (HR) and/or nonhomologous end-joining (NHEJ). Genome editing effectors include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), the Clustered Regularly Interspaced Short Palindromic Repeats/CAS9 (CRISPR/Cas9) system, and meganucleases (e.g., meganucleases re-engineered as homing endonucleases). The term also includes the use of transgenic processes and techniques, including, for example, where the change is a deletion or relatively small insertion (typically less than 20 nt) and/or does not introduce DNA from a foreign species. The term also includes progeny animals, such as those produced by sexual or asexual reproduction from an initial genome-edited animal.

As used herein, "heterologous" with respect to a nucleic acid is a nucleic acid that is either from a foreign species or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by intended human intervention. For example, a promoter operably linked to a heterologous structural gene may be from a different species than that which drives the structural gene, or, if from the same species, one or both are substantially modified from their native form. A heterologous protein may be from a foreign species or, if from the same species, is substantially modified from its native form by intended human intervention.

As used herein, “homing DNA technology”, “homing technology” and “homing endonuclease” apply to any mechanism that allows a specific molecule to be targeted to a specific DNA sequence, including zinc finger (ZF) proteins, transcription activator-like effector (TALE) meganucleases, and the CRISPR/Cas9 system.

As used herein, the terms "increased resistance" and "reduced susceptibility" mean, but are not limited to, a statistically significant reduction in the incidence and/or severity of clinical signs or symptoms associated with infection by a pathogen. For example, "increased resistance" or "reduced susceptibility" can refer to a statistically significant reduction in the incidence and/or severity of clinical signs or symptoms associated with infection by PRRSV in an animal comprising at least one altered chromosomal sequence in a gene encoding the CD163 protein, as compared to a control animal having the unaltered chromosomal sequence. The term "statistically significant reduction in clinical symptoms" means, but is not limited to, that the incidence of at least one clinical symptom in an edited group of subjects is at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70% lower after challenge with an infectious agent than in a non-edited control group.

The term "knock-in" as used herein means replacing an endogenous gene with a transgene or with the same endogenous gene with slight structural modifications while maintaining transcriptional regulation of the endogenous gene.

"Knock-out" refers to a disruption of the structure or regulatory mechanism of a gene. Knock-outs can be produced by homologous recombination of targeting vectors, replacement vectors, or hit-and-run vectors, or by random insertion of gene trap vectors that result in complete, partial, or conditional loss of gene function.

The term "animal" includes any non-human animal, such as farmed animals (e.g., livestock animals). The term "livestock animal" includes any animal traditionally raised on a ranch, such as porcine animals, bovine animals (e.g., beef or dairy cattle), ovine animals, caprine animals, equine animals (e.g., horses or donkeys), buffalo, camels, or fowl animals (e.g., chickens, turkeys, ducks, geese, guinea fowl, or hatchlings). The term "livestock animal" does not include rats, mice, or other rodents.

The term "mutation" as used herein includes a change in the nucleotide sequence of a polynucleotide, such as a gene or a coding DNA sequence (CDS), relative to the wild-type sequence. The term includes, without limitation, substitutions, insertions, frameshifts, deletions, inversions, translocations, duplications, splice-donor site mutations, point mutations, and the like.

As used herein, "operably linked" includes reference to a functional linkage between two nucleic acid sequences, e.g., a promoter sequence and a secondary sequence, wherein the promoter sequence initiates and mediates transcription of a DNA sequence corresponding to the secondary sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous, and, if necessary, link two protein coding regions so that they are contiguous and in the same reading frame.

As used herein, "polynucleotide" includes reference to a deoxyribopolynucleotide, a ribopolynucleotide, or a conservatively modified variant; the term may also refer to analogs thereof that have the essential properties of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to a nucleotide sequence substantially identical to a naturally occurring nucleotide and/or are translated into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide may be a full-length or subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to a specific sequence as well as its complementary sequence. Thus, a DNA or RNA having a backbone modified for stability or other reasons is a "polynucleotide" as the term is intended herein. Moreover, a DNA or RNA containing an unusual base, such as inosine, or a modified base, such as a tritylated base, to name just two examples, is a polynucleotide as the term is used herein. It will be appreciated by those skilled in the art that a wide variety of modifications have been made to DNA and RNA, serving a number of useful purposes.

The term polynucleotide, as used herein, includes such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as, among others, chemical forms of DNA and RNA characteristic of cells and viruses, including simple and complex cells.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms may also apply to amino acid polymers that are conservatively modified variants and artificial chemical analogs of one or more amino acid residues of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. The essential property of such analogs of naturally occurring amino acids is that, when incorporated into a protein, the protein is specifically reactive to antibodies directed to the same protein comprised entirely of naturally occurring amino acids.

The terms "polypeptide", "peptide" and "protein" also include modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. As is well known and noted above, it will be appreciated that polypeptides are not always entirely linear. For example, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, as a result of post-translational events, including both natural processing events and events caused by human manipulation that do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translational natural processes and entirely synthetic methods. The present invention also contemplates the use of both methionine-containing and non-methionine-containing amino terminal variants of the proteins of the invention.

As used herein, "reduction in the incidence and/or severity of clinical signs" or "reduction in clinical symptoms" means, but is not limited to, a reduction in the number of infected subjects in a group, a reduction or elimination of the number of subjects exhibiting clinical signs of infection, or a reduction in the severity of any clinical symptom present in one or more subjects, as compared to a wild-type infection. For example, these terms include a reduction in any clinical sign of infection, pulmonary pathology, viremia antibody production, a reduction in pathogen load, pathogen shedding, a reduction in pathogen transmission, or a reduction in any clinical sign symptomatic of PRRSV. Preferably, these clinical signs are reduced by at least 10% in one or more animals of the invention as compared to infected subjects without a mutation in the CD163 gene. More preferably, the clinical signs are reduced by at least 20% in the subjects of the invention, preferably by at least 30%, more preferably by at least 40%, and even more preferably by at least 50%.

The terms "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively, "protein"). The amino acid may be a naturally occurring amino acid, and may include non-natural analogs of natural amino acids that can function in a similar manner to the naturally occurring amino acid, unless otherwise limited.

The term "selectively hybridizes" includes reference to the hybridization of a nucleic acid sequence to another nucleic acid sequence or to another biological agent under stringent hybridization conditions. When using a hybridization-based detection system, a nucleic acid probe complementary to a reference nucleic acid sequence is selected, and then, by selection of appropriate conditions, the probe and reference sequence hybridize or bind to each other to form a duplex molecule.

The term "stringent conditions" or "stringent hybridization conditions" includes reference to conditions under which a probe hybridizes to its target sequence to a detectably greater extent (e.g., at least 2-fold greater than background) than to other sequences. Stringent conditions are sequence-dependent and will vary in different situations. By adjusting the stringency of the hybridization and/or wash conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing).

Alternatively, stringent conditions can be adjusted to allow some mismatches in the sequences so that lower levels of similarity can be detected (heterologous probing). Typically, probes are less than about 1000 nucleotides in length, and optionally less than 500 nucleotides in length.

Typically, stringent conditions will be those at pH 7.0 to 8.3, with a salt concentration of less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salt), and a temperature of at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions can also be achieved with the addition of a destabilizing agent, such as formamide. Specificity is typically a function of the post-hybridization wash, the critical factors being the ionic strength and the temperature of the final wash solution. For DNA/DNA hybrids, the thermal melting point (Tm) is given by the equation of Meinkoth and Wahl [Anal. Biochem., 138: 267-284 (1984)] can be approximated as: Tm [℃] = 81.5 + 16.6 (log M) + 0.41(% GC)-0.61 (% form)-500/L; where M is the molar concentration of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. T _m is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. T m decreases by about 1℃ for each 1% mismatch; thus, T m, hybridization and/or wash conditions can be adjusted to hybridize to sequences of desired identity. For example, if a sequence having > 90% identity is sought, the Tm may be reduced by 10°C. Typically, stringent conditions are selected to be about 5°C lower than the Tm for the particular sequence and its complement at a defined ionic strength and pH. However, highly stringent conditions may utilize hybridization and/or washing at temperatures from 1 to 4°C lower than the Tm; moderately stringent conditions may utilize hybridization and/or washing at temperatures from 6 to 10°C lower than the Tm; and low stringency conditions may utilize hybridization and/or washing at temperatures from 11 to 20°C lower than the Tm. Using the equations, hybridization and wash compositions, and the desired Tm, one of ordinary skill in the art will appreciate that variations in the stringency of the hybridization and/or wash solutions are essentially accounted for. Extensive guidance on the hybridization of nucleic acids can be found in the literature [see; Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).

A "TALE DNA binding domain" or "TALE" is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains are involved in binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also called a "repeat") is typically 33-35 amino acids in length and exhibits at least some sequence homology to other TALE repeat sequences in naturally occurring TALE proteins. The zinc finger and TALE binding domains can be "engineered" to bind to a given nucleotide sequence by engineering (changing one or more amino acids) the recognition helix region of a naturally occurring zinc finger or TALE protein. Thus, the engineered DNA binding protein (zinc finger or TALE) is a non-naturally occurring protein. Non-limiting examples of methods for engineering DNA-binding proteins are design and selection. An engineered DNA binding protein is a non-naturally occurring protein whose design/composition is primarily based on rational criteria. Reasonable criteria for design include application of computerized algorithms and substitution rules to process information from existing ZFP and/or TALE design and database storage of combined data information. See, e.g., U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Publication No. 20110301073.

As used herein, "vector" refers to a nucleic acid that can be used to transfect a host cell and can be inserted into a polynucleotide. The vector is often a replicon. An expression vector allows for transcription of a nucleic acid inserted therein.

“Wild type” means an animal, and blastocysts, embryos or cells derived therefrom, that has not been genetically edited or otherwise genetically modified and is typically an inbred or outbred variant developed from a naturally occurring variant.

A "zinc finger DNA-binding protein" (or binding domain) is a protein, or domain within a larger protein, that binds DNA in a sequence-specific manner via one or more zinc fingers, a region of amino acid sequence within the binding domain whose structure is stabilized by coordination of a zinc ion. The term zinc finger DNA-binding protein is often abbreviated as zinc finger protein or ZFP.

A "selected" zinc finger protein or TALE is a protein not found in nature, often produced from experimental procedures such as phage display, interaction traps, or hybrid selection. See, e.g., U.S. Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988; U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197, WO 02/099084, and U.S. Publication No. See 20110301073.

The following terms are used to describe the sequence relationship between a polynucleotide/polypeptide of the present invention and a reference polynucleotide/polypeptide: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", and (d) "percent sequence identity".

(a) As used herein, a "reference sequence" is a defined sequence that is used as a basis for sequence comparison with the polynucleotides/polypeptides of the present invention. The reference sequence may be a subset of a specified sequence or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence.

(b) As used herein, a "comparison window" includes reference to a contiguous specific segment of a polynucleotide/polypeptide sequence, wherein the polynucleotide/polypeptide sequence can be compared to a reference sequence, and wherein the portion of the polynucleotide/polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) relative to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. Typically, the comparison window is at least 20 contiguous nucleotides/amino acid residues in length, and optionally may be 30, 40, 50, 100, or longer. Those skilled in the art will appreciate that in order to avoid high similarity to the reference sequence due to the inclusion of gaps in the polynucleotide/polypeptide sequence, a gap penalty is typically introduced, which is subtracted from the number of matches.

Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed by computerized implementations of these algorithms, including but not limited to the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444 (1988); and the following: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, California; GAP, BESTFIT, BLAST, FASTA, and TFASTA, and related programs in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). The CLUSTAL program is described in Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al., Computer Applications in the Biosciences 8: 155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24: 307-331 (1994).

Programs in the BLAST family that can be used for database similarity searches include: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Altschul et al., J. Mol. Biol., 215: 403-410 (1990); and, Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). Software for performing BLAST analyses is publicly available, for example, through the National Center for Biotechnology Information (ncbi.nlm.nih.gov/). These algorithms are fully described in numerous publications. See, for example, Altschul SF et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, 25 NUCLEIC ACIDs Res. 3389 (1997); National Center for Biotechnology Information , The NCBI Handbook [Internet], Chapter 16: The BLAST Sequence Analysis Tool (McEntyre J, Ostell J, eds., 2002), available at http://www.ncbi.nlm.nih.gov/books/NBK21097/pdf/ch16.pdf. The BLASTP program for amino acid sequences is also well described (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5877 (1993)). Several low-complexity filter programs can be used to reduce these low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17: 149-163 (1993)) and XNU (Claverie and States, Comput. Chem., 17: 191-201 (1993)) low-complexity filters can be used alone or in combination.

Unless otherwise specified, nucleotide and protein identity/similarity values provided herein are calculated using GAP (GCG Version 10) under default values. GAP (Global Alignment Program) can also be used to compare polynucleotides or polypeptides of the present invention to reference sequences. GAP uses the algorithm of Needleman and Bunsch (J. Mol. Biol. 48: 443-453, 1970) to find an alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP represents one member of a family of best alignments. There may be multiple members in this family, but no other member has better quality. GAP represents four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. Quality is a metric that is maximized for aligning sequences. The ratio is the quality divided by the number of bases in the shorter segment. The percent identity is the percentage of symbols that actually match. The percent similarity is the percentage of similar symbols. Symbols directly across a gap are ignored. Similarity is scored if the scoring matrix value for a pair of symbols is greater than or equal to the similarity threshold of 0.50. The scoring matrix used in version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (cf. Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915).

Multiple alignment of sequences can be performed using the CLUSTAL alignment method (Higgins and Sharp (1989) CABIOS. 5: 151-153) with default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). The default parameters for pairwise alignment using the CLUSTAL method include KTUPLE 1, GAP PENALTY=3, WINDOW=5, and DIAGONALS SAVED=5.

(c) As used herein, "sequence identity" or "identity" of two nucleic acid or polypeptide sequences in the context of the two sequences includes reference to residues in the two sequences that are identical when aligned for maximum correspondence over a specified comparison window. When percent sequence identity is used for proteins, it is recognized that the non-identical residue positions often differ by conservative amino acid substitutions, where the amino acid residue is replaced by another amino acid residue that has similar chemical properties (e.g. charge or hydrophobicity) and thus does not alter the functional properties of the molecule. When the sequences differ by a conservative substitution, the percent sequence identity can be adjusted to correct for the conservative nature of the substitution. The sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Methods for making such adjustments are well known to those skilled in the art. Typically, this involves scoring the conservative substitution as a partial mismatch rather than a full mismatch, thereby increasing the percent sequence identity. Thus, for example, a conservative substitution receives a score between 0 and 1, while an identical amino acid receives a score of 1 and a non-conservative substitution receives a score of 0. Scoring of conservative substitutions can be computed, for example, according to the algorithm of Meyers and Miller [Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988)], as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).

(d) As used herein, "percentage of sequence identity" means a value determined by comparing two optimally aligned sequences over a comparison window, wherein portions of the polynucleotide sequences in the comparison window may include additions or deletions (i.e., gaps) as compared to a reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percent is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity.

Animals and cells having a chromosomal sequence altered in the gene encoding CD163 protein

CD163 has 17 exons and the protein consists of an extracellular region with nine scavenger receptor cysteine-rich (SRCR) domains, a transmembrane segment, and a short cytoplasmic tail. Several different variants arise from differential splicing of a single gene (Ritter et al. 1999a; Ritter et al. 1999b). Most of these variations are in the length of the cytoplasmic tail.

CD163 has several important functions, including acting as a haptoglobin-hemoglobin scavenger receptor. Because the heme group can be highly toxic, removal of free hemoglobin from the blood is an important function of CD163 (Kristiansen et al. 2001). CD163 has a cytoplasmic tail that promotes endocytosis. Mutations in this tail result in reduced haptoglobin-hemoglobin complex uptake (Nielsen et al. 2006). Additional functions of C163 include erythroblast adhesion (SRCR2), TWEAK receptors (SRCR1-4 & 6-9), bacterial receptor (SRCR5), African swine virus receptor (Sanchez-Tones et al. 2003), and a potential role as an immune-modulator (discussed in the literature (Van Gorp et al. 2010a)). Given this important function, it was previously thought that complete knock-out of CD163 would produce non-viable or severely impaired animals (see, e.g., PCT Publication No. 2012/158828).

CD163 is a member of the scavenger receptor cysteine-rich (SRCR) superfamily and consists of an intracellular domain and nine extracellular SRCR domains. In humans, CD163-mediated endocytosis of hemoglobin-heme uptake via SRCR3 protects cells from oxidative stress (Schaer et al., 2006a; Schaer et al., 2006b; Schaer et al., 2006c). CD163 also functions as a receptor for tumor necrosis factor-like weak inducer of apoptosis (TWEAK: SRCR1-4 & 6-9), a pathogen receptor (African Swine Fever Virus; bacteria: SRCR2), and erythroblast binding (SRCR2).

CD163 plays a role in infection by several different pathogens and thus the present invention is not limited to animals having reduced susceptibility to PRRSV infection, but also encompasses animals having reduced susceptibility to any pathogen that relies on CD163 for infection of cells or for later replication and/or persistence in cells. The infection pathway of PRRSV begins with initial binding to heparan sulfate on the surface of alveolar macrophages. Prior to 2013, it was thought that definitive binding then occurred to sialoadhesin (SIGLEC1, also called CD169 or SN). The virus is then internalized via clatherin-mediated endocytosis. Another molecule, CD163, then promotes uncoating of the virus in endosomes (Van Breedam et al. 2010a). The viral genome is released and the cell is infected.

Described herein are animals and offspring and cells thereof comprising at least one altered chromosomal sequence, e.g., insertion or deletion ("INDEL"), in a gene encoding CD163 protein, which confers improved or complete resistance to infection by a pathogen (e.g., PRRSV). Applicants have demonstrated that CD163 is a critical gene for PRRSV infection and have created pioneer resistant animals and strains.

The present invention provides a genetically modified animal, offspring thereof, or animal cell comprising at least one altered chromosomal sequence in a gene encoding CD163 protein. The present invention does not encompass inactivation or editing of the SIGLEC1 (CD169) gene, which has previously been postulated to be important for PRRSV resistance.

The edited chromosomal sequence can comprise an integrated sequence that is (1) inactivated, (2) altered, or (3) causes a silent mutation. The inactivated chromosomal sequence is altered to impair, reduce, or eliminate CD163 protein function as it relates to PRRSV infection. Thus, a gene-edited animal comprising an inactivated chromosomal sequence can be referred to as a "knock out" or a "conditional knock out." Similarly, a gene-edited animal comprising an integrated sequence can be referred to as a "knock in" or a "conditional knock in." Furthermore, a gene-edited animal comprising an altered chromosomal sequence can comprise targeted point mutation(s) or other modifications that result in the production of an altered protein product. Briefly, the process can comprise introducing into an embryo or cell at least one RNA molecule encoding a target zinc finger nuclease and, optionally, at least one accessory polynucleotide. The method further comprises culturing the embryo or cell to allow for expression of the zinc finger nuclease, wherein the double-stranded break introduced into the targeted chromosomal sequence by the zinc finger nuclease is repaired by an error-prone non-homologous end-joining DNA repair process or a homology-directed DNA repair process. The method for editing a chromosomal sequence encoding a protein involved in germline development using the targeted zinc finger nuclease technology is rapid, accurate, and highly efficient.

Alternatively, the process may involve using the CRISPR/Cas9 system to modify the genome sequence. When using the CRISPR/Cas9 system to modify the genome sequence, the protein may be delivered directly into the cell, mRNA encoding Cas9 may be delivered into the cell, or a gene providing for expression of mRNA encoding Cas9 may be delivered into the cell. Additionally, target-specific crRNA and tracrRNA may be delivered directly into the cell, or target-specific gRNA(s) may be delivered into the cell (which RNAs may alternatively be produced by genes engineered to express such RNAs). The design of crRNA/gRNA and selection of target sites are well known in the art. Construction and cloning of gRNAs may be found at http://www.genome-engineering.org/crispr/wp-content/uploads/2014/05/CRISPR-Reagent-Description-Rev20140509.pdf.

At least one CD163 locus can be used as a target site for site-specific editing. Site-specific editing can involve insertion of an exogenous nucleic acid (e.g., a nucleic acid comprising a nucleotide sequence encoding a polypeptide of interest) or deletion of nucleic acid from the locus. For example, integration of an exogenous nucleic acid and/or deletion of a portion of a genomic nucleic acid can modify the locus to produce a disrupted (i.e., reduced activity of the CD163 protein) CD163 gene.

Provided herein are non-human animals, offspring of such animals, and animal cells comprising at least one altered chromosomal sequence in a gene encoding CD163 protein.

An alteration in the chromosomal sequence in the gene encoding the CD163 protein reduces the susceptibility of the animal, offspring, or cells to infection by a pathogen (e.g., a virus such as PRRSV) compared to the susceptibility of the animal, offspring, or cells to infection by the pathogen that do not contain the altered chromosomal sequence in the gene encoding the CD163 protein.

The animal or offspring may be an embryo, a juvenile, or an adult. Similarly, the cell may comprise an embryonic cell, a cell derived from a juvenile animal, or a cell derived from an adult animal.

The animal or offspring may comprise a domestic animal. Likewise, the cell may comprise a cell derived from a domestic animal. The domestic animal may comprise a livestock animal, such as a porcine animal, a bovine animal (e.g., a beef or dairy cow), an ovine animal, a caprine animal, an equine animal (e.g., a horse or a donkey), a buffalo, a camel, or a avian animal (e.g., a chicken, turkey, duck, goose, guinea fowl, or a hatchling). The livestock animal is preferably a bovine or porcine animal, and most preferably a porcine animal.

The animal or offspring may comprise a gene-edited animal. The cell may comprise a gene-edited cell.

The animal or cell can be gene edited using a homing endonuclease. The homing endonuclease can be a naturally occurring endonuclease, but it is preferred that the endonuclease is a rationally designed non-naturally occurring homing endonuclease having a DNA recognition sequence designed to target the chromosomal sequence of the gene encoding the CD163 protein. Thus, the homing endonuclease can be a designed homing endonuclease. The homing endonuclease can include, for example, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a recombinase fusion protein, a meganuclease, or a combination thereof. The animal or cell is preferably an animal or cell that has been gene edited using the CRISPR/Cas9 system.

The gene-edited animal, its offspring, or gene-edited cells preferably exhibit increased resistance to pathogens (e.g., viruses such as PRRSV) compared to a non-edited animal.

The animal, offspring, or cell may be heterozygous for the altered chromosomal sequence. Alternatively, the animal, offspring, or cell may be homozygous for the altered chromosomal sequence.

In any animal, offspring or cell, the altered chromosomal sequence can comprise an insertion of a gene encoding a CD163 protein, a deletion of a gene encoding a CD163 protein, or a combination thereof. For example, the altered chromosomal sequence can comprise a deletion (e.g., an in-frame deletion) of a gene encoding a CD163 protein. Alternatively, the altered chromosomal sequence can comprise an insertion of a gene encoding a CD163 protein.

The insertion or deletion may result in decreased CD163 protein production or activity compared to CD163 protein production or activity in an animal, offspring, or cell lacking the insertion or deletion.

The insertion or deletion can result in the production of substantially non-functional CD163 protein by the animal, offspring, or cell. By "substantially non-functional CD163 protein" is meant that the level of CD163 protein in the animal, offspring, or cell is undetectable, or, if detectable, at least about 90% lower than the level observed in an animal, offspring, or cell that does not contain the insertion or deletion.

Where the animal, offspring, or cell comprises a porcine animal, offspring, or cell, the altered chromosomal sequence can comprise an alteration in exon 7 of the gene encoding CD163 protein, exon 8 of the gene encoding CD163 protein, an intron adjacent to exon 7 or exon 8 of the gene encoding CD163 protein, or a combination thereof. The altered chromosomal sequence suitably comprises an alteration in exon 7 of the gene encoding CD163 protein.

An alteration in exon 7 of the gene encoding the CD163 protein may comprise a deletion (e.g., an in-frame deletion in exon 7). Alternatively, an alteration in exon 7 of the gene encoding the CD163 protein may comprise an insertion.

In any porcine animal, offspring, or cell, the alteration comprises an 11 base pair deletion from nucleotides 3,137 to 3,147 relative to reference sequence SEQ ID NO: 47; a 2 base pair insertion between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47 and, on the same allele, a 377 base pair deletion from nucleotides 2,573 to nucleotides 2,949 relative to reference sequence SEQ ID NO: 47; a 124 base pair deletion from nucleotides 3,024 to 3,147 relative to reference sequence SEQ ID NO: 47; a 123 base pair deletion from nucleotides 3,024 to 3,146 relative to reference sequence SEQ ID NO: 47; a 1 base pair insertion between nucleotides 3,147 and 3,148 relative to reference sequence SEQ ID NO: 47; A 130 base pair deletion from nucleotides 3,030 to 3,159 relative to reference sequence SEQ ID NO: 47; a 132 base pair deletion from nucleotides 3,030 to 3,161 relative to reference sequence SEQ ID NO: 47; a 1,506 base pair deletion from nucleotides 1,525 to nucleotides 3,030 relative to reference sequence SEQ ID NO: 47; a 7 base pair insertion between nucleotides 3,148 and nucleotides 3,149 relative to reference sequence SEQ ID NO: 47; a 1,280 base pair deletion from nucleotides 2,818 to 4,097 relative to reference sequence SEQ ID NO: 47; a 1,373 base pair deletion from nucleotides 2,724 to 4,096 relative to reference sequence SEQ ID NO: 47; A 1467 base pair deletion from nucleotides 2,431 to 3,897 relative to reference sequence SEQ ID NO: 47; a 1930 base pair deletion from nucleotides 488 to 2,417 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insertion starting at nucleotide 488 and an additional 129 base pair deletion in exon 7 from nucleotides 3,044 to 3,172 relative to reference sequence SEQ ID NO: 47; a 28 base pair deletion from nucleotides 3,145 to 3,172 relative to reference sequence SEQ ID NO: 47; a 1387 base pair deletion from nucleotides 3,145 to 4,531 relative to reference sequence SEQ ID NO: 47; A 1382 base pair deletion from nucleotides 3113 to 4494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3113; a 1720 base pair deletion from nucleotides 2440 to 4160 relative to reference sequence SEQ ID NO: 47; or a combination thereof.

SEQ ID NO: 47 provides the nucleotide sequence for a region beginning 3000 base pairs (bp) upstream of exon 7 of the wild-type porcine CD163 gene to the last base of exon 10 of that gene. SEQ ID NO: 47 is used herein as a reference sequence and is illustrated in FIG. 16 .

Where the porcine animal or cell comprises a two base pair insertion between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47, the two base pair insertion can comprise an insertion of the dinucleotide AG.

Where the porcine animal or cell comprises a one base pair insertion between nucleotides 3,147 and 3,148 relative to reference sequence SEQ ID NO: 47, the one base pair insertion may comprise the insertion of a single adenine residue.

Where the porcine animal or cell comprises a 7 base pair insertion between nucleotides 3,148 and 3,149 relative to reference sequence SEQ ID NO: 47, the 7 base pair insertion can comprise an insertion of the sequence TACTACT (SEQ ID NO: 115).

Wherein the porcine animal or cell comprises a 1930 base pair deletion from nucleotide 488 to nucleotide 2,417 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insertion beginning at nucleotide 488, and there is an additional 129 base pair deletion in exon 7 from nucleotides 3,044 to nucleotides 3,172 relative to reference sequence SEQ ID NO: 47, the 12 base pair insertion can comprise an insertion of the sequence TGTGGAGAATTC (SEQ ID NO: 116).

Wherein the porcine animal or cell comprises a 1382 base pair deletion from nucleotide 3,113 to nucleotide 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3,113, the 11 base pair insertion can comprise an insertion of the sequence AGCCAGCGTGC (SEQ ID NO: 117).

When the altered chromosomal sequence of the gene encoding the CD163 protein comprises a deletion, the deletion preferably comprises an in-frame deletion. An in-frame deletion is a deletion that does not result in a change in the triplet reading frame and thus results in a protein product having an internal deletion of one or more amino acids but not truncated. If splicing occurs correctly, deletions of three base pairs or multiples of three base pairs within an exon can result in an in-frame mutation.

The following INDELs described herein for porcine animals and cells are expected to result in in-frame deletions because the deletions within exon 7 of the porcine CD163 gene are a multiple of 3: a 1506 base pair deletion from nucleotides 1525 to nucleotides 3030 relative to reference sequence SEQ ID NO: 47; a 1930 base pair deletion from nucleotides 488 to nucleotides 2417 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insertion beginning at nucleotide 488 and there is an additional 129 base pair deletion in exon 7 from nucleotides 3044 to nucleotides 3172 relative to reference sequence SEQ ID NO: 47; A 1373 base pair deletion from nucleotides 2,724 to nucleotides 4,096 relative to reference sequence SEQ ID NO: 47; a 123 base pair deletion from nucleotides 3,024 to nucleotides 3,146 relative to reference sequence SEQ ID NO: 47; a 1467 base pair deletion from nucleotides 2,431 to nucleotides 3,897 relative to reference sequence SEQ ID NO: 47; a 1387 base pair deletion from nucleotides 3,145 to nucleotides 4,531 relative to reference sequence SEQ ID NO: 47; a 1382 base pair deletion from nucleotides 3,113 to nucleotides 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3,113; And a 1720 base pair deletion from nucleotide 2,440 to nucleotide 4,160 compared to the reference sequence SEQ ID NO: 47.

Thus, in the porcine animal and cell, the insertion or deletion in the gene encoding the CD163 protein is a 1506 base pair deletion from nucleotides 1525 to nucleotides 3030 relative to reference sequence SEQ ID NO: 47; a 1930 base pair deletion from nucleotides 488 to nucleotides 2417 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insertion starting at nucleotide 488 and an additional 129 base pair deletion from nucleotides 3044 to nucleotides 3172 in exon 7 relative to reference sequence SEQ ID NO: 47; a 1373 base pair deletion from nucleotides 2724 to nucleotides 4096 relative to reference sequence SEQ ID NO: 47; A 123 base pair deletion from nucleotides 3,024 to 3,146 relative to reference sequence SEQ ID NO: 47; a 1,467 base pair deletion from nucleotides 2,431 to 3,897 relative to reference sequence SEQ ID NO: 47; a 1,382 base pair deletion from nucleotides 3,113 to 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3,113; a 1,720 base pair deletion from nucleotides 2,440 to 4,160 relative to reference sequence SEQ ID NO: 47; and combinations thereof may include an in-frame deletion in exon 7 selected from the group consisting of:

The porcine animal or cell can comprise an insertion or deletion selected from the group consisting of: a 2 base pair insertion between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47 and a 377 base pair deletion from nucleotides 2,573 to nucleotides 2,949 relative to reference sequence SEQ ID NO: 47 on the same allele; a 28 base pair deletion from nucleotides 3,145 to nucleotides 3,172 relative to reference sequence SEQ ID NO: 47; and combinations thereof. For example, the animal or cell can comprise a 2 base pair insertion between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47 and a 377 base pair deletion from nucleotides 2,573 to nucleotides 2,949 relative to reference sequence SEQ ID NO: 47 on the same allele. The animal or cell can comprise a 28 base pair deletion from nucleotide 3,145 to nucleotide 3,172 relative to reference sequence SEQ ID NO: 47.

The porcine animal or cell can comprise a 7 base pair insertion between nucleotides 3,148 and 3,149 relative to reference sequence SEQ ID NO: 47 and an 11 base pair deletion from nucleotides 3,137 to 3,147 relative to reference sequence SEQ ID NO: 47.

A porcine animal or cell comprising any of the insertions or deletions described above can comprise a chromosomal sequence having a high degree of sequence identity to SEQ ID NO: 47 other than the insertion or deletion. Thus, for example, the porcine animal or cell can comprise a chromosomal sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 47 in a region of the chromosomal sequence other than the insertion or deletion.

The porcine animal or cell can comprise a chromosomal sequence comprising SEQ ID NO: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, or 114. As further described in the Examples below, SEQ ID NOs: 98-114 provide nucleotide sequences for regions corresponding to the regions of wild type porcine CD163 provided in SEQ ID NO: 47, and include insertions or deletions in the porcine CD163 chromosomal sequences described herein.

For example, the porcine animal or cell can comprise a chromosomal sequence comprising SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, or 114. SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, or 114 provides the nucleotide sequence for an in-frame deletion in exon 7 of the porcine CD163 chromosomal sequence.

As another example, a porcine animal or cell may comprise a chromosome sequence comprising sequence ID NO: 103 or 111.

The porcine animal or cell can comprise an 11 base pair deletion in one allele of a gene encoding CD163 protein and a 2 base pair insertion and a 377 base pair deletion in another allele of the gene encoding CD163 protein.

The porcine animal or cell can comprise a 124 base pair deletion in one allele of a gene encoding CD163 protein and a 123 base pair deletion in another allele of the gene encoding CD163 protein.

The pig animal or cell may contain a single base pair insertion.

The porcine animal or cell can comprise a 130 base pair deletion in one allele of a gene encoding CD163 protein and a 132 base pair deletion in another allele of the gene encoding CD163 protein.

A pig animal or cell may contain a 1506 base pair deletion.

A pig animal or cell may contain a 7 base pair insertion.

The porcine animal or cell can comprise a 1280 base pair deletion in one allele of a gene encoding CD163 protein and a 1373 base pair deletion in another allele of the gene encoding CD163 protein.

A pig animal or cell may contain a 1467 base pair deletion.

The porcine animal or cell can comprise a 1930 base pair intron 6 deletion from nucleotide 488 to nucleotide 2,417, a 12 base pair addition to nucleotide 4,488, and an additional 129 base pair deletion in exon 7.

The porcine animal or cell can comprise a 28 base pair deletion in one allele of a gene encoding CD163 protein and a 1387 base pair deletion in another allele of the gene encoding CD163 protein.

The porcine animal or cell can contain a 1382 base pair deletion and an 11 base pair insertion in one allele of a gene encoding CD163 protein and a 1720 base pair deletion in another allele of the gene encoding CD163 protein.

Any of the cells comprising at least one altered chromosomal sequence in a gene encoding CD163 protein may comprise a sperm cell. Alternatively, any of these cells may comprise an egg cell (e.g., a fertilized egg).

Any of the cells comprising at least one altered chromosomal sequence in a gene encoding CD163 protein may comprise a somatic cell. For example, any of the cells may comprise a fibroblast (e.g., a fetal fibroblast).

Targeted integration of nucleic acids at the CD163 locus

Site-specific integration of an exogenous nucleic acid into the CD163 locus can be accomplished by any technique known to those skilled in the art. For example, integration of an exogenous nucleic acid into the CD163 locus can comprise contacting a cell (e.g., an isolated cell or a cell of a tissue or organism) with a nucleic acid molecule comprising the exogenous nucleic acid. The nucleic acid molecule can comprise a nucleotide sequence flanking the exogenous nucleic acid that promotes homologous recombination between the nucleic acid molecule and at least one CD163 locus. The nucleotide sequence flanking the exogenous nucleic acid that promotes homologous recombination can be complementary to an endogenous nucleotide of the CD163 locus. Alternatively, the nucleotide sequence flanking the exogenous nucleic acid that promotes homologous recombination can be complementary to a previously integrated exogenous nucleotide. Multiple exogenous nucleic acids can be integrated into a single CD163 locus, such as in gene stacking.

Integration of the nucleic acid at the CD163 locus can be facilitated (e.g., catalyzed) by endogenous cellular machinery of the host cell, such as, but not limited to, endogenous DNA and endogenous recombinase enzymes. Alternatively, integration of the nucleic acid at the CD163 locus can be facilitated by one or more factors (e.g., polypeptides) provided to the host cell. For example, the nuclease(s), recombinase(s), and/or ligase polypeptides can be provided by contacting the polypeptide with the host cell, or by expressing the polypeptide within the host cell (either independently or as part of a chimeric polypeptide). Thus, a nucleic acid comprising a nucleotide sequence encoding at least one nuclease, recombinase, and/or ligase polypeptide can be introduced into a host cell simultaneously or sequentially with a nucleic acid that site-specifically integrates into the CD163 locus, wherein the at least one nuclease, recombinase, and/or ligase polypeptide is expressed from the nucleotide sequence in the host cell.

DNA-binding polypeptide

Site-specific integration can be accomplished, for example, by utilizing factors that can recognize and bind to specific nucleotide sequences in the genome of a host organism. For example, many proteins contain a polypeptide domain that can recognize and bind DNA in a site-specific manner. A DNA sequence that is recognized by a DNA-binding polypeptide can be referred to as a "target" sequence. A polypeptide domain that can recognize and bind DNA in a site-specific manner generally folds correctly and functions independently to bind DNA in a site-specific manner, even when expressed in a polypeptide other than the protein from which the domain was originally isolated. Similarly, a target sequence for recognition and binding by a DNA-binding polypeptide can generally be recognized and bound by such a polypeptide even when it is present in a large DNA structure (e.g., a chromosome), particularly if the site at which the target sequence is located is known to be accessible to soluble cellular proteins (e.g., a gene).

DNA-binding polypeptides identified from proteins that occur in nature typically bind to distinct nucleotide sequences or motifs (e.g., consensus recognition sequences), but methods exist and are known in the art for modifying many of these DNA-binding polypeptides to recognize different nucleotide sequences or motifs. DNA-binding polypeptides include, but are not limited to, zinc finger DNA-binding domains; leucine zippers; UPA DNA-binding domains; GAL4; TAL; LexA; Tet repressors; LacI; and steroid hormone receptors.

For example, the DNA-binding polypeptide may be a zinc finger. Individual zinc finger motifs can be designed to target and bind specifically to any of a wide variety of DNA sites. Canonical Cys ₂ His ₂ (as well as non-canonical Cys ₃ His) zinc finger polypeptides bind DNA by inserting their α-helices into the major groove of the target DNA double helix. Recognition of DNA by zinc fingers is modular; each finger primarily contacts three conserved base pairs in the target, and several key residues in the polypeptide mediate recognition. By including multiple zinc finger DNA-binding domains in a targeting endonuclease, the DNA-binding specificity of the targeting endonuclease can be further increased (and thus the specificity of any gene regulatory effect imparted thereby). See, e.g., Urnov et al. (2005) Nature 435:646-51. Thus, one or more zinc finger DNA-binding polypeptides can be engineered and utilized to cause a targeting endonuclease introduced into a host cell to interact with a unique DNA sequence within the genome of the host cell.

Preferably, the zinc finger protein is non-naturally occurring in that it is engineered to bind to a target site of choice. See, e.g., Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; See, e.g., 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528; 2005/0267061.

Engineered zinc finger binding domains can have novel binding specificities compared to naturally-occurring zinc finger proteins. Methods of engineering include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using a database comprising triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, wherein each triplet or quadruple nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to a particular triplet or quadruple sequence. See, e.g., U.S. Pat. Nos. 6,453,242 and 6,534,261.

Exemplary selection methods including phage display and two-hybrid systems are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB 2,338,237. Enhancement of binding specificity for zinc finger binding domains is also described, for example, in WO 02/077227.

Additionally, as disclosed in these and other references, zinc finger domains and/or multi-fingered zinc finger proteins may be linked together using any suitable linker sequence, including, for example, linkers that are at least 5 amino acids in length. See also U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences that are at least 6 amino acids in length. The proteins described herein may comprise any combination of suitable linkers between the individual zinc fingers of the protein.

Selection of Target Sites: Methods for designing and constructing ZFPs and fusion proteins (and polynucleotides encoding them) are known to those skilled in the art and are described in U.S. Pat. Nos. 6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

Additionally, as disclosed in these and other references, zinc finger domains and/or multi-fingered zinc finger proteins may be linked together using any suitable linker sequence, including, for example, linkers of at least 5 amino acids in length. See also U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences of at least 6 amino acids in length. The proteins described herein may comprise any combination of suitable linkers between the individual zinc fingers of the protein.

Alternatively, the DNA-binding polypeptide is a DNA-binding domain from GAL4. GAL4 is a modular transactivator in Saccharomyces cerevisiae , but it also functions as a transactivator in several other organisms. See, e.g., Sadowski et al. (1988) Nature 335:563-4. In this regulatory system, expression of genes encoding enzymes of the galactose mesa pathway in S. cerevisiae is tightly regulated by available carbon sources. See, e.g., Johnston (1987) Microbiol. Rev. 51:458-76. Transcriptional regulation of these metabolic enzymes is mediated by the interaction between a positive regulatory protein, GAL4, and a 17 bp symmetric DNA sequence to which GAL4 specifically binds (the upstream activating sequence (UAS)).

Native GAL4 consists of 881 amino acid residues with a molecular weight of 99 kDa. GAL4 contains functionally autonomous domains, the combined activities of which account for the activity of GAL4 in vivo [reviewed in; Ma and Ptashne (1987) Cell 48:847-53; Brent and Ptashne (1985) Cell 43(3 Pt 2):729-36]. The N-terminal 65 amino acids of GAL4 contain the GAL4 DNA-binding domain [reviewed in; Keegan et al. (1986) Science 231:699-704; Johnston (1987) Nature 328:353-5]. Sequence-specific binding requires the presence of a divalent cation coordinated by six Cys residues in the DNA-binding domain. The coordinated cation-containing domain recognizes and interacts with the conserved CCG triad at each end of the 17 bp UAS through direct contact with the major groove of the DNA helix [reviewed in; Marmorstein et al. (1992) Nature 356:408-14]. The DNA-binding function of the protein places the C-terminal transcription activation domain close to the promoter so that the activation domain can initiate transcription.

Additional DNA-binding polypeptides that may be used include, but are not limited to, binding sequences from an AVRBS3-inducible gene; a consensus binding sequence from an AVRBS3-inducible gene or a synthetic binding sequence engineered therefrom (e.g., a UPA DNA-binding domain); TAL; LexA (see, e.g., Brent & Ptashne (1985), supra); LacR (see, e.g., Labow et al. (1990) Mol. Cell. Biol. 10:3343-56; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88(12):5072-6); steroid hormone receptors (Ellliston et al. (1990) J. Biol. Chem. 265:11517-121); A Tet repressor (U.S. Pat. No. 6,271,341) and a mutated Tet repressor that binds to a Tet effector sequence in the presence but not the absence of tetracycline (Tc); the DNA-binding domain of NF-kappaB; and components of the regulatory system described in the literature [reference; Wang et al. (1994) Proc. Natl. Acad. Sci. USA 91(17):8180-4] utilizing a fusion of GAL4, a hormone receptor, and VP16.

The DNA-binding domain of one or more nucleases used in the methods and compositions described herein may comprise a naturally occurring or engineered (non-naturally occurring) TAL effector DNA binding domain. See, e.g., U.S. Patent Publication No. 2011/0301073.

Alternatively, the nuclease can comprise a CRISPR/Cas system. Such a system comprises a CRISPR (clustered regularly interspaced short palindromic repeats) locus, which encodes the RNA component of the system, and a Cas (CRISPR-associated) locus, which encodes a protein (Jansen et al., 2002. Mol. Microbiol. 43: 1565-1575; Makarova et al., 2002. Nucleic Acids Res. 30: 482-496; Makarova et al., 2006. Biol. Direct 1: 7; Haft et al., 2005. PLoS Comput. Biol. 1: e60). In a microbial host, the CRISPR locus contains a combination of Cas genes as well as non-coding RNA elements that can program the specificity of CRISPR-mediated nucleic acid cleavage.

Type II CRISPR is one of the best characterized systems and essentially performs targeted DNA double-strand breaks in four sequential steps. First, two non-coding RNAs, the pre-crRNA array and the tracrRNA, are transcribed from the CRISPR locus. Second, the tracrRNA hybridizes to the repeat region of the pre-crRNA and mediates processing of the pre-crRNA into mature crRNA containing an individual spacer sequence. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Wastson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA immediately flanked by the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of the target DNA to generate a double-strand break within the protospacer.

For the use of the CRISPR/Cas system to generate targeted insertions and deletions, the two non-coding RNAs (crRNA and TracrRNA) can be replaced by a single RNA, called a guide RNA (gRNA). The activity of the CRISPR/Cas system consists of three steps: (i) insertion of an exogenous DNA sequence into the CRISPR array to prevent future attacks in a process called "adaptation", (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the foreign nucleic acid. In bacterial cells, several Cas proteins are associated with the natural function of the CRISPR/Cas system and play a role in functions such as insertion of foreign DNA.

The Cas protein may be a "functional derivative" of a naturally occurring Cas protein. A "functional derivative" of a native sequence polypeptide is a compound that has qualitative biological properties in common with the native sequence polypeptide. A "functional derivative" includes, but is not limited to, fragments of the native sequence and derivatives of the native sequence polypeptide and fragments thereof, so long as they have biological activity in common with the corresponding native sequence polypeptide. The biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" includes both amino acid sequence variants of the polypeptide, covalent modifications and fusions thereof. Suitable derivatives of a Cas polypeptide or fragment thereof include, but are not limited to, mutants, fusions, covalent modifications of the Cas protein or fragment thereof. Cas proteins, including Cas proteins or fragments thereof, as well as derivatives of Cas proteins or fragments thereof, may be obtained from cells or may be synthesized chemically or by a combination of these two processes. The cell can be a cell that naturally produces the Cas protein, or a cell that naturally produces the Cas protein and has been genetically engineered to produce the Cas protein at a higher level of expression than the endogenous Cas protein or to produce the Cas protein from an exogenously introduced nucleic acid (wherein the nucleic acid encodes a Cas that is identical to or different from the endogenous Cas). In some cases, the cell does not naturally produce the Cas protein and has been genetically engineered to produce the Cas protein.

The DNA-binding polypeptide can specifically recognize and bind to a target nucleotide sequence contained within the genomic nucleic acid of a host organism. Multiple distinct instances of the target nucleotide sequence can be found in the host genome, in some instances. The target nucleotide sequence can be rare in the genome of the organism (e.g., less than about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 copy(s) of the target sequence can be present in the genome). For example, the target nucleotide sequence can be located at a unique site in the genome of the organism. The target nucleotide sequences can be, for example, without limitation, randomly distributed throughout the genome with respect to one another; located in different linkage groups in the genome; located in the same linkage group; located on different chromosomes; located on the same chromosome; located in the genome at sites that are expressed under similar conditions in the organism; may be located in close proximity to one another in the genome (e.g., the target sequence may be contained within a nucleic acid integrated as a concatemer at a genomic locus).

Targeting endonuclease

A DNA-binding polypeptide that specifically recognizes and binds to a target nucleotide sequence can be included in the chimeric polypeptide so as to confer specific binding to the target sequence to the chimeric polypeptide. In an example, such a chimeric polypeptide can include, for example, but not limited to, a nuclease, recombinase, and/or ligase polypeptide, as described above. A chimeric polypeptide comprising a DNA-binding polypeptide and a nuclease, recombinase, and/or ligase polypeptide can also include other functional polypeptide motifs and/or domains, such as, but not limited to: a spacer sequence located between the functional polypeptides in the chimeric protein; a leader peptide; a peptide that targets the fusion protein to an organelle (e.g., a nucleus); a polypeptide that is cleaved by a cellular enzyme; a peptide tag (e.g., Myc, His, etc.); and other amino acid sequences that do not interfere with the function of the chimeric polypeptide.

The functional polypeptides (e.g., the DNA-binding polypeptide and the nuclease polypeptide) in the chimeric polypeptide can be operably linked. The functional polypeptides of the chimeric polypeptide can be operably linked by their expression from a single polynucleotide encoding at least one functional polypeptide, each of which is ligated in-frame to produce a chimeric gene encoding the chimeric protein. Alternatively, the functional polypeptides of the chimeric polypeptide can be operably linked by other means, such as by cross-linking of independently expressed polypeptides.

A DNA-binding polypeptide, or guide RNA, that specifically recognizes and binds to a target nucleotide sequence can be comprised within a native isolated protein (or a mutant thereof), wherein the native isolated protein or mutant thereof also comprises a nuclease polypeptide (and can also comprise a recombinase and/or ligase polypeptide). Examples of such isolated proteins include TALENs, recombinases (e.g., Cre, Hin, Tre, and FLP recombinases), RNA-guided CRISPR/Cas9, and meganucleases.

The term "targeting endonuclease" as used herein refers to natural or engineered isolated proteins and mutants thereof comprising a DNA-binding polypeptide or guide RNA and a nuclease polypeptide, as well as chimeric polypeptides comprising a DNA-binding polypeptide or guide RNA and a nuclease. Any targeting endonuclease comprising a DNA-binding polypeptide or guide RNA that specifically recognizes and binds to a target nucleotide sequence comprised within the CD163 locus (e.g., because the target sequence is comprised within the native sequence in the locus or because the target sequence was introduced into the locus, e.g., by recombination) can be used.

Some examples of suitable chimeric polypeptides include, without limitation, combinations of the following polypeptides: zinc finger DNA-binding polypeptides; FokI nuclease polypeptides; TALE domains; leucine zippers; transcription factor DNA-binding motifs; and, for example, without limitation, DNA recognition and/or cleavage domains isolated from TALENs, recombinases (e.g., Cre, Hin, RecA, Tre, and FLP recombinases), RNA-guided CRISPR/Cas9, meganucleases; and others known in the art. Particular examples include chimeric proteins comprising a site-specific DNA binding polypeptide and a nuclease polypeptide. The chimeric polypeptide can be manipulated by methods known to those of skill in the art to alter the recognition sequence of the DNA-binding polypeptide included in the chimeric polypeptide so as to target the chimeric polypeptide to a particular nucleotide sequence of interest.

The chimeric polypeptide can comprise a DNA-binding domain (e.g., a zinc finger, a TAL-effector domain, etc.) and a nuclease (cleavage) domain. The cleavage domain can be heterologous to a DNA-binding domain, such as a zinc finger DNA-binding domain and cleavage domain from a nuclease, or a TALEN DNA-binding domain and cleavage domain, or a meganuclease DNA-binding domain and cleavage domain from a different nuclease. The heterologous cleavage domain can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which the cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, e.g., 2002-2003 Catalog, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes that cleave DNA are known (e.g., 51 nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of the cleavage domain and cleavage half-domain.

Similarly, the cleavage half-domains can be derived from any of the nucleases described above, or portions thereof, that require dimerization for cleavage activity. Typically, two fusion proteins are required for cleavage if the fusion protein comprises a cleavage half-domain. Alternatively, a single protein comprising two cleavage half-domains can be used. The two cleavage half-domains can be derived from the same endonuclease (or functional fragment thereof), or each cleavage half-domain can be derived from a different endonuclease (or functional fragment thereof). Furthermore, the target sites for the two fusion proteins are positioned relative to each other such that binding of the two fusion proteins to their respective target sites positions the cleavage half-domains in a spatial orientation that allows the cleavage half-domains to form a functional cleavage domain, for example, by dimerization. Thus, the edges of the target sites can be separated by 5-8 nucleotides, or by 15-18 nucleotides. However, any integer number of nucleotides, or nucleotide pairs, may intervene between the two target sites (e.g., from 2 to 50 nucleotide pairs or more). Typically, the site of cleavage is between the target sites.

Restriction endonucleases (restriction enzymes) are present in many species and can sequence-specifically bind DNA (at a recognition site) and cleave DNA at or near the ligation site, for example, to allow one or more exogenous sequences (donor/transgenes) to be incorporated at or near the ligation (target) site. Certain restriction enzymes (e.g., type IIS) cleave DNA at a site removed from the recognition site and have separable binding and cleavage domains. For example, the type IIS enzyme Fok I catalyzes double-strand cleavage of DNA 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. See, e.g., U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31,978-31,982]. Thus, the fusion protein will comprise a cleavage domain (or cleavage half-domain) from at least one type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered.

An exemplary type IIS restriction enzyme in which the cleavage domain is separable from the binding domain is Fok I. This particular enzyme is active as a dimer [see; Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575]. Thus, for the purposes of the present invention, a portion of the Fok I enzyme used in the disclosed fusion proteins is considered a cleavage half-domain. Thus, for targeted replacement of cellular sequences and/or targeted double-strand cleavage using zinc finger-Fok I fusions, two fusion proteins, each containing a Fok I cleavage half-domain, may be used to reconstitute a catalytically active cleavage domain. Alternatively, a single polypeptide molecule containing a DNA binding domain and two Fok I cleavage half-domains may also be used.

A cleavage domain or cleavage half-domain can be any portion of a protein that possesses cleavage activity or the ability to multimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in U.S. Patent Publication No. 2007/0134796. Additional restriction enzymes also contain separable binding and cleavage domains and are contemplated by the present invention. See, e.g., Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

The cleavage domain may comprise one or more engineered cleavage half-domains (also called dimerization domain mutants) that minimize or prevent homodimerization, as described, for example, in U.S. Patent Publication Nos. 2005/0064474; 2006/0188987 and 2008/0131962.

Alternatively, the nuclease can be assembled in vivo at the nucleic acid target site using so-called "split-enzyme" technology (see, e.g., U.S. Patent Publication No. 20090068164). The components of such a split enzyme can be expressed on separate expression constructs, or the individual components can be conjugated in a single open reading frame separated by, for example, a self-cleaving 2A peptide or an IRES sequence. The components can be individual zinc finger binding domains or domains of a meganuclease nucleic acid binding domain.

Zinc finger nuclease

The chimeric polypeptide can include a custom-designed zinc finger nuclease (ZFN) that can be designed to deliver targeted site-specific double-stranded DNA cleavage to allow incorporation of an exogenous nucleic acid, or donor DNA (see US Patent Publication 2010/0257638). ZFNs are chimeric polypeptides containing a non-specific cleavage domain from a restriction endonuclease (e.g., FokI) and a zinc finger DNA-binding domain polypeptide. See, e.g., Huang et al. (1996) J. Protein Chem. 15:481-9; Kim et al. (1997a) Proc. Natl. Acad. Sci. USA 94:3616-20; Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93:1156-60; Kim et al. (1994) Proc Natl. Acad. Sci. USA 91:883-7; Kim et al. (1997b) Proc. Natl. Acad. Sci. USA 94:12875-9; Kim et al. (1997c) Gene 203:43-9; Kim et al. (1998) Biol. Chem. 379:489-95; Nahon and Raveh (1998) Nucleic Acids Res. 26:1233-9; Smith et al. (1999) Nucleic Acids Res. 27:674-81]. ZFNs can comprise a non-canonical zinc finger DNA binding domain (see US Patent Publication 2008/0182332). The FokI restriction endonuclease must dimerize through the nuclease domain to cleave DNA and introduce double-strand breaks. Thus, ZFNs containing nuclease domains from these endonucleases also require dimerization of the nuclease domains to cleave target DNA (see; Mani et al. (2005) Biochem. Biophys. Res. Commun. 334:1191-7; Smith et al. (2000) Nucleic Acids Res. 28:3361-9). Dimerization of ZFNs can be promoted by two adjacent, oppositely oriented DNA-binding sites. Id.

A method for site-specific integration of an exogenous nucleic acid into at least one CD163 locus of a host can comprise introducing a ZFN into a cell of the host, wherein the ZFN recognizes and binds to a target nucleotide sequence, wherein the target nucleotide sequence is comprised within at least one CD163 locus of the host. In certain instances, the target nucleotide sequence is not comprised within the genome of the host at a location other than the at least one CD163 locus. For example, the DNA-binding polypeptide of the ZFN can be engineered to recognize and bind to a target nucleotide sequence identified within the at least one CD163 locus (e.g., by sequencing the CD163 locus). Methods for site-specific integration of an exogenous nucleic acid into at least one CD163 performance locus of a host, comprising introducing a ZFN into a cell of the host, can also comprise introducing the exogenous nucleic acid into the cell, wherein recombination of the exogenous nucleic acid into a nucleic acid of the host comprising at least one CD163 locus is facilitated by site-specific recognition and binding of the ZFN to the target sequence (and subsequent cleavage of the nucleic acid comprising the CD163 locus).

Any exogenous nucleic acid for integration into the CD163 locus

Exogenous nucleic acids for integration at the CD163 locus include: an exogenous nucleic acid for site-specific integration at at least one CD163 locus, such as, but not limited to, an ORF; a nucleic acid comprising a nucleotide sequence encoding a targeting endonuclease; and a vector comprising at least one or both of the foregoing. Thus, certain nucleic acids include a nucleotide sequence encoding a polypeptide, a structural nucleotide sequence, and/or a DNA-binding polypeptide recognition and binding site.

Any exogenous nucleic acid molecule for site-specific integration

As noted above, insertion of an exogenous sequence (also referred to as a "donor sequence" or "donor" or "transgene") provides, for example, for expression of a polypeptide, for correction of a mutant gene, or for increased expression of a nocturnal gene. It will be readily apparent that the donor sequence is typically not identical to the genomic sequence into which it is placed. The donor sequence may contain non-homologous sequences flanked by two regions of homology to enable efficient homology-directed repair (HDR) at the site of interest. In addition, the donor sequence may comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin. The donor molecule may contain several discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of a sequence that is not normally present in the region of interest, such sequence may be present in the donor nucleic acid molecule and may be flanked by sequences of homology to the region of interest.

The donor polynucleotide may be DNA or RNA, single-stranded or double-stranded, and may be introduced into the cell in a linear or circular form. See, e.g., U.S. Patent Publication Nos. 2010/0047805, 2011/0281361, 2011/0207221, and 2013/0326645. If introduced linearly, the termini of the donor sequence may be protected (e.g., from degradation by nucleotide termini) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues are added to the 3' end of the linear molecule and/or a self-complementary oligonucleotide is ligated to one or both of the termini. See, e.g., Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; See Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and use of modified internucleotide linkages, such as phosphorothionates, phosphoramidates, and O-methyl ribose or deoxyribose moieties.

The polynucleotide may be introduced into the cell as part of a vector molecule having additional sequences, such as genes encoding, for example, a replication origin, a promoter, and antibiotic resistance. Additionally, the donor polynucleotide may be introduced as a naked nucleic acid, as a nucleic acid complexed with an agent such as a liposome or a poloxamer, or may be delivered by a virus (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus, and integrase defective lentivirus (IDLV)).

The donor is generally integrated such that its expression is driven by an endogenous promoter at the site of integration, i.e., by a promoter (e.g., CD163) that drives expression of the endogenous gene into which the donor has been integrated. However, it will be appreciated that the donor may comprise promoters and/or enhancers, e.g., constitutive promoters or inducible or tissue-specific promoters.

Furthermore, although not required for expression, the exogenous sequence may also include sequences encoding transcriptional or translational regulatory sequences, such as a promoter, enhancer, insulator, internal ribosome entry site, 2A peptide and/or polyadenylation signal.

Exogenous nucleic acids that can be integrated into at least one CD163 locus in a site-specific manner to modify the CD163 locus include, for example and without limitation, a nucleic acid comprising a nucleotide sequence encoding a polypeptide of interest; a nucleic acid comprising a crop gene; a nucleic acid comprising a nucleotide sequence encoding an RNAi molecule; or a nucleic acid that cleaves a CD163 gene.

An exogenous nucleic acid can be incorporated into the CD163 locus to modify the CD163 locus, wherein the nucleic acid comprises a nucleotide sequence encoding a polypeptide of interest such that the nucleotide sequence is expressed from the CD163 locus in a host. In some instances, the polypeptide of interest (e.g., a foreign protein) is expressed from a nucleotide sequence encoding the polypeptide of interest in commercial quantities. In such instances, the polypeptide of interest can be extracted from a host cell, tissue, or biomass.

A nucleic acid molecule comprising a nucleotide sequence encoding a targeting endonuclease

The nucleotide sequence encoding the targeting endonuclease can be manipulated by manipulation (e.g., ligation) of the native nucleotide sequence encoding the polypeptide comprised within the targeting endonuclease. For example, the nucleotide sequence of a gene encoding a protein comprising a DNA-binding polypeptide can be examined to identify a nucleotide sequence of the gene corresponding to the DNA-binding polypeptide, and the nucleotide sequence can be used as a component of the nucleotide sequence encoding the targeting endonuclease comprising the DNA-binding polypeptide. Alternatively, the amino acid sequence of the targeting endonuclease can be used to deduce the nucleotide sequence encoding the targeting endonuclease, for example, by degeneracy of the genetic code.

In an exemplary nucleic acid molecule comprising a nucleotide sequence encoding a targeting endonuclease, the last codon of the first polynucleotide sequence encoding the nuclease polypeptide and the first codon of the second polynucleotide sequence encoding the DNA-binding polypeptide can be separated by any number of nucleotide triplets, e.g., without coding for an intron or a "STOP." Likewise, the last codon of the nucleotide sequence encoding the first polynucleotide sequence encoding the DNA-binding polypeptide and the first codon of the second polynucleotide sequence encoding the nuclease polypeptide can be separated by any number of nucleotide triplets. The last codon (i.e., usually 3' in the nucleic acid sequence) of a first polynucleotide sequence encoding a nuclease polypeptide and a second polynucleotide sequence encoding a DNA-binding polypeptide can be fused in phase register with the first codon of an immediately adjacent additional polynucleotide coding sequence, or can be separated therefrom only by a short peptide sequence, such as encoded by a synthetic nucleotide linker (e.g., a nucleotide linker that can be used to effect the fusion). Examples of such additional polynucleotide sequences include, without limitation, tags, targeting peptides, and enzymatic cleavage sites. Likewise, the first codon, usually 5' (in the nucleic acid sequence), of the first and second polynucleotide sequences can be fused in phase register with the last codon of an immediately adjacent additional polynucleotide coding sequence, or can be separated therefrom only by a short peptide sequence.

The sequence separating the polynucleotide sequence encoding the functional polypeptide (e.g., the DNA-binding polypeptide and the nuclease polypeptide) from the targeting endonuclease can be comprised of any sequence that does not significantly alter the translation of the encoded amino acid sequence of the targeting endonuclease, for example. Because of the autonomous nature of the known nuclease polypeptide and the known DNA-binding polypeptide, the intervening sequence will not interfere with the respective function of these structures.

How to knock out other guitars

Various other techniques known in the art can be used to inactivate genes to create knock-out animals and/or to introduce nucleic acid constructs into animals to produce founder animals and create animal lines, wherein the knockout or nucleic acid construct is integrated into the genome. These techniques include, but are not limited to, pronuclear microinjection (U.S. Pat. No. 4,873,191), retroviral-mediated gene transfer to the germ line (Van der Putten et al. (1985) Proc. Natl. Acad. Sci. USA 82, 6148-1652), gene targeting to embryonic stem cells (Thompson et al. (1989) Cell 56, 313-321), electroporation of embryos (Lo (1983) Mol. Cell. Biol. 3, 1803-1814), sperm-mediated gene transfer (Lavitrano et al. (2002) Proc. Natl. Acad. Sci. USA 99, 14230-14235; Lavitrano et al. (2006) Reprod. Fert. Develop. 18, 19-23), and somatic cells, such as ovarian or mammary cells, or adult, fetal, or embryonic stem cells followed by nuclear transfer (Wilmut et al. (1997) Nature 385, 810-813; and Wakayama et al. (1998) Nature 394, 369-374). Pronuclear microinjection, sperm-mediated gene transfer, and somatic cell nuclear transfer are particularly useful techniques. A genomically modified animal is one in which all of its cells, including its germline cells, have the genetic modification. If a method is used to produce mosaic animals in their genetic modification, the animals can be inbred and the genomically modified offspring selected for. For example, cloning can be used to produce mosaic animals if the cells are modified in the blastocyst stage, or if a single cell is modified, the genomic modification can occur. The modified animal that fails to reach sexual maturity can be either homozygous or heterozygous for the modification, depending on the particular approach used. If a particular gene is inactivated by a knockout mutation, homozygosity will normally be required. If a particular gene is inactivated by RNA interference or a dominant negative strategy, heterozygosity is often appropriate.

Typically, in embryo/zygote microinjection, a nucleic acid construct or mRNA is introduced into the zygote; a one- or two-cell zygote is used as a nuclear structure containing genetic material from the sperm head, and the oocyte is visible within the protoplasm. The zygote in the pronuclear state can be obtained in vitro or in vivo (i.e., surgically recovered from the oviduct of a donor animal). In vitro zygotes can be generated as follows. For example, porcine ovaries can be collected from an slaughterhouse and maintained at 22-28°C during transport. The ovaries can be washed and isolated for follicular aspiration, and follicles measuring 4-8 mm can be aspirated into a 50 mL conical centrifuge tube using an 18 gauge needle under vacuum. The follicular fluid and aspirated oocytes can be washed through a pre-filter with commercially available TL-HEPES (Minitube, Verona, Wis.). Oocytes surrounded by a compact cumulus mass can be selected and placed in _TCM -199 Oocyte MATURATION MEDIUM (Minitube, Verona, Wis.) supplemented with 0.1 mg/mL cysteine, 10 ng/mL epidermal growth factor, 10% porcine follicular fluid, 50 μM 2-mercaptoethanol, 0.5 mg/mL cAMP, and 10 IU/mL each of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) in humidified air at 38.7°C and 5% CO2 for approximately 22 hours. Oocytes can then be transferred to fresh TCM-199 maturation medium (which will not contain cAMP, PMSG, or hCG) and cultured for an additional 22 hours. Mature oocytes can be stripped from their cumulus cells by vortexing in 0.1% hyaluronidase for 1 minute.

In pigs, mature oocytes can be fertilized in 500 μl Minitube PORCPRO IVF MEDIUM SYSTEM (Minitube, Verona, Wis.) in a Minitube 5-well fertilization dish. In preparation for in vitro fertilization (IVF), freshly-collected or frozen collected sperm can be washed and resuspended in PORCPRO IVF Medium to a concentration of 400,000 sperm. Sperm concentration can be analyzed by computer-assisted semen analysis (SPERMVISION, Minitube, Verona, Wis.). Final in vitro In vitro insemination can be performed with a final concentration of approximately 40 motile sperm/oocyte in a volume of 10 μL, depending on the boar. All fertilized oocytes are incubated for 6 h at 38.7°C in a 5.0% CO ₂ atmosphere. Six hours after insemination, putative zygotes can be washed twice in NCSU-23 and transferred to 0.5 mL of the same medium. This system can routinely produce 20-30% blastocysts across most boars with a polyspermic insemination rate of 10-30%.

The linearized nucleic acid construct or mRNA can be injected into one of the pronuclei or into the cytoplasm. The injected eggs can then be transferred to a recipient female (e.g., into the oviduct of the recipient female) and grown in the recipient female to produce transgenic or gene-edited animals. In particular, the in vitro fertilized embryos can be centrifuged at 15,000 x g for 5 minutes to sediment lipids and allow visualization of the pronuclei. The embryos can be injected using an Eppendorf FEMTOJET syringe and cultured until blastocyst formation. The rate and quality of embryo cleavage and blastocyst formation can be recorded.

Embryos can be surgically transferred into the uterus of an asynchronous recipient. Typically, 100-200 (e.g., 150-200) embryos are placed into the ampulla-isthmus junction of the fallopian tubes using a 5.5-inch TOMCAT® catheter. After the procedure, real-time ultrasound testing of pregnancy can be performed.

In somatic cell nuclear transfer, a combined cell can be established by introducing a transgenic or gene-edited cell, such as an embryonic blastomere, fetal fibroblast, adult ear fibroblast, or granulosa cell, containing the nucleic acid construct described above into an enucleated oocyte. The oocyte can be enucleated by making a partial section incision near the polar body followed by squeezing the cytoplasm from the incision. Typically, a syringe pipette with a sharp beveled tip is used to inject the transgenic or gene-edited cell into an enucleated oocyte that has been arrested in meiosis II. In some practices, the oocyte that has been arrested in meiosis II is referred to as an oocyte. After producing a porcine or bovine embryo (e.g., by fusing and activating the oocyte), the embryo is transferred into the oviduct of the recipient female about 20 to 24 hours after activation. See, for example, Cibelli et al. (1998) Science 280, 1256-1258 and U.S. Pat. Nos. 6,548,741, 7,547,816, 7,989,657, or 6,211,429. In pigs, pregnancy can be confirmed in the recipient female approximately 20-21 days after embryo transfer.

Standard breeding practices can be used to produce animals homozygous for the inactivated gene from an initial heterozygous progenitor animal. However, homozygosity may not be necessary. The gene-edited pigs described herein can be bred with other pigs of interest.

Once the gene-edited animal is created, inactivation of the endogenous nucleic acid can be assessed using standard techniques. Initial screening can be accomplished by Southern blot analysis to determine whether inactivation has occurred or not. For a description of Southern analysis, see sections 9.37-9.52 of Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Press, Plainview; N.Y. Polymerase chain reaction (PCR) techniques can also be used for initial screening. PCR refers to a process or technique for amplifying a target nucleic acid. Typically, sequence information from the end of or beyond the region of interest is used to design oligonucleotide primers that are identical or similar in sequence to the opposite strand of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers are typically 14 to 40 nucleotides long, but can range from 10 nucleotides to several hundred nucleotides in length. PCR is described, for example, in PCR Primer: A Laboratory Manual, ed. Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. Nucleic acids can also be amplified by ligase chain reaction, strand displacement amplification, self-sustaining sequence replication, or nucleic acid sequence-based amplification. See, for example, Lewis (1992) Genetic Engineering News 12,1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874; and Weiss (1991) Science 254:1292. At the blastocyst stage, embryos can be individually processed for analysis by PCR, Southern hybridization and splinkerette PCR (see, e.g., Dupuy et al. Proc Natl Acad Sci USA (2002) 99:4495).

Interfering RNAs

A variety of RNA interference (RNAi) systems are known. Double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous gene transcripts. The RNA-induced silencing complex (RISC) metabolizes the dsRNA into small 21-23 nucleotide small interfering RNAs (siRNAs). The RISC contains a double-stranded RNAse (dsRNase, e.g., Dicer) and an ssRNase (e.g., Argonaut 2 or Ago2). The RISC uses the antisense strand as a guide to find a cleavable target. Both siRNAs and microRNAs (miRNAs) are known. A method of inactivating a gene in a gene-edited animal comprises inducing RNA interference against a target gene and/or nucleic acid such that expression of the target gene and/or nucleic acid is reduced.

For example, the exogenous nucleic acid sequence can induce RNA interference against a nucleic acid encoding a polypeptide. For example, double-stranded small interfering RNA (siRNA) or small hairpin RNA (shRNA) homologous to the target DNA can be used to reduce expression of that DNA. Constructs for siRNA can be prepared as described, for example, in the literature [see, e.g., Fire et al. (1998) Nature 391:806; Romano and Masino (1992) Mol. Microbiol. 6:3343; Cogoni et al. (1996) EMBO J. 15:3153; Cogoni and Masino (1999) Nature 399:166; Misquitta and Paterson (1999) Proc. Natl. Acad. Sci. USA 96:1451; and Kennerdell and Carthew (1998) Cell 95:1017]. Constructs for shRNAs can be prepared as described in the literature [reference; McIntyre and Fanning (2006) BMC Biotechnology 6:1]. Generally, shRNAs are transcribed as single-stranded RNA molecules containing complementary regions that can anneal to form short hairpins.

The probability of finding a single, individual functional siRNA or miRNA directed to a specific gene is high. For example, the predictability of the specific sequence of an siRNA is about 50%, but multiple interfering RNAs can be made with good confidence that at least one of them will be effective.

In vitro cells, in vivo cells, or gene-edited animals, for example, livestock animals expressing RNAi directed against a gene encoding CD163, can be used. The RNAi can be selected from the group consisting of, for example, siRNA, shRNA, dsRNA, RISC and miRNA.

Inductive system

Inducible systems can be used to inactivate the CD163 gene. A variety of inducible systems are known that allow spatial and temporal control of gene inactivation. Some have been shown to be functional in vivo in porcine animals.

An example of an inducible system is the tetracycline (tet)-on promoter system, which can be used to control the transcription of nucleic acids. In this system, a mutated Tet repressor (TetR) is fused to the activation domain of the herpes simplex virus VP 16 transcription-activator protein to produce a tetracycline-regulated transcription activator (tTA), which is regulated by tet or doxycycline (dox). In the absence of the antibiotic, transcription is minimal, whereas in the presence of tet or dox, transcription is induced. Alternative inducible systems include the ecdysone or rapamycin systems. Ecdysone is an insect moulting hormone whose production is controlled by heterodimerization of the ecdysone receptor with the product of the ultraspiracle gene (USP). Expression is induced by treatment with ecdysone or an analog of ecdysone, such as muristerone A. The agent administered to the animal to trigger the inducible system is called an inducer.

Among the more commonly used inducible systems are the tetracycline-inducible system and the Cre/loxP recombinase system (either constitutive or inducible). The tetracycline-inducible system comprises a tetracycline-regulated transcription activator (tTA)/reverse tTA (rtTA). A method for using this system in vivo involves generating two strains of gene-edited animals. One strain of animals expresses the activator (tTA, rtTA, or Cre recombinase) under the control of a selected promoter. The other strain of animals expresses the receptor, wherein expression of the gene of interest (or the gene to be modified) is under the control of a target sequence for the tTA/rtTA transcription activator (or is flanked by loxP sequences). Mating the two animals provides control of gene expression.

The tetracycline-dependent regulatory system (tet system) relies on two components, a tetracycline-regulated transcription activator (tTA or rtTA) and a tTA/rtTA-dependent promoter that controls expression of downstream cDNA in a tetracycline-dependent manner. In the absence of tetracycline or its derivatives (e.g. doxycycline), tTA binds to the tetO sequence, allowing transcriptional activation of the tTA-dependent promoter. However, in the presence of doxycycline, tTA cannot interact with its target and transcription does not occur. The tet system using tTA is referred to as tet-OFF because tetracycline or doxycycline allows transcriptional down-regulation. Administration of tetracycline or its derivatives allows temporal control of transgene expression in vivo. rtTA is a mutant of tTA that is nonfunctional in the absence of doxycycline but requires the presence of a ligand for transcriptional activation. Therefore, this tet system is called tet-ON. The tet system has been used in vivo for the inducible expression of several transgenes, for example, encoding reporter genes, oncogenes, or proteins involved in signaling cascades.

The Cre/lox system uses Cre recombinase to catalyze site-specific recombination by crossing over between two separate Cre recognition sequences, i.e., loxP sites. The DNA sequence introduced between the two loxP sequences (called floxed DNA) is excised by Cre-mediated recombination. Control of Cre expression in transgenic and/or gene-edited animals is achieved by spatial control (via tissue- or cell-specific promoters) or temporal control (via inducible systems), allowing controlled excision of DNA between the two loxP sites. One application is for conditional gene inactivation (conditional knockout). Another approach is for protein overexpression, where a floxed stop codon is inserted between the promoter sequence and the DNA of interest. The gene-edited animal does not express the transgene until Cre is expressed, resulting in excision of the floxed stop codon. This system has been applied to tissue-specific tumorigenesis and controlled antigen receptor expression in B lymphocytes. Inducible Cre recombinases have also been developed. Inducible Cre recombinase is activated only by the administration of an exogenous ligand. Inducible Cre recombinase is a fusion protein containing the native Cre recombinase and a specific ligand-binding domain. The functional activity of Cre recombinase depends on the ability of the exogenous ligand to bind to this specific domain in the fusion protein.

In vitro cells, in vivo cells, or gene-edited animals, such as domestic animals, containing the CD163 gene under the control of an inducible system can be used. The genetic modification of the animal can be genomic or mosaic. The inducible system can be selected from the group consisting of, for example, Tet-On, Tet-Off, Cre-lox, and Hif1 alpha.

Vectors and Nucleic Acids

A variety of nucleic acids can be introduced into cells for knockout purposes, for inactivation of a gene, for obtaining expression of a gene, or for other purposes. As used herein, the term nucleic acid includes DNA, RNA, and nucleic acid analogs, and double-stranded or single-stranded (i.e., sense or antisense single-stranded) nucleic acids. Nucleic acid analogs can be modified at the base moiety, the sugar moiety, or the phosphate backbone, for example, to improve the stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine and 5-bromo-2'-doxycytidine for deoxycytidine. Modifications at the sugar moiety include modification of the 2' hydroxyl of the ribose sugar to form a 2'-O-methyl or 2'-O-allyl sugar. The deoxyribose phosphate backbone can be modified to produce a morpholino nucleic acid, wherein each base moiety is linked to a six-atom, a morpholino ring, or a peptide nucleic acid, and the deoxyphosphate backbone is replaced with a pseudopeptide backbone and the four bases are retained. See Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7(3):187; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5. Additionally, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.

The target nucleic acid sequence can be operably linked to a regulatory region, such as a promoter. The regulatory region can be a porcine regulatory region or can be from another species. As used herein, positioning a regulatory region relative to a nucleic acid sequence in such a way that it permits or promotes transcription of the target nucleic acid to which it is operably linked.

Any type of promoter can be operably linked to the target nucleic acid sequence. Examples of promoters include, but are not limited to, tissue-specific promoters, constitutive promoters, inducible promoters, and promoters that are responsive or unresponsive to a particular stimulus. Suitable tissue-specific promoters can cause preferential expression of the nucleic acid transcript in beta cells, and include, for example, the human insulin promoter. Other tissue-specific promoters can cause preferential expression in hepatocytes or cardiac tissue, and include, for example, the albumin or alpha-myosin heavy chain promoters, respectively. Promoters that promote expression of a nucleic acid molecule without significant tissue or time-specificity can be used (i.e., constitutive promoters). For example, a beta-actin promoter, such as the chicken beta-actin gene promoter, a ubiquitin promoter, a miniCAGs promoter, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, or a 3-phosphoglycerate kinase (PGK) promoter, as well as a viral promoter, such as the herpes simplex virus thymidine kinase (HSV-TK) promoter, the SV40 promoter, or the cytomegalovirus (CMV) promoter, can be used. For example, a fusion of the chicken beta-actin gene promoter and the CMV enhancer can be used as a promoter. See, e.g., Xu et al. (2001) Hum. Gene Ther. 12:563; and Kiwaki et al. (1996) Hum. Gene Ther. 7:821.

Additional regulatory regions that may be useful in the nucleic acid construct include, but are not limited to, polyadenylation sequences, translational regulatory sequences (e.g., internal ribosome entry segments, IRES), enhancers, inducer elements, or introns. Although such regulatory regions may increase expression by affecting transcription, mRNA stability, translation efficiency, etc., they may not be essential. Such regulatory regions may be included in the nucleic acid construct as desired to obtain optimal expression of the nucleic acid in the cell(s). However, sufficient expression can sometimes be obtained without these additional elements.

Nucleic acid constructs encoding a single peptide or selectable marker can be used. A single peptide can be used that directs the encoded polypeptide to a specific cellular location, such as the cell surface. Non-limiting examples of selectable markers include puromycin, ganciclovir, adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase, thymidine kinase (TK), and xanthine-guanine phosphoribosyltransferase (XGPRT). Such markers are useful for selecting stable transformants in culture. Other selectable markers include fluorescent polypeptides, such as green fluorescent protein or yellow fluorescent protein.

The sequence encoding the selectable marker may be flanked by recognition sequences for a recombinase, such as Cre or Flp. For example, the selectable marker may be flanked by loxP recognition sites (a 34-bp recognition site recognized by Cre recombinase) or FRT recognition sites, such that the selectable marker can be excised from the construct. See Orban, et al., Proc. Natl. Acad. Sci. (1992) 89:6861, for a review of Cre/lox technology, and Brand and Dymecki, Dev. Cell (2004) 6:7. Transposons containing a Cre- or Flp-activatable transgene interrupted by a selectable marker gene may also be used to obtain animals with conditional expression of the transgene. For example, promoter-driven expression of the marker/transgene can be ubiquitous or tissue-specific, which will result in ubiquitous or tissue-specific expression of the marker in F0 animals (e.g., pigs). Tissue-specific activation of the transgene can be achieved, for example, by breeding pigs that ubiquitously express the marker-blocked transgene to pigs that express Cre or Flp in a tissue-specific manner, or by breeding pigs that express the marker-blocked transgene in a tissue-specific manner to pigs that ubiquitously express Cre or Flp recombinase. Controlled expression of the transgene or controlled excision of the marker allows for expression of the transgene.

The exogenous nucleic acid can encode a polypeptide. The nucleic acid sequence encoding the polypeptide can include a tag sequence encoding a "tag" designed to facilitate subsequent manipulation of the encoded polypeptide (e.g., to facilitate localization or detection). The tag sequence can be inserted into the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at the carboxyl or amino terminus of the polypeptide. Non-limiting examples of encoded tags include glutathione S-transferase (GST) and the FLAG ^™ tag (Kodak, New Haven, Conn.).

Nucleic acid constructs can be methylated using SssI CpG methylase (New England Biolabs, Ipswich, Mass.). Typically, the nucleic acid construct is incubated with S-adenosylmethionine and SssI CpG-methylase in buffer at 37°C. Hypermethylation can be confirmed by incubating the construct with one unit of HinP1I endonuclease at 37°C for 1 hour and analyzing by agarose gel electrophoresis.

Nucleic acid constructs can be introduced into any type of embryonic, fetal, or adult animal cell, including, for example, germ cells, e.g., oocytes or egg cells, progenitor cells, adult or embryonic stem cells, primordial germ cells, kidney cells, e.g., PK-15 cells, islet cells, beta cells, liver cells, or fibroblasts, e.g., skin fibroblasts, using a variety of techniques. Non-limiting examples of techniques include the use of transposon systems, recombinant viruses capable of infecting cells or liposomes, or other non-viral methods for delivering nucleic acids to cells, e.g., electroporation, microinjection, or calcium phosphate precipitation.

In a transposon system, the transcriptional unit of a nucleic acid construct, i.e., a regulatory region operably linked to an exogenous nucleic acid sequence, is flanked by inverted repeats of the transposon. Several transposon systems have been developed to introduce nucleic acids into cells, including mouse, human, and porcine cells, including Sleeping Beauty (see U.S. Pat. No. 6,613,752 and U.S. Publication No. 2005/0003542); Frog Prince (Miskey et al. (2003) Nucleic Acids Res. 31:6873); Tol2 (Kawakami (2007) Genome Biology 8(Suppl. 1):S7; Minos (Pavlopoulos et al. (2007) Genome Biology 8(Suppl. 1):S2); Hsmar1 (Miskey et al. (2007)) Mol Cell Biol. 27:4589); and Passport. Sleeping Beauty transposons are particularly useful. The transposase can be delivered as a protein, encoded on the same nucleic acid construct as an exogenous nucleic acid, introduced on a separate nucleic acid construct, or provided as mRNA (e.g., in vitro transcribed and capped mRNA).

Insulator elements may also be included in the nucleic acid construct to maintain expression of the exogenous nucleic acid and to suppress unwanted transcription of the host gene. See, e.g., U.S. Publication No. 2004/0203158. Typically, the insulator elements flank each side of the transcription unit and are internal to the inverted repeats of the transposon. Non-limiting examples of insulator elements include matrix attachment region- (MAR) type insulator elements and border-type insulator elements. See, e.g., U.S. Pat. Nos. 6,395,549, 5,731,178, 6,100,448, and 5,610,053, and U.S. Publication No. 2004/0203158.

Nucleic acids can be inserted into a vector. A vector is a broad term that includes any specific DNA segment designed to move from a carrier to a target DNA. A vector can refer to an expression vector, or a vector system, which is a set of components necessary to effect DNA insertion into a genome or other targeted DNA sequence, such as an episome, a plasmid, or even a viral/phage DNA segment. Vector systems, such as viral vectors (e.g., retroviruses, adeno-associated viruses, and integrating phage viruses) used for gene transfer in animals, or non-viral vectors (e.g., transposons), have two basic components: 1) a vector consisting of DNA (or RNA that is reverse transcribed into cDNA) and 2) a transposase, recombinase, or other integrase enzyme that recognizes both the vector and the DNA target sequence and inserts the vector into the target DNA sequence. The vector most often contains one or more expression cassettes, which include one or more expression control sequences, wherein the expression control sequences are DNA sequences that control and regulate the transcription and/or translation of a different DNA sequence or mRNA, respectively.

Many different types of vectors are known. For example, plasmids and viral vectors, e.g., retroviral vectors, are known. Mammalian expression plasmids typically have an origin of replication, a suitable promoter and optionally an enhancer, essential ribosome binding sites, a polyadenylation site, splicing donor and acceptor sites, a transcription termination sequence, and 5' flanking non-transcribed sequences. Examples of vectors include: plasmids (which may be carriers for other types of vectors), adenoviruses, adeno-associated viruses (AAV), lentiviruses (e.g., modified HIV-1, SIV or FIV), retroviruses (e.g., ASV, ALV or MoMLV), and transposons (e.g., Sleeping Beauty, P-element, Tol-2, Frog Prince, piggyBac).

As used herein, the term nucleic acid refers to both naturally occurring and chemically modified nucleic acids, such as RNA and DNA, including synthetic bases or alternative backbones, as well as, for example, cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA. Nucleic acid molecules may be double-stranded or single-stranded (i.e., sense or antisense single-stranded).

Ancestor animals, animal lineage, traits, and reproduction

The founder animal can be produced by cloning and other methods described herein. The founder can be homozygous for the genetic modification, such as when a zygote or primary cell undergoes a homozygous modification. Similarly, a heterozygous founder can also be produced. In the case of an animal comprising at least one altered chromosomal sequence in a gene encoding the CD163 protein, the founder is preferably heterozygous. The founder can be genomically modified, meaning that all cells of the genome undergo the modification. The founder can be mosaic for the modification, as can occur when the vector is introduced into one of a number of cells in an embryo, typically at the blastocyst stage. The progeny of the mosaic animal can be tested to identify progeny with the genomic modification. An animal line is established when it is possible to produce a herd of animals that can be sexually reproduced or propagated by assisted reproductive techniques with heterozygous or homozygous progeny that consistently express the modification.

In livestock, multiple alleles are known to be associated with various traits, such as production traits, type traits, workability traits, and other functional traits. Skilled practitioners are familiar with monitoring and characterizing these traits [see, e.g., Visscher et al., Livestock Production Science, 40 (1994) 123-137, U.S. Pat. No. 7,709,206, US 2001/0016315, US 2011/0023140, and US 2005/0153317]. An animal line may include a trait selected from the group consisting of production traits, type traits, workability traits, fertility traits, mothering traits, and disease resistance traits. Additional traits include expression of recombinant gene products.

Animals having a desired trait or traits can be modified to prevent their sexual maturation. Since the animals are sterile until maturity, sexual maturation can be controlled as a means of controlling dissemination of the animals. Thus, animals bred or modified to have one or more traits can be provided to recipients with reduced risk of the recipient breeding the animals and assessing the value of the traits to themselves. For example, the genome of an animal can be genetically modified, wherein the modification comprises the inactivation of a sexual maturation gene, wherein the sexual maturation gene in a wild-type animal expresses a factor selective for sexual maturation. The animal can be treated by administering a compound that rescues the defect caused by the loss of expression of the gene that induces sexual maturation in the animal.

Breeding of animals requiring administration of compounds that induce sexual maturation can be advantageously accomplished in a treatment facility. The treatment facility can implement standardized protocols for well-controlled strains to efficiently produce consistent animals. The animal offspring can be distributed to multiple locations where it is desired to be raised. Thus, farms and ranchers (a term that includes ranches and ranchers) can order a desired number of offspring of a given range of ages and/or weights and/or traits and deliver them at a desired time and/or location. The recipient, i.e., the rancher, can then raise the animals and deliver them to the market when desired.

Genetically modified livestock animals having an inactivated sexual maturation gene can be delivered (e.g., to one or more locations, to multiple farms). The animals can have an age of about 1 day to about 180 days. The animals can have one or more traits (e.g., expressing a desired trait or a high-value trait or a novel trait or a recombinant trait).

Methods for increasing the resistance of animals to breeding methods and infections and animal populations

Provided herein are methods of breeding animals or lineages having reduced susceptibility to infection by a pathogen. The methods include genetically modifying an oocyte or sperm cell to introduce a modified chromosomal sequence in a gene encoding a CD163 protein into at least one of an oocyte and a sperm cell, and fertilizing the oocyte with a sperm cell to produce a fertilized egg containing the modified chromosomal sequence in a gene encoding a CD163 protein. Alternatively, the methods include genetically modifying a fertilized egg to introduce a modified chromosomal sequence in a gene encoding a CD163 protein into the fertilized egg. The methods further include transferring the fertilized egg into a surrogate female animal, wherein pregnancy and term delivery produce progeny animals, screening the progeny animals for susceptibility to the pathogen, and selecting the progeny animals having reduced susceptibility to the pathogen compared to animals that do not contain the modified chromosomal sequence in a gene encoding a CD163 protein.

The pathogen preferably includes a virus, e.g. PRRSV.

The animal or offspring may be an embryo, juvenile, or adult.

The animal or offspring may comprise a domestic animal. The domestic animal may comprise a livestock animal, such as a porcine animal, a bovine animal (e.g., a beef or dairy cow), an ovine animal, a caprine animal, an equine animal (e.g., a horse or a donkey), a buffalo, a camel, or a fowl animal (e.g., a chicken, turkey, duck, goose, guinea fowl, or a hatchling). The livestock animal is preferably a bovine or porcine animal, and most preferably a porcine animal.

The step of genetically modifying an oocyte, sperm cell, or fertilized egg can comprise gene editing the oocyte, sperm cell, or fertilized egg. The gene editing can comprise use of a homing endonuclease. The homing endonuclease can be a naturally occurring endonuclease, but it is preferred that the endonuclease is a rationally designed non-naturally occurring homing endonuclease having a DNA recognition sequence designed to target a chromosomal sequence of a gene encoding the CD163 protein. Thus, the homing endonuclease can be a designed homing endonuclease. Thus, the homing endonuclease can be a designed homing endonuclease. The homing endonuclease can include, for example, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a recombinase fusion protein, a meganuclease, or a combination thereof. The gene editing preferably involves the use of a CRISPR/Cas9 system.

The oocyte, sperm cell, or fertilized egg may be heterozygous for the altered chromosomal sequence. Alternatively, the oocyte, sperm cell, or fertilized egg may be homozygous for the altered chromosomal sequence.

The altered chromosomal sequence can comprise an insertion of a gene encoding a CD163 protein, a deletion of a gene encoding a CD163 protein, or a combination thereof. For example, the altered chromosomal sequence can comprise a deletion of a gene encoding a CD163 protein (e.g., an in-frame deletion). Alternatively, the altered chromosomal sequence can comprise an insertion of a gene encoding a CD163 protein.

An insertion or deletion may result in decreased CD163 protein production or activity compared to CD163 protein production or activity in an animal without the insertion or deletion.

The insertion or deletion can result in the production of substantially non-functional CD163 protein by the animal. By "substantially non-functional CD163 protein" is meant that the level of CD163 protein in the animal, offspring, or cell is undetectable, or, if detectable, at least about 90% lower than the level observed in an animal, offspring, or cell that does not contain the insertion or deletion.

Where the animal is a porcine animal, the altered chromosomal sequence may comprise an alteration in exon 7 of the gene encoding CD163 protein, exon 8 of the gene encoding CD163 protein, an intron adjacent to exon 7 or exon 8 of the gene encoding CD163 protein, or a combination thereof. The altered chromosomal sequence suitably comprises an alteration in exon 7 of the gene encoding CD163 protein.

An alteration in exon 7 of the gene encoding the CD163 protein may comprise a deletion (e.g., an in-frame deletion in exon 7). Alternatively, an alteration in exon 7 of the gene encoding the CD163 protein may comprise an insertion.

If the animal is a porcine animal, the modification comprises an 11 base pair deletion from nucleotides 3,137 to 3,147 relative to reference sequence SEQ ID NO: 47; a 2 base pair insertion between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47 and, on the same allele, a 377 base pair deletion from nucleotides 2,573 to nucleotides 2,949 relative to reference sequence SEQ ID NO: 47; a 124 base pair deletion from nucleotides 3,024 to nucleotides 3,147 relative to reference sequence SEQ ID NO: 47; a 123 base pair deletion from nucleotides 3,024 to 3,146 relative to reference sequence SEQ ID NO: 47; a 1 base pair insertion between nucleotides 3,147 and 3,148 relative to reference sequence SEQ ID NO: 47; A 130 base pair deletion from nucleotides 3,030 to 3,159 relative to reference sequence SEQ ID NO: 47; a 132 base pair deletion from nucleotides 3,030 to 3,161 relative to reference sequence SEQ ID NO: 47; a 1,506 base pair deletion from nucleotides 1,525 to nucleotides 3,030 relative to reference sequence SEQ ID NO: 47; a 7 base pair insertion between nucleotides 3,148 and nucleotides 3,149 relative to reference sequence SEQ ID NO: 47; a 1,280 base pair deletion from nucleotides 2,818 to 4,097 relative to reference sequence SEQ ID NO: 47; a 1,373 base pair deletion from nucleotides 2,724 to 4,096 relative to reference sequence SEQ ID NO: 47; A 1467 base pair deletion from nucleotides 2,431 to 3,897 relative to reference sequence SEQ ID NO: 47; a 1930 base pair deletion from nucleotides 488 to 2,417 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insertion starting at nucleotide 488 and an additional 129 base pair deletion in exon 7 from nucleotides 3,044 to 3,172 relative to reference sequence SEQ ID NO: 47; a 28 base pair deletion from nucleotides 3,145 to 3,172 relative to reference sequence SEQ ID NO: 47; a 1387 base pair deletion from nucleotides 3,145 to 4,531 relative to reference sequence SEQ ID NO: 47; A 1382 base pair deletion from nucleotides 3113 to 4494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3113; a 1720 base pair deletion from nucleotides 2440 to 4160 relative to reference sequence SEQ ID NO: 47; or a combination thereof.

If the porcine animal comprises a two base pair insertion between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47, the two base pair insertion may comprise an insertion of the dinucleotide AG.

Where the porcine animal comprises a one base pair insertion between nucleotides 3,147 and 3,148 relative to reference sequence SEQ ID NO: 47, the one base pair insertion may comprise the insertion of a single adenine residue.

Where the porcine animal comprises a 7 base pair insertion between nucleotides 3,148 and 3,149 relative to reference sequence SEQ ID NO: 47, the 7 base pair insertion can comprise an insertion of the sequence TACTACT (SEQ ID NO: 115).

Wherein the porcine animal comprises a 1930 base pair deletion from nucleotide 488 to nucleotide 2,417 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insertion beginning at nucleotide 488, and there is an additional 129 base pair deletion in exon 7 from nucleotides 3,044 to nucleotides 3,172 relative to reference sequence SEQ ID NO: 47, the 12 base pair insertion can comprise an insertion of the sequence TGTGGAGAATTC (SEQ ID NO: 116).

Wherein the porcine animal comprises a 1382 base pair deletion from nucleotide 3,113 to nucleotide 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3,113, the 11 base pair insertion can comprise an insertion of the sequence AGCCAGCGTGC (SEQ ID NO: 117).

When the altered chromosomal sequence of the gene encoding CD163 protein comprises a deletion, the deletion preferably comprises an in-frame deletion. Thus, when the animal is a porcine animal, the insertion or deletion in the gene encoding CD163 protein comprises a 1506 base pair deletion from nucleotide 1,525 to nucleotide 3,030 relative to reference sequence SEQ ID NO: 47; a 1930 base pair deletion from nucleotide 488 to nucleotide 2,417 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insertion starting at nucleotide 488 and an additional 129 base pair deletion from nucleotide 3,044 to nucleotide 3,172 in exon 7 relative to reference sequence SEQ ID NO: 47; A 1373 base pair deletion from nucleotides 2,724 to nucleotides 4,096 relative to reference sequence SEQ ID NO: 47; a 123 base pair deletion from nucleotides 3,024 to nucleotides 3,146 relative to reference sequence SEQ ID NO: 47; a 1467 base pair deletion from nucleotides 2,431 to nucleotides 3,897 relative to reference sequence SEQ ID NO: 47; a 1387 base pair deletion from nucleotides 3,145 to nucleotides 4,531 relative to reference sequence SEQ ID NO: 47; a 1382 base pair deletion from nucleotides 3,113 to nucleotides 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3,113; A 1720 base pair deletion from nucleotide 2,440 to nucleotide 4,160 relative to the reference sequence SEQ ID NO: 47; and an in-frame deletion selected from the group consisting of combinations thereof.

Where the animal is a porcine animal, the insertion or deletion can be selected from the group consisting of a two base pair insertion between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47 and a 377 base pair deletion from nucleotides 2,573 to nucleotides 2,949 relative to reference sequence SEQ ID NO: 47 on the same allele; a 28 base pair deletion from nucleotides 3,145 to nucleotides 3,172 relative to reference sequence SEQ ID NO: 47; and combinations thereof. For example, the oocyte, sperm cell, or fertilized egg can comprise a two base pair insertion between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47 and a 377 base pair deletion from nucleotides 2,573 to nucleotides 2,949 relative to reference sequence SEQ ID NO: 47 on the same allele. The oocyte, sperm cell, or fertilized egg can comprise a 28 base pair deletion from nucleotide 3,145 to nucleotide 3,172 relative to reference sequence SEQ ID NO: 47.

The oocyte, sperm cell, or fertilized egg can comprise a 7 base pair insertion between nucleotides 3148 and 3149 relative to reference sequence SEQ ID NO: 47 and an 11 base pair deletion from nucleotides 3137 to 3147 relative to reference sequence SEQ ID NO: 47.

An oocyte, sperm cell, or fertilized egg comprising any of the insertions or deletions described above can comprise a chromosomal sequence having a high degree of sequence identity to SEQ ID NO: 47 other than the insertion or deletion. Thus, for example, the oocyte, sperm cell, or fertilized egg can comprise a chromosomal sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 47 in a region of the chromosomal sequence other than the insertion or deletion.

The oocyte, sperm cell, or fertilized egg can comprise a chromosomal sequence comprising SEQ ID NO: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, or 114. As further described in the Examples below, SEQ ID NOs: 98-114 provide nucleotide sequences for a region corresponding to the region of wild type porcine CD163 provided in SEQ ID NO: 47, and comprise insertions or deletions in the porcine CD163 chromosomal sequence described herein.

For example, the oocyte, sperm cell, or fertilized egg can comprise a chromosomal sequence comprising SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, or 114. SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, or 114 provides the nucleotide sequence for an in-frame deletion in exon 7 of the porcine CD163 chromosomal sequence.

As another example, an oocyte, sperm cell, or fertilized egg may comprise a chromosome sequence comprising SEQ ID NO: 103 or 111.

An oocyte, sperm cell, or fertilized egg may contain an 11 base pair deletion in one allele of the gene encoding the CD163 protein and a 2 base pair insertion and a 377 base pair deletion in the other allele of the gene encoding the CD163 protein.

The oocyte, sperm cell, or fertilized egg can contain a 124 base pair deletion in one allele of the gene encoding the CD163 protein and a 123 base pair deletion in the other allele of the gene encoding the CD163 protein.

An oocyte, sperm cell, or fertilized egg may contain a single base pair insertion.

An oocyte, sperm cell, or fertilized egg can contain a 130 base pair deletion in one allele of the gene encoding the CD163 protein and a 132 base pair deletion in another allele of the gene encoding the CD163 protein.

Oocytes, sperm cells, or fertilized eggs can contain a 1506 base pair deletion.

Oocytes, sperm cells, or fertilized eggs may contain seven base pair insertions.

An oocyte, sperm cell, or fertilized egg can contain a 1280 base pair deletion in one allele of the gene encoding the CD163 protein and a 1373 base pair deletion in the other allele of the gene encoding the CD163 protein.

Oocytes, sperm cells, or fertilized eggs can contain a 1467 base pair deletion.

The oocyte, sperm cell, or fertilized egg can contain a 1930 base pair intron 6 deletion from nucleotides 488 to 2,417, a 12 base pair addition to nucleotide 4,488, and an additional 129 base pair deletion in exon 7.

An oocyte, sperm cell, or fertilized egg can contain a 28 base pair deletion in one allele of the gene encoding the CD163 protein and a 1387 base pair deletion in another allele of the gene encoding the CD163 protein.

An oocyte, sperm cell, or fertilized egg may contain a 1382 base pair deletion and an 11 base pair insertion in one allele of the gene encoding the CD163 protein and a 1720 base pair deletion in another allele of the gene encoding the CD163 protein.

In any of the breeding methods, a selected animal can be used as a progenitor animal.

In any of the breeding methods, the conception step may include artificial insemination.

A population of animals produced by any of the breeding methods is also provided. The population of animals is preferably resistant to infection by pathogens, for example viruses such as PRRSV.

Also provided is a method of increasing the resistance of a livestock animal to infection with a pathogen. The method comprises gene editing at least one chromosomal sequence from a gene encoding a CD163 protein such that production or activity of the CD163 protein is reduced compared to production or activity of the CD63 protein in a livestock animal that does not comprise the edited chromosomal sequence in the gene encoding the CD163 protein. The pathogen preferably comprises a virus (e.g., PRRSV).

Isolated nucleic acid

An isolated nucleic acid is provided. The isolated nucleic acid molecule can comprise a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence comprising SEQ ID NO: 47; (b) a nucleotide sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 47, wherein the nucleotide sequence contains at least one substitution, insertion, or deletion relative to SEQ ID NO: 47; and (c) a cDNA sequence of (a) or (b).

For example, the isolated nucleic acid can comprise a nucleotide sequence comprising SEQ ID NO: 47.

Alternatively, the isolated nucleic acid can comprise a nucleotide sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 47, wherein said nucleotide sequence contains at least one substitution, insertion, or deletion relative to SEQ ID NO: 47. The isolated nucleic acid can comprise a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.9% sequence identity to the sequence of SEQ ID NO: 47, wherein said nucleotide sequence contains at least one substitution, insertion, or deletion relative to SEQ ID NO: 47.

The substitution, insertion, or deletion preferably reduces or eliminates CD163 protein production or activity compared to a nucleic acid that does not contain the substitution, insertion, or deletion.

The isolated nucleic acid can comprise sequence ID NO: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, or 114.

For example, the isolated nucleic acid can comprise sequence ID NO: 98, 101, 105, 109, 110, 112, 113, or 114.

For example, the isolated nucleic acid can comprise sequence ID NO: 103 or 111.

The isolated nucleic acid may comprise cDNA.

Additional isolated nucleic acids are provided. The isolated nucleic acids can comprise SEQ ID NO: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, or 114. For example, the isolated nucleic acids can comprise SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, or 114. As another example, the isolated nucleic acids can comprise SEQ ID NO: 103 or 111.

The present invention has been described in detail, so it will be obvious that modifications and variations are possible without departing from the scope of the invention as defined in the appended claims.

Example

The following non-limiting examples are provided to further illustrate the present invention.

Example 1: Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro derived oocytes and embryos

Recent reports describing homing endonucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and components of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas9) system, suggest that genetic engineering (GE) in pigs may now be more efficient. Targeted homing endonucleases can induce double-strand breaks (DSBs) at specific locations in the genome and, if donor DNA is provided, can stimulate homologous recombination (HR) or induce random mutations via nonhomologous end joining (NHEJ). Targeted modifications of the genome via HR can be achieved with homing endonucleases if donor DNA is provided together with the targeted nuclease. After inducing specific modifications in somatic cells, these cells have been used to produce GE pigs for various purposes via SCNT. Thus, homing endonucleases are useful tools for producing GE pigs. Among the different homing endonucleases, the CRISPR/Cas9 system adapted from prokaryotes appears to be an effective approach when used as a defense mechanism. In fact, the Cas9 system requires three components: an RNA (~20 bases) containing a region complementary to the target sequence (cis-repressed RNA [crRNA]), an RNA containing a region complementary to the crRNA (transcription-activating crRNA [tracrRNA]), and the enzyme protein component of this complex, Cas9. A single guide RNA (gRNA) can be engineered to perform the role of the base-pairing crRNA and tracrRNA. The gRNA/protein complex can scan the genome and catalyze a DSB in the region complementary to the crRNA/gRNA. Unlike other engineered nucleases, only short oligomers need to be designed to construct the reagents needed to target a gene of interest, whereas a series of cloning steps are required to assemble ZFNs and TALENs.

Unlike the current standard methods for gene disruption, the use of engineered nucleases offers the opportunity to use zygotes as starting material for GE. The standard methods for gene disruption in livestock involve HR in cultured cells and subsequent restoration of embryos by somatic cell nuclear transfer (SCNT). Since cloned animals produced via SCNT sometimes exhibit signs of developmental defects, progeny of SCNT/GE progenitors are typically used in research to avoid confounding SCNT abnormalities and phenotypes that may arise if progenitor animals are used for experiments. Given the longer gestation period and higher housing costs of pigs compared to rodents, there are time and cost advantages for reduced breeding requirements. Recent reports have demonstrated that direct injection of ZFNs and TALENs into pig zygotes can disrupt endogenous genes and produce piglets with the desired mutations. However, only about 10% of piglets exhibited biallelic alterations of the target gene, with some exhibiting mosaic genotypes. A recent article demonstrated that the CRISPR/Cas9 system can induce mutations in developing embryos and produce GE pigs with higher efficiency than ZFNs or TALENs. However, GE pigs produced by the CRISPR/Cas9 system also had mosaic genotypes. In addition, all of the above studies used in vivo-induced zygotes for experiments, which requires intensive labor and numerous sows to obtain a sufficient number of zygotes.

This example describes an efficient approach using the CRISPR/Cas9 system to produce GE pigs via SCNT followed by injection of in vitro derived zygotes and modification of somatic cells. Two endogenous genes (CD163 and CD1D) and one transgene (eGFP) were targeted, and only in vitro derived oocytes or zygotes were used for SCNT or RNA injection, respectively. CD163 appears to be required for proliferative infection by porcine reproductive and respiratory syndrome virus, a virus known to cause significant economic losses to the swine industry. CD1D is considered a non-classical major histocompatibility complex protein and is involved in the presentation of lipid antigens to invariant natural killer T cells. Pigs lacking these genes were designed as models for agricultural and biomedical applications. The eGFP transgene was used as a target for preliminary proof-of-concept experiments and optimization of the method.

Materials and Methods

Chemicals and reagents . Unless otherwise specified, all chemicals used in this study were purchased from Sigma.

Design of gRNAs to construct specific CRISPRs

To ensure that CRISPR induces DSBs within wild-type CD163 but not within the domain swap targeting vector, the guide RNA was designed to a region within exon 7 of CD163 that is unique to wild-type CD163 and absent from the domain swap targeting vector (described below). The targeting vector was designed to be S. pyogenes ( Spy ) There are only four sites that introduce single nucleotide polymorphisms (SNPs) that alter the protospacer adjacent motif (PAM). All four targets were selected, including:

(SEQ ID NO. 1) GGAAACCCAGGCTGGTTGGAgGG (CRISPR 10),

(SEQ ID NO: 2) GGAACTACAGTGCGGCACTGtGG (CRISPR 131),

(SEQ ID NO: 3) CAGTAGCACCCCGCCCTGACgGG (CRISPR 256) and

(SEQ ID NO: 4) TGTAGCCACAGCAGGGACGTcGG (CRISPR 282). PAM is identified in bold in each gRNA.

For CD1D mutations, the search for CRISPR targets was arbitrarily restricted to the coding strand within the first 1000 bp of the primary transcript. However, RepeatMasker [26] (a “porcine” repeat library) identified a repeat element starting at base 943 of the primary transcript. The search for CRISPR targets was then restricted to the first 942 bp of the primary transcript. Since the last Spy PAM is located at base 873, the search was further restricted to the first 873 bp of the primary transcript. The first target (CRISPR 4800) was chosen because it overlaps with the initiation codon located at base 42 in the primary transcript (CCAGCCTCGCCCAGCGACATgGG (SEQ ID NO: 5)). Two additional targets (CRISPR 5620 and 5626) were chosen because they were furthest from our first selection within the randomly selected region (CTTTCATTTATCTGAACTCAgGG (SEQ ID NO:6) and TTATCTGAACTCAGGGTCCCcGG (SEQ ID NO:7)). These targets overlap. With respect to the start codon, the most proximal Spy PAM was located in a simple sequence that contained extensive homopolymer sequences as determined by visual assessment. A fourth target (CRISPR 5350) was chosen because, with respect to the first target selection, it was the most proximal target that did not contain extensive homopolymer sequences (CAGCTGCAGCATATATTTAAgGG (SEQ ID NO:8)). The specificity of the designed crRNA was confirmed by searching for similar pig sequences in GenBank. Oligonucleotides (Table 1) were annealed and cloned into the p330X vector containing two expression cassettes, human codon-optimized S. pyogenes ( hSpy ) Cas9 and a chimeric guide RNA. P330X was digested with BbsI (New England Biolabs) according to the Zhang laboratory protocol (http://www.addgene.org/crispr/zhang/).

To target eGFP , two specific gRNAs targeting the eGFP coding sequence were designed within the first 60 bp of the eGFP start codon: eGFP 1 and eGFP 2. gRNAs, both on the antisense strand, directly targeted the initiation codon of eGFP1. The eGFP 1 gRNA sequence was CTCCTCGCCCTTGCTCACCAtGG (SEQ ID NO: 9) and the eGFP 2 gRNA sequence was GACCAGGATGGGCACCACCCcGG (SEQ ID NO: 10).

Table 1. Designed crRNAs. Primer 1 and primer 2 were annealed according to the Zhang protocol.

Synthesis of donor DNA for CD163 and CD1D genes

Both porcine CD163 and CD1D were amplified by PCR from DNA isolated from fetal fibroblasts used for later transfections to ensure an isogenic match between the targeting vector and the transfected cell line. Briefly, LA Tag (Clontech) using forward primer CTCTCCCTCACTCTAACCTACTT (SEQ ID NO: 11) and reverse primer TATTTCTCTCACATGGCCAGTC (SEQ ID NO: 12) was used to amplify a 9538 bp fragment of CD163. The fragment was DNA sequenced and used to construct a domain-swap targeting vector (Fig. 1). This vector contained 33 point mutations within exon 7 to encode an amino acid sequence identical to human CD163L from exon 11. The replacement exon was 315 bp. Additionally, a modified myostatin intron B was substituted that accommodates a selectable marker gene whose subsequent introns can be removed by Cre recombinase (Cre), and which has previously been demonstrated to result in normal splicing when harboring retained loxP sites (Wells, unpublished results). The long arm of the construct was 3469 bp and contained the domain-swapped DS exon. The short arm was 1578 bp and contained exons 7 and 8 (Fig. 1B ). This plasmid was used to replace the coding region of exon 7 in primary transfection experiments and allowed selection of targeting events via the selectable marker (G418). If targeting was desired, the marker could be deleted by Cre recombinase. The CD163 DS-targeting vector was then modified for use in cell lines that already contained the SIGLEC1 gene disrupted with Neo, which cannot be Cre deleted. In this targeting vector, the Neo cassette, loxP, and myomycin intron B are removed, leaving only the DS exons in the WT long and short arms (Fig. 1c).

The genomic sequence for porcine CD1D was amplified by LA taq using forward primer CTCTCCCTCACTCTAACCTACTT (SEQ ID NO: 13) and reverse primer GACTGGCCATGTGAGAGAAATA (SEQ ID NO: 14), resulting in a 8729 bp fragment. The fragment was DNA sequenced and used to construct the targeting vector shown in Fig. 2. The Neo cassette was under the control of the phosphoglycerol kinase (PGK) promoter and was flanked by loxP sequences, which were introduced for selection. The long arm of the construct was 4832 bp and the short arm was 3563 bp, containing exons 6 and 7. If successful HR occurs, exons 3, 4, and 5 will be removed and replaced by the Neo cassette. If NHEJ repair occurs incorrectly, exon 3 will be destroyed.

Collection of fetal fibroblast cells

To generate cell lines, porcine fetal tissues were collected on day 35 of gestation. Two wild-type (WT) male and female fetal fibroblast cell lines were established from large white domestic crosses. Male and female fetal fibroblasts previously modified to contain the Neo cassette (SIGLEC1-/- genetics) were also used in this study. Fetal fibroblasts were collected as described with minor modifications; pulverized tissue from each fetus was digested in 20 ml of digestion medium (Dulbecco's modified Eagle's medium [DMEM] containing 1 g/L D-glucose [Cellgro] and L-glutamine, supplemented with 200 units/ml collagenase and 25 Kunitz units/ml DNaseI) at 38.5°C for 5 h. After digestion, fetal fibroblasts were washed and cultured with DMEM, 15% fetal bovine serum (FBS), and 40 μg/ml gentamicin. After overnight culture, cells were trypsinized, aliquoted in FBS with 10% dimethyl sulfoxide, frozen at -80°C, and stored in liquid nitrogen.

Cell transfection and genotyping

Transfection conditions were essentially as previously reported. Donor DNA was always used at a constant amount of 1 μg together with various amounts of CRISPR/Cas9 plasmids (listed below). Donor DNA was linearized with MLUI (CD163) (NEB) or AFLII (CD1D) (NEB) prior to transfection. The sex of the established cell lines was determined by PCR prior to transfection as previously described. Both male and female cell lines were transfected, and genomic modification data were analyzed concurrently between transfections. Fetal fibroblast cell lines of similar passage numbers (2-4) were cultured for 2 days and grown to 75-85% confluency in DMEM containing 1 g/L D-glucose (Cellgro) and L-glutamine supplemented with 15% FBS, 2.5 ng/ml basic fibroblast growth factor, and 10 mg/ml gentamicin. Fibroblasts were washed with phosphate-buffered saline (PBS) (Life Technologies) and trypsinized. Upon detachment, cells were rinsed with electroporation medium (75% cytosalts [120 mM KCl, 0.15 mM CaCl ₂ , 10 mM K ₂ HPO ₄ , pH 7.6, 5 Mm MgCl ₂ ]) and 25% Opti-MEM (LifeTechnologies). Cell concentration was quantified using a hemocytometer. Cells were pelleted at 600 X g for 5 min and resuspended in electroporation medium to a concentration of 1 X 10 ⁶ . Each electroporation used 200 μL of cells in a 2 mm gap cuvette with three (1 msec) square wave pulses administered through a BTX ECM 2001 at 250 V. After electroporation, cells were resuspended in DMEM as described above. For selection, 600 μg/ml G418 (Life Technologies) was added 24 h after transfection, and the medium was changed on day 7. Colonies were picked 14 days after transfection. Fetal fibroblasts were plated at 10,000 cells/plate if G418 selection was used and 50 cells/plate if G418 selection was not used. Fetal fibroblast colonies were harvested by applying a 10 mm autoclavable cloning cylinder sealed around each colony with autoclave vacuum grease. Colonies were washed with PBS, harvested via trypsin; and resuspended in DMEM culture medium. A portion (1/3) of the resuspended colonies were transferred to 96-well PCR plates, and the remaining (2/3) cells were cultured in wells of 24-well plates. The cell pellet was resuspended in 6 μl of lysis buffer (40 mM Tris, pH 8.9, 0.9% Triton X-100, 0.4 mg/ml proteinase K [NEB]) and incubated at 65°C for 30 min to lyse cells, then at 85°C for 10 min to inactivate proteinase K.

PCR screening for DS and large and small deletions

Detection of HR-directed repair . Mutations in CD163 or CD1D were identified using long-range PCR. HR events were identified using three different PCR assays: PCR amplification of a region extending from the CD163 or CD1D sequence of the donor DNA to the left or right endogenous CD163 or CD1D sequence, and long-range PCR amplifying a large region of CD163 or CD1D encompassing the designed donor DNA. An increase in the size of the PCR product of 1.8 kb (CD1D) or 3.5 kb (CD163) resulting from the addition of the exogenous Neo sequence was considered evidence of HR-directed repair of the gene. All PCR conditions included an initial denaturation at 95°C for 2 min followed by 33 cycles of 94°C for 30 sec, 50°C for 30 sec, and 68°C for 7-10 min. LA taq was used in all assays according to the manufacturer's recommendations. Primers are listed in Table 2.

Table 2. Primers used to confirm HR-directed repair of CD163 and CD1D

Small deletion assay (NHEJ) . Small deletions were detected by PCR amplification of CD163 or CD1D flanked by protruding cleavage sites introduced by the CRISPR/Cas9 system. The amplicon sizes were 435 bp and 1244 bp for CD163 and CD1D, respectively. Lysates from both embryonic and fetal fibroblasts were PCR amplified with LA taq. The PCR conditions for the assay were an initial denaturation at 95°C for 2 min followed by 33 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 1 min. For genotyping of transfected cells, insertions and deletions (INDELs) were identified by separating PCR amplicons by agarose gel electrophoresis. For embryo genotyping, the resulting PCR products were subsequently DNA sequenced to confirm the small deletion using the forward primer used in the PCR. Primer information is shown in Table 3.

Table 3. Primers used to identify mutations via NHEJ on CD163 and CD1D.

Somatic Cell Nuclear Transfer (SCNT)

To produce SCNT embryos, sow-derived oocytes (ART, Inc.) from a local slaughterhouse or gilt-derived oocytes that had never borne litters were used. Sow-derived oocytes were cultured overnight in maturation medium (TCM-199 with 2.9 mM Hepes, 5 μg/ml insulin, 10 ng/ml epidermal growth factor [EGF], 0.5 μg/ml porcine follicle-stimulating hormone [p-FSH], 0.91 mM pyruvate, 0.5 mM cysteine, 10% porcine follicular fluid, and 25 ng/ml gentamicin) and transferred to fresh medium after 24 h. After 40-42 h of maturation, cumulus cells were removed from the oocytes by vortexing in the presence of 0.1% hyaluronidase. Sow-derived oocytes that had never conceived offspring were matured for in vitro fertilization (IVF) as described below. During manipulation, oocytes were placed in manipulation medium (TCM-199 [Life Technologies] with 0.6 mM NaHCO ₃ , 2.9 mM Hepes, 30 mM NaCl, 10 ng/ml gentamicin, and 3 mg/ml BSA; osmolarity 305 mOsm) supplemented with 7.0 μg/ml cytochalasin B. The polar body together with some of the adjacent cytoplasm presumed to contain the metaphase II plate were removed, and donor cells were placed into the perivitelline space using a fine glass capillary. Afterwards, the reconstituted embryos were fused in fusion medium (0.3 M mannitol, 0.1 mM CaCl ₂ , 0.1 mM MgCl ₂ , and 0.5 mM Hepes) with two DC pulses (1-sec apart) at 1.2 kV/cm for 30 lsec using a BTX Electro cell manipulator (Harvard Apparatus). After fusion, the fused embryos were fully activated with 200 μM thimerosal for 10 min in the dark and 8 mM dithiothreitol for 30 min. The embryos were then cultured for 14–16 h in modified porcine zygote medium PZM3-MU1 with 0.5 μM Scriptaid (S7817; Sigma-Aldrich), a histone deacetylase inhibitor, as described above.

In vitro fertilization (IVF)

For IVF, ovaries from never-breeding prepubertal sows were obtained from an abattoir (Farmland Foods Inc.). Immature oocytes were aspirated from medium-sized (3-6 mm) follicles using an 18-gauge hypodermic needle attached to a 10 ml syringe. Oocytes with uniformly dark cytoplasm and intact surrounding cumulus cells were then selected for maturation. Approximately 50 cumulus oocyte complexes were placed in wells containing 500 μl of maturation medium containing 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 10 ng/ml epidermal growth factor (EGF), 0.5 μg/ml luteinizing hormone (LH), 0.5 μg/ml FSH, 10 ng/ml gentamicin (APP Pharm), and TCM-199 (Invitrogen) with 0.1% polyvinyl alcohol at 38.5°C, 5% CO ₂ in humidified air for 42-44 h. At the end of maturation, surrounding cumulus cells were removed from the oocytes by vortexing for 3 min in the presence of 0.1% hyaluronidase. In vitro matured oocytes were then plated in groups of 25–30 oocytes in 50 μl aliquots of IVF medium (modified Tris-buffered medium containing 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2, 11 mM glucose, 20 mM Tris, 2 mM caffeine, 5 mM sodium pyruvate, and 2 mg/ml bovine serum albumin [BSA]). One 100 μl frozen sperm pellet was thawed in 3 ml of Dulbecco's PBS supplemented with 0.1% BSA. Frozen WT or fresh eGFP sperm were washed by centrifugation at 650 × g for 20 min in 60% Percoll and for 10 min in modified Tris-buffered medium. In some cases, freshly collected sperm heterozygous for a previously described eGFP transgene were washed three times in PBS. The sperm pellet was then resuspended in IVF medium to a density of 0.5 X 10 ⁶ cells/ml. Fifty microliters of the sperm suspension was introduced into the spot containing the oocytes. The gametes were co-cultured for 5 hours at 38.5°C in an atmosphere of 5% CO ₂ in air. After fertilization, the embryos were cultured in PZM3-MU1 at 38.5°C in air and 5% CO ₂ .

Embryo transfer

Embryos generated to produce GE CD163 or CD1D pigs were transferred to surrogate mothers on day 1 (SCNT) or day 6 (injected zygotes) after first standing estrus. For day 6 transfers, zygotes were cultured in PZM3-MU1 in the presence of 10 ng/ml ps48 (Stemgent, Inc.) for an additional 5 days. Embryos were surgically transferred into the ampulla-isthmus-junction of the oviduct of the surrogate mother.

In vitro synthesis of RNA for the CRISPR/Cas9 system

Template DNA for in vitro transcription was amplified using PCR (Table 4). The CRISPR/Cas9 plasmid used in all transfection experiments served as the template for PCR. To express Cas9 in zygotes, Cas9 mRNA was produced using the mMESSAGE mMACHINE Ultra kit (Ambion). Poly A signal was then added to Cas9 mRNA using the poly (A) tailing kit (Ambion). CRISPR guide RNA was produced by MEGAshortscript (Ambion). The quality of the synthesized RNA was visualized on a 1.5% agarose gel, then diluted to a final concentration of 10 ng/μL (both gRNA and Cas9) and distributed into 3 μL aliquots.

Table 4. Primers used to amplify templates for in vitro transcription.

Microinjection of the CRISPR/Cas9 system designed in the zygote

Messenger RNA encoding Cas9 and gRNA were injected into the cytoplasm of fertilized oocytes 14 h post-fertilization (putative zygotes) using a FemtoJet microinjector (Eppendorf). Microinjections were performed in manipulation medium on the heated stage of a Nikon inverted microscope (Nikon Corporation; Tokyo, Japan). Injected zygotes were then transferred to PZM3-MU1 with 10 ng/ml ps48 until further use.

Statistical Analysis

The number of colonies with altered genomes was classified as 1, and colonies without genome alterations were classified as 0. Differences were measured using PROC GLM (SAS), and a P value of 0.05 was considered significant. Means were calculated as least-squares means. Data are presented as mean ± SEM.

result

CRISPR/Cas9-mediated knockout of CD163 and CD1D in somatic cells

The efficiency of four different CRISPR plasmids targeting CD163 (guides 10, 131, 256, and 282) was tested in 2 μg/μl donor DNA amounts (Table 5). CRISPR 282 resulted in significantly higher mean colony formation than CRISPR 10 and 256 treatments (P < 0.05). From the long-range PCR assays described above, large deletions ranging from 503 bp to 1506 bp were found instead of DS via HR as originally intended (Fig. 3a). This was unexpected since previous reports using other DNA-editing systems have shown much smaller deletions of 6-333 bp using ZFNs in pigs. CRISPR 10 and the mix of all four CRISPRs resulted in a greater number of colonies with altered genomes than CRISPR 256 and 282 (Table 5, P < 0.002). Transfection with a plasmid containing Neo but not homologous to CD163 and CRISPR 10 did not result in colonies exhibiting large deletions. Interestingly, one monoallelic deletion was also detected when the donor DNA was introduced without any CRISPR. This assay likely represents an underestimation of the mutation rate, because any possible small deletions that would not be detectable on an agarose gel in the transfected somatic cells were not screened for by sequencing.

Table 5. Efficiency of four different CRISPR plasmids targeting CD163 (guides 10, 131, 256, and 282). The four different CRISPRs were tested in amounts ranging from 2 μg to 1 μg of donor DNA (as shown in Figure 1 ).

* The 4 + donor DNA mix represents an equal mix of 0.5 μg of each CRISPR and 1 μg of donor DNA. Donor DNA treatment serves as a non-CRISPR control and the 10 + Neo treatment illustrates that the large deletions observed in the CRISPR treatment were present only when CD163 donor DNA was also present.

† ANOVA was performed comparing the mean number of colonies/plate and the percentage of colonies with altered genomes to estimate CRISPR toxicity. P values were 0.025 and 0.0002, respectively. n/a = No replicates were observed for this treatment, so no statistical analysis was performed.

‡ A single colony with HR represents a partial HR event.

Superscript letters in ^ac indicate significant differences between treatments for both mean number of colonies/plate and percentage of colonies with altered genomes (P <0.05).

The initial goal was to obtain domain swap (DS)-targeting events by HR for CD163, but CRISPR did not increase the efficiency of targeting CD163. It should be noted that various combinations of these targeting vectors have been used to modify CD163 by HR by conventional transfection, resulting in zero targeting events after screening 3399 colonies (Whitworth and Prather, unpublished results). We obtained two pigs with partial DS resulting from HR containing 16 of the 32 mutations we aimed to introduce by transfection using CRISPR 10 and the DS-targeting vector as donor DNA.

We then tested the efficiency of CRISPR/Cas9-induced mutagenesis without drug selection; the fetal fibroblast cell line used in this study already had integration of the Neo resistance cassette and a knockout of SIGLEC1. We also tested whether the ratio of CRISPR/Cas9 and donor DNA increased genome modification or caused toxic effects at higher concentrations. CRISPR 131 was chosen for this test because in previous experiments it resulted in a high number of total colonies and an increased percentage of colonies with modified genomes. Increasing amounts of CRISPR 131 DNA from 3:1 to 20:1 had no significant effect on fetal fibroblast viability. The percentage of colonies with genomes modified by NHEJ did not differ significantly across the different CRISPR concentrations, but the 10:1 ratio had the highest number of NHEJs (Table 6, P = 0.33). Even at the highest ratio of CRISPR DNA to donor DNA (20:1), no HR was observed.

Table 6. Efficiency of CRISPR/Cas9-induced mutagenesis without drug selection. Four different ratios of donor DNA to CRISPR 131 DNA were compared in previously transformed cell lines without the use of G418 selection.

^a Significant difference between treatment groups for the percentage of colonies with NHEJ repair (P>0.05).

^b There was no significant difference in the number of genome-modified colonies with increasing concentrations of CRISPR (P>0.33).

Based on this experiment, we attempted targeted disruption of CD1D in somatic cells. Four different CRISPRs were designed and tested in both male and female cells. We could not detect any alteration of CD1D from three of the applied CRISPRs, but the use of CRISPR 5350 did not result in alteration of CD1D with deletions large enough to be detected by agarose gel electrophoresis (Table 7). Interestingly, we did not obtain any genetic alteration through HR, even when donor DNA was provided. However, we observed large deletions similar to the CD163 knockout experiments (Fig. 3b). Targeted alteration of CD1D with large deletions was not detected when CRISPR/Cas9 was not used with donor DNA. Alterations of CD1D from CRISPR/Cas9-guided targeting were 4/121 and 3/28 of male and female colonies, respectively. Only INDELs detectable by agarose gel electrophoresis were included in the transfection data.

Table 7. Four different CRISPRs were tested in amounts ranging from 2 μg to 1 μg of donor DNA (as shown in Figure 2 ). Donor DNA treatment served as a non-CRISPR control.

Production of CD163 and CD1D pigs via SCNT using GE cells

CD163 and CD1D knockout pigs were produced using SCNT with cells expressing alterations in CD163 or CD1D (Fig. 3). Seven embryo transfers (CD163 Table 8), six embryo transfers (CD163-No Neo), and five embryo transfers (CD1D) were performed with SCNT embryos from male and female fetal fibroblasts transfected with the CRISPR/Cas9 system into nulliparous recipient sows. Of the nulliparous recipient sows, six (CD163), two (CD163-No Neo), and four (CD1D) (Table 9) carried pregnancies to term, resulting in pregnancy rates of 85.7%, 33.3%, and 80%, respectively. Among the CD163 recipients, five delivered healthy piglets by cesarean section. One (O044) delivered naturally. Litter sizes ranged from 1 to 8 piglets. Four piglets were euthanized due to postnatal growth failure. One piglet was euthanized due to severe cleft palate. All remaining piglets appeared healthy (Fig. 3c). Two littermate male piglets generated from donor DNA and CRISPR 10-transfected fetal fibroblasts described in Fig. 3b had a 30 bp deletion in exon 7 adjacent to CRISPR 10 and an additional 1476 bp deletion in the preceding intron, thus removing the intron 6/exon 7 junction of CD163 (Fig. 3e). Genotypes and predicted translations are summarized in Table 10 . One male and one female littermate (4 piglets) were obtained from CD163-No Neo transfection of previously transformed SIGLEC1 cells. All five piglets were double knockouts for SIGLEC1 and CD163. The male piglet had a biallelic alteration in CD163 with a 28 bp deletion in exon 7 on one allele and a partial deletion of exon 7 and a complete deletion of exon 8 and a 1387 bp deletion on the other allele including the preceding intron, thus resulting in intron-exon splicing. The female piglet had a biallelic mutation in CD163 with a 1382 bp deletion and an 11 bp insertion on one allele and a 1720 bp deletion in CD163 on the other allele. A summary of the CD163 alterations and predicted translations can be found in Table 10 . A summary of the CD1D alterations and predicted translations due to the CRISPR alterations can be found in Table 11 . Briefly, one female and two male litters were born, producing 13 piglets. One piglet died immediately after birth. Twelve of the 13 piglets contained biallelic or homozygous deletions of CD1D (Fig. 3f). One piglet was WT.

Table 8. Embryo transplantation data for CD163 .

* CD163 CRISPR NT line represents embryos generated by NT from a transfection-modified fetal fibroblast line. CRISPR-injected embryos were IVF embryos injected at the 1-cell stage with CD163 guide RNA and CAS9 RNA. CD163 CRISPR NT-no Neo fetal line represents embryos generated by NT from previously modified fetal fibroblasts that were already Neo -resistant and modified by transfection without the use of a selection marker.

† MU refers to never-bearing sow oocytes aspirated and matured at the University of Missouri as described in the IVF section of Materials and Methods. ART refers to sow oocytes purchased and matured as described in the SCNT section of Materials and Methods.

Table 9. Embryo transfer data for CD1D .

* CD1D CRISPR NT line represents embryos generated by NT, a transgenic fetal fibroblast line. CRISPR-injected embryos were IVF embryos injected at the 1-cell stage with CD1D guide RNA and CAS9 RNA.

† MU refers to never-bearing sow oocytes aspirated and matured at the University of Missouri as described in the IVF section of Materials and Methods. ART refers to sow oocytes purchased and matured as described in the SCNT section of Materials and Methods.

Table 10. Genotype and translation prediction for CD163 mutant pigs. Some pigs contained not only biallelic variants but also one allele described and another mutant allele that was not amplified by PCR.

*KO, knock-out

** Not included because piglets were euthanized.

† The sequence numbers in this column refer to the sequence numbers for sequences that exhibit INDELs with respect to sequence number 47.

^a The inserted sequence was TACTACT (SEQ ID NO: 115).

^b The inserted sequence was AG.

^c The inserted sequence was a single adenine (A) residue.

^d The inserted sequence was TGTGGAGAATTC (SEQ ID NO: 116).

^e The inserted sequence was AGCCAGCGTGC (SEQ ID NO: 117).

Table 11. Genotype and translation predictions for CD1D mutant pigs

*KO, knock-out

Efficiency of the CRISPR/Cas9 system in pig zygotes

Based on the targeted disruption of CD163 and CD1D in somatic cells using the CRISPR/Cas9 system, the method was applied to porcine embryogenesis. First, we tested the effectiveness of the CRISPR/Cas9 system in developing embryos. The CRISPR/Cas9 system targeting eGFP was introduced into the semen of a boar heterozygous for the eGFP transgene and fertilized zygotes. After injection, the subsequent embryos expressing eGFP were monitored. We tested various concentrations of the CRISPR/Cas9 system and observed the cytotoxicity of the delivered CRISPR/Cas9 system (Fig. 4a); the embryo development after CRISPR/Cas9 injection was lower than that of the control group. However, all tested concentrations of CRISPR/Cas9 were effective in generating modifications of eGFP, as no embryos with eGFP expression were found in the CRISPR/Cas9-injected group (Fig. 4b); Of the non-injected control embryos, 67.7% were green, indicating expression of eGFP. When individual blastocysts were genotyped, we were able to identify a small mutation near the CRISPR binding site (Fig. 4c). Based on toxicity and efficacy, 10 ng/μl of gRNA and Cas9 mRNA were used in the experiments below.

When we introduced CRISPR/Cas9 components designed to target CD163 into putative zygotes, we observed targeted alterations of the gene in subsequent blastocysts. When individual blastocysts were genotyped for mutations in CD163, the specific mutation was detected in all embryos (100% GE efficiency). More importantly, we were able to find embryos with homozygous or biallelic mutations (8/18 and 3/18, respectively) (Fig. 5 ), while also detecting mosaic (monallelic mutation) genotypes (4/18 embryos). Some embryos from the pool (8/10) were injected with 2 ng/μl Cas9 and 10 ng/μl CRISPR, and no difference in mutagenesis efficiency was observed. Next, based on the in vitro results, we introduced two CRISPRs expressing different gRNAs that disrupt CD163 or CD1D during embryogenesis to induce specific deletions of the target gene. As a result, we were able to successfully induce designed deletions of CD163 and CD1D by introducing two guides. The designed deletion is defined as a deletion that removes the genomic sequence between the two introduced guides. Among the embryos that received two CRISPRs targeting CD163, all but one embryos induced targeted alterations of CD163. In addition, we found 5/13 embryos with designed deletions in CD163 (Fig. 6a), and 10/13 embryos appeared to have alterations in CD163 in a homozygous or biallelic manner. Targeting CD1D with two CRISPRs was also effective, as all embryos (23/23) showed alterations in CD1D. However, we could detect the designed deletion of CD1D in only two embryos (2/23) (Fig. 6b). We also detected 5/23 embryos with a mosaic genotype, whereas the remaining embryos had homozygous or biallelic alterations of CD1D. Finally, we tested whether multiple genes could be targeted by the CRISPR/Cas9 system within the same embryo. For this purpose, targeting both CD163 and eGFP was performed in heterozygous eGFP sperm and fertilized zygotes. When blastocysts from the injected embryos were genotyped for CD163 and eGFP, we found that CD163 and eGFP were successfully targeted during embryogenesis. The sequence analysis results demonstrated that multiple genes could be targeted by introducing multiple CRISPR and Cas9 (Fig. 6c).

Production of CD163 and CD1D mutants from CRISPR/Cas9-injected zygotes

Based on the success from our previous in vitro studies, we produced several CRISPR/Cas9-injected zygotes and transferred 46-55 blastocysts per recipient (as this number has been shown to be effective for producing pigs from our in vitro-derived embryos). We performed four embryo transfers, two each for CD163 and CD1D, and obtained pregnancies for each alteration. We produced four healthy piglets carrying alterations in CD163 (Table 8). All piglets, litter 67 from recipient sow ID O083, displayed homozygous or biallelic alterations in CD163 (Fig. 7 ). Two piglets displayed the designed deletion of CD163 by both CRISPRs transferred. All piglets were healthy. For CD1D, one pregnancy also produced four piglets (litter 166 from recipient sow ID O165): one female and three males (Table 9). One piglet (166-1) carried a mosaic mutation in CD1D including a 362 bp deletion that completely removed exon 3 containing the initiation codon (Fig. 8). One piglet contained a 2 bp mismatch and a 6 bp insertion in one allele and a large deletion in the other allele. Two additional piglets had biallelic single bp insertions. No mosaic mutations were detected for CD163.

Reflection

Increased efficiency in GE pig production could have broad implications by providing more GE pigs for agriculture and biomedicine. The above data demonstrate that GE pigs with specific mutations can be produced with high efficiency using the CRISPR/Cas9 system. The CRISPR/Cas9 system has been successfully applied to genetic modification in somatic cells and preimplantation embryos.

When the CRISPR/Cas9 system was introduced into somatic cells, it successfully induced targeted disruption of our target genes by NHEJ, but did not enhance their ability to target by HR. The targeting efficiency of individual CRISPR/Cas9s in somatic cells was variable, suggesting that the design of the guide affects targeting efficiency. In particular, we did not detect any targeted modification of CD1D when CRISPR 5350 and Cas9 were introduced into somatic cells. This suggests that it may be advantageous to design multiple gRNAs and validate their efficiency prior to producing pigs. The reason for the lack of HR-directed repair in the presence of donor DNA remains unclear. After screening 886 colonies (both CD163 and CD1D) transfected with CRISPR and donor DNA, only one colony had evidence for a partial HR event. Our results demonstrate that the CRISPR/Cas9 system can work with donor DNA to induce unexpected large deletions in the target gene, but does not increase HR efficiency for these two specific targeting vectors. However, the specific mechanism for the large deletions observed is unknown. Previous reports from our group have suggested that donor DNA can be effectively used with ZFNs to induce HR-directed repair. Similarly, we observed increased targeting efficiency when donor DNA was used with the CRISPR/Cas9 system, but complete HR-directed repair was not observed. In our previous studies using ZFNs, we observed partial recombination of the induced donor DNA after DSBs were induced by ZFNs, suggesting that targeted modifications can occur through a combination of HR and NHEJ. One explanation is that the HR and NHEJ pathways are not independent, but rather can work together to complete the repair process after DSBs are induced by homing endonucleases. Although higher concentrations of CRISPR may improve targeting efficiency in somatic cells, no statistical difference was found in these experimental results. This may suggest that CRISPR is a limiting factor in the CRISPR/Cas9 system, but further validation is needed. Targeted cells were successfully used to produce GE pigs via SCNT, indicating that the application of CRISPR/Cas9 does not affect the ability of the cells to replicate. Some piglets were euthanized for health reasons; however, this is a common occurrence in SCNT-derived piglets.

When the CRISPR/Cas9 system was introduced into developing embryos by zygote injection, nearly 100% of embryos and pigs contained INDELs in the targeted gene, demonstrating that the technique is highly efficient during embryogenesis. The efficiencies we observed during our study exceeded the frequencies reported in other studies using homing endonucleases during embryogenesis. The reduced number of embryos reaching the blastocyst stage suggested that the concentration of CRISPR/Cas9 we introduced in this study may have been toxic to the embryos. Further optimization of the delivery system may increase the viability of the embryos and thus improve the overall efficiency of the process. The nearly 100% mutagenesis rate observed here differs from previous reports of CRISPR/Cas9-mediated knockout in pigs; however, the differences in efficiency between studies may be due to the combination of targets and guides selected. In our study, lower concentrations of CRISPR/Cas9 (10 ng/μl each) were effective in generating mutations in developing embryos and producing GE pigs. The concentrations are lower than those previously reported in pig zygotes (125 ng/μl of Cas9 and 12.5 ng/μl of CRISPR). Lower concentrations of CRISPR/Cas9 components may be beneficial for developing embryos, as introducing excessive amounts of nucleic acids into developing embryos can be toxic. We observed some mosaic genotypes in CRISPR/Cas9-injected embryos from our in vitro assays; however, only one piglet produced via our approach had a mosaic genotype. Potentially, injection of CRISPR/Cas9 components may be more effective than introduction of other homing endonucleases, as mosaic genotypes have been considered a major obstacle to the use of the CRISPR/Cas9 system in zygotes. Another advantage of using the CRISPR/Cas9 system, as evidenced by our results, is that we did not lose any CD163 knockout pigs produced from IVF-derived zygotes injected with the CRISPR/Cas9 system, whereas several piglets resulting from SCNT were euthanized after a few days. This suggests that the technique may not only avoid the need for SCNT to produce knockout pigs, but may also overcome common health problems associated with SCNT. Since the injection of CRISPR/Cas9 mRNA into zygotes has been optimized, future experiments will also include co-injection of donor DNA.

We demonstrate that introducing two CRISPRs and Cas9 into a zygote can induce chromosomal deletions in the developing embryo, producing pigs with the intended deletion, i.e., the specific deletion between the two CRISPR guides. Such designed deletions may be advantageous because we can specify the size of the deletion, rather than relying on random events induced by NHEJ. In particular, if the insertion/deletion of nucleotides is a multiple of 3 of that induced by a homing endonuclease, the mutation may rather result in a hypomorphic mutation, since no frameshift occurs. However, by introducing two CRISPRs, we can induce larger deletions that have a higher chance of producing a non-functional protein. Interestingly, the CD1D CRISPR was designed to span a larger region of the genome than CD163; There was a 124 bp gap between CD163 CRISPRs 10 and 131, whereas for CD1D there was a 550 bp gap between CRISPRs 4800 and 5350. Longer gaps between CRISPRs were not as effective in generating deletions as shown in our study. However, since we only have a limited number of observations and need to consider the efficiency of individual CRISPRs, which are not addressed here, further studies are needed to verify the relationship between the distance between CRISPRs and their ability to induce the intended deletions.

The CRISPR/Cas9 system was also effective in targeting two genes simultaneously in the same medium, where the only additional step was the introduction of one additional CRISPR and crRNA. This illustrates the ease of disrupting multiple genes compared to other homing endonucleases. These results suggest that this technology can be used to target gene clusters or gene families that may have complementary effects, and thus the role of individual genes may prove difficult to determine unless all genes are disrupted. Our results demonstrate that the CRISPR/Cas9 technology can be applied to increase the efficiency of gene targeting in somatic cells and to produce GE pigs by direct zygotic injection.

References

1. Whyte JJ, Prather RS. Genetic modifications of pigs for medicine and agriculture. Mol Reprod Dev 2011; 78:879-891.

2. Walters EM, Prather RS. Advancing swine models for human health and diseases. Mo Med 2013; 110:212-215.

3.U.S. Department of Agriculture and U.S. Department of Health and Human Services. Dietary Guidelines for Americans, 2010. 7th Edition,

Washington, D.C.: U.S. Government Printing Office; December 2010.

4. Hammer RE, Pursel VG, Rexroad CE Jr, Wall RJ, Bolt DJ, Ebert KM, Palmiter RD, Brinster RL. Production of transgenic rabbits, sheep and pigs by microinjection. Nature 1985; 315:680-683.

5. Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, et al. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002; 295:1089-1092.

6. Dai Y, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, Cowell-Lucero JL, Wells KD, et al. Targeted disruption of the alpha1,3-galactosyltransferase genes in cloned pigs. Nat Biotechnol 2002; 20:251-255.

7. Gaj T, Gersbach CA, Barbas CF III. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 2013; 31: 397-405.

8. Hauschild J, Petersen B, Santiago Y, Queisser AL, Carnwath JW, Lucas-Hahn A, Zhang L, Meng X, Gregory PD, Schwinzer R, Cost GJ, Niemann.

H. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc Natl Acad Sci U S A 2011; 108:12013-12017.

9. Yang D, Yang H, Li W, Zhao B, Ouyang Z, Liu Z, Zhao Y, Fan N, Song J, Tian J, Li F, Zhang J, et al. Generation of PPARgamma mono-allelic

knockout pigs via zinc-finger nucleases and nuclear transfer cloning. Cell Res 2011; 21:979-982.

10. Whyte JJ, Zhao J, Wells KD, Samuel MS, Whitworth KM, Walters EM, Laughlin MH, Prather RS. Gene targeting with zinc finger nucleases to

produce cloned eGFP knockout pigs. Mol Reprod Dev 2011; 78:2.

11. Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea. Nature 2012; 482:331-338.

12. Terns MP, Terns RM. CRISPR-based adaptive immune systems. Curr Opin Microbiol 2011; 14:321-327.

13. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK. Efficient genome editing in zebrafish using a

CRISPR-Cas system. Nat Biotechnol 2013; 31:227-229.

14. Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, et al. Generation of gene-modified Cynomolgus monkey via Cas9/RNA -mediated gene targeting in one-cell embryos. Cell 2014; 156:836-843.

15. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 2013; 153: 910-918.

16. Li D, Qiu Z, Shao Y, Chen Y, Guan Y, Liu M, Li Y, Gao N, Wang L, Lu Cas system. Nat Biotechnol 2013; 31:681-683.

17. Hai T, Teng F, Guo R, Li W, Zhou Q. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res 2014; 24: 372-375.

18. Carter DB, Lai L, Park KW, Samuel M, Lattimer JC, Jordan KR, Estes DM, Besch-Williford C, Prather RS. Phenotyping of transgenic cloned piglets. Cloning Stem Cells 2002; 4:131-145.

19. Shimozawa N, Ono Y, Kimoto S, Hioki K, Araki Y, Shinkai Y, Kono T, Ito M. Abnormalities in cloned mice are not transmitted to the progeny. Genesis 2002; 34:203-207.

20. Lillico SG, Proudfoot C, Carlson DF, Stverakova D, Neil C, Blain C, King TJ, Ritchie WA, Tan W, Mileham AJ, McLaren DG, Fahrenkrug SC, et al. Live pigs produced from genome edited zygotes. Sci Rep 2013; 3:2847.

21. Van Breedam W, Delputte PL, Van Gorp H, Misinzo G, Vanderheijden N, Duan X, Nauwynck HJ. Porcine reproductive and respiratory syndrome virus entry into the porcine macrophage. J Gen Virol 2010; 91:1659-1667.

22. Prather RS, Rowland RR, Ewen C, Trible B, Kerrigan M, Bawa B, Teson JM, Mao J, Lee K, Samuel MS, Whitworth KM, Murphy CN, et al. An intact sialoadhesin (Sn/SIGLEC1/CD169) is not required for attachment/ internalization of the porcine reproductive and respiratory syndrome virus. J Virol 2013; 87:9538-9546.

23. Neumann EJ, Kliebenstein JB, Johnson CD, Mabry JW, Bush EJ, Seitzinger AH, Green AL, Zimmerman JJ. Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States. J Am Vet Med Assoc 2005; 227:385-392.

24. Borg NA, Wun KS, Kjer-Nielsen L, Wilce MC, Pellicci DG, Koh R, Besra GS, Bharadwaj M, Godfrey DI, McCluskey J, Rossjohn J. CD1d-lipid-antigen recognition by the semi-invariant NKT T -cell receptor. Nature 2007; 448:44-49.

25. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339:819-823.

26. Smit, AFA, Hubley, R, Green, P. RepeatMasker Open-3.0. 1996-2010.

Current Version: open-4.0.5 (RMLib: 20140131 and Dfam: 1.2). http://www.repeatmasker.org&gt. CD163: accessed July 25, 2014; CD1D: accessed August 27, 2013.

27. Lai L, Prather RS. Creating genetically modified pigs by using nuclear transfer. Reprod Biol Endocrinol 2003; 1:82.

28. Ross JW, Whyte JJ, Zhao J, Samuel M, Wells KD, Prather RS. Optimization of square-wave electroporation for transfection of porcine fetal fibroblasts. Transgenic Res 2010; 19:611-620.

29. Whitworth KM, Spate LD, Li R, Rieke A, Sutovsky P, Green JA, Prather RS. Activation method does not alter abnormal placental gene expression and development in cloned pigs. Mol Reprod Dev 2010; 77:1016-1030.

30. van den Hoff MJ, Moorman AF, Lamers WH. Electroporation in 'intracellular' buffer increases cell survival. Nucleic Acids Res 1992; 20:2902.

31. Beaton BP, Wells, Kevin D. Compound transgenics: recombinase-mediated gene stacking. In: Pinkert CA (ed.). Transgenic Animal Technology: A Laboratory Handbook, 3rd ed. Waltham, MA: Elsevier; 2014:565-578.

32. Lai L, Prather RS. Production of cloned pigs by using somatic cells as donors. Cloning Stem Cells 2003; 5:233-241.

33. Machaty Z, Wang WH, Day BN, Prather RS. Complete activation of porcine oocytes induced by the sulfhydryl reagent, thimerosal. Biol Reprod 1997; 57:1123-1127.

34. Yoshioka K, Suzuki C, Tanaka A, Anas IM, Iwamura S. Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol Reprod 2002; 66:112-119.

35. Bauer BK, Spate LD, Murphy CN, Prather RS. Arginine supplementation in vitro increases porcine embryo development and affects mRNA transcript expression. Reprod Fertil Dev 2011; 23:107.

36. Zhao J, Hao Y, Ross JW, Spate LD, Walters EM, Samuel MS, Rieke A, Murphy CN, Prather RS. Histone deacetylase inhibitors improve in vitro and in vivo developmental competence of somatic cell nuclear transfer porcine embryos. Cell Reprogram 2010; 12:75-83.

37. Zhao J, Ross JW, Hao Y, Spate LD, Walters EM, Samuel MS, Rieke A, Murphy CN, Prather RS. Significant improvement in cloning efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer. Biol Reprod 2009; 81:525-530.

38. Whitworth KM, Zhao J, Spate LD, Li R, Prather RS. Scriptaid corrects gene expression of a few aberrantly reprogrammed transcripts in nuclear transfer pig blastocyst stage embryos. Cell Reprogram 2011; 13:191-204.

39. Whitworth KM, Li R, Spate LD, Wax DM, Rieke A, Whyte JJ, Manandhar G, Sutovsky M, Green JA, Sutovsky P, Prather RS. Method of oocyte activation affects cloning efficiency in pigs. Mol Reprod Dev 2009;

76:490-500.

40. Lee K, Redel BK, Spate L, Teson J, Brown AN, Park KW, Walters E, Samuel M, Murphy CN, Prather RS. Piglets produced from cloned blastocysts cultured in vitro with GM-CSF. Mol Reprod Dev 2013; 80: 145-154.

41. Kwon DN, Lee K, Kang MJ, Choi YJ, Park C, Whyte JJ, Brown AN, Kim JH, Samuel M, Mao J, Park KW, Murphy CN, et al. Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs . Sci Rep 2013; 3:1981.

42. Lee K, Kwon DN, Ezashi T, Choi YJ, Park C, Ericsson AC, Brown AN, Samuel MS, Park KW, Walters EM, Kim DY, Kim JH, et al. Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency. Proc Natl Acad Sci U S A 2014; 111:7260-7265.

43. Brinster RL, Chen HY, Trumbauer ME, Yagle MK, Palmiter RD. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci U S A 1985; 82:4438-4442.

Example 2: Increased resistance to PRRSV in pigs having a chromosomal sequence altered in the gene encoding the CD163 protein

Porcine reproductive and respiratory syndrome virus (PRRSV) has invaded the swine industry for the past quarter century. Speculations on the mode of virus entry have involved both SIGLEC1 and CD163. While knockout of SIGLEC1 did not affect the response to virus challenge, we show here that CD163 null animals do not display clinical signs of infection, lung lesions, viremia, or antibody production, all of which are hallmarks of PRRSV infection. In addition to identifying a PRRSV entry vector; we describe a strategy that could prevent significant economic losses and animal suffering if similarly generated animals were allowed to enter the food supply.

Materials and Methods

Genotyping . Genotyping was based on both DNA and mRNA sequence analysis. The sire had an 11 bp deletion in one allele that, when translated, predicted 45 amino acids into domain 5, resulting in a premature stop codon at amino acid 64. Another allele had a 2 bp addition in exon 7 and a 377 bp deletion in the intron preceding exon 7, which, when translated, predicted the first 49 amino acids of domain 5, resulting in a premature stop codon at amino acid 85. One sow had a 7 bp addition in one allele that, when translated, predicted the first 48 amino acids of domain 5, resulting in a premature stop codon at amino acid 70. The other allele was uncharacterized because it did not yield any band from exon 7 by PCR or by a distant 6.3 kb PCR (A). The other three sows were clonal and had a 129 bp deletion in exon 7 predicted to result in a deletion of 43 amino acids from domain 5. The other allele was uncharacterized (B).

Growth of PRRSV in culture and production of virus inoculum for infection of pigs were performed under approved IBC protocol 973. The reference strain of PRRSV, isolate NVSL 97-7895 (GenBank # AF325691 2001-02-11), was grown as described in the approved IBC protocol 973. This laboratory isolate has been used in experimental studies for approximately 20 years (Ladinig et al., 2015). A secondary isolate was used for the 2 ^nd test, KS06-72109, as previously described (Prather et al., 2013).

Infection of pigs with PRRSV . A standardized infection protocol for PRRSV was used to infect pigs. Three-week-old piglets were inoculated with approximately 10^4 TCID50 of PRRS virus administered intramuscularly (IM) and intranasally (IN). Pigs were monitored daily and pigs showing signs of disease were treated as recommended by the CMG veterinarian. Pigs showing significant distress and at risk of succumbing to infection were humanely euthanized and samples collected. Staff and veterinarians were blinded to the genetic status of the pigs to eliminate bias in evaluation or treatment. PRRSV is present in body fluids during infection; therefore, blood samples were collected and stored at -80°C until assayed to determine the amount or degree of viremia in each pig. At the end of the experiment, pigs were weighed and humanely euthanized, and tissues were collected, fixed in 10% buffered formalin, embedded in paraffin, and processed for histopathology by a licensed pathologist.

Phenotypic scoring of challenged pigs . The phenotypes of the pigs were scored blindly daily as follows: What is the attitude of the pig? Attitude score: 0: BAR, 1: QAR, 2: Slightly depressed, 3: Depressed, 4: Moribund. What is the health status of the pig? Health score: 1: Thin, 2: Thin, 3: Ideal, 4: Fat, 5: Excessive obesity/obese. What is the rectal temperature of the pig? Normal body temperature 101.6-103.6 (fever is considered ≥ 104). Is the leg lame (grade)? Which leg? Assess the legs for joint swelling and hoof lesions (check the sole and lateral aspect of the hoof). Lameness Score: 1: No limping, 2: Slightly uneven gait, some joints appear stiff but no limping, 3: Mild limping, slight limping while walking, 4: Moderate limping, obvious limping including toe touching lame, 5: Severe limping, unable to bear weight on leg, requires encouragement to stand/walk. Is there difficulty breathing (grading)? Does the animal breathe with its mouth open? Is there nasal discharge (color of discharge, amount: mild/moderate/severe)? Have you noticed the animal coughing? Is there eye discharge? Respiratory Score: 0: Normal, 1: Mild dyspnea and/or tachypnea when stressed (when handled), 2: Mild dyspnea and/or tachypnea at rest, 3: Moderate dyspnea and/or tachypnea when stressed (when handled), 4: Moderate dyspnea and/or tachypnea at rest, 5: Severe dyspnea and/or tachypnea when stressed (when handled), 6: Severe dyspnea and/or tachypnea at rest. Is there any evidence of diarrhea (grade) or vomiting? Is there blood or mucus? Diarrhea Score: 0: No visible stool, 1: Normal stool, 2: Soft but formed stool (consistency of soft-serve yogurt, resembles cow dung), 3: Brown/tan liquid diarrhea with particulate fecal matter, 4: Brown/tan liquid diarrhea without particulate fecal matter, 5: Liquid diarrhea that looks similar to water.

This scoring system was developed by Dr. Megan Niederwerder at KSU and is based on the following publications (Halbur et al., 1995; Merck; Miao et al., 2009; Patience and Thacker, 1989; Winckler and Willen, 2001). Scores and temperature were analyzed using separate ANOVAs based on genotype as treatment.

Measurement of PRRSV viremia . Viremia was determined by two approaches. Viral titration was performed by adding serial 1:10 dilutions of serum to confluent MARC-145 cells in 96-well plates. Serum was diluted in Eagle's minimal essential medium supplemented with 8% fetal bovine serum, penicillin, streptomycin, and amphotericin B as previously described (Prather et al., 2013). Cells were examined for the presence of cytopathic effect after 4 days of culture using microscopy. The highest dilution showing cytopathic effect was scored as the titration endpoint. Total RNA was isolated from serum using the Life Technologies MagMAX-96 Viral RNA Isolation Kit for viral nucleic acid measurement. Reverse transcription polymerase chain reaction was performed using the EZ-PRRSV MPX 4.0 Kit from Tetracore on a CFX-96 Real-Time PCR System (Bio-Rad) according to the manufacturer's instructions. Each reaction (25 μl) contained 5.8 μl of RNA from serum. Standard curves were prepared by preparing serial dilutions of the RNA control supplied with the kit (Tetracore). The number of templates per PCR is reported.

SIGLEC1 and CD163 staining of PAM cells . Porcine alveolar macrophages (PAM) were harvested by excising the lungs and flushing them with ~100 ml ice-cold phosphate-buffered saline. After recovering the phosphate-buffered saline washes, cells were pelleted, resuspended in 5 ml ice-cold phosphate-buffered saline, and stored on ice. Approximately 10 ⁷ PAM were incubated in 5 ml of various antibodies (anti-swine CD169 (clone 3B11/11; AbD Serotec); anti-swine CD163 (clone 2A10/11; AbD Serotec) diluted in phosphate-buffered saline with 5% fetal bovine serum and 0.1% sodium azide) for 30 min on ice. Cells were washed and resuspended in a 1/100 dilution of fluorescein isothiocyanate (FITC) conjugated to goat anti-mouse IgG (Life Technologies) diluted in staining buffer and incubated on ice for 30 min. At least 10 ⁴ cells were analyzed using a FACSCalibur flow cytometer and Cell Quest software (Becton Dickinson).

Measurement of PRRSV-specific Ig. To measure PRRSV-specific Ig, recombinant PRRSV N protein was expressed in bacteria (Trible et al., 2012) and conjugated to magnetic Luminex beads using a kit (Luminex Corporation). N protein-coupled beads were diluted to 2,500 beads/50 μl in phosphate-buffered saline containing 10% goat serum and placed into wells of a 96-well round-bottom polystyrene plate. Serum was diluted 1:400 in phosphate-buffered saline containing 10% goat serum, and 50 μl was added to duplicate wells and incubated for 30 min with gentle shaking at room temperature. The plates were then washed (3X) with phosphate-buffered saline containing 10% goat serum and 50 μl of biotin-SP-conjugated affinity-purified goat anti-swine secondary antibody (IgG, Jackson ImmunoResearch) or biotin-labeled affinity-purified goat anti-swine IgM (KPL) diluted to 2 μg/ml in phosphate-buffered saline containing 10% goat serum was added. After 30 min incubation, the plates were washed (3X) and 50 μl of streptavidin-conjugated phycoerythrin (2 μg/ml in phosphate-buffered saline containing 10% goat serum (Moss, Inc.)) was added. The plates were washed after 30 min and the microspheres were resuspended in 100 μl of phosphate-buffered saline containing 10% goat serum and analyzed using MAGPIX and Luminex xPONENT 4.2 software. The mean fluorescence intensity (MFI) is recorded.

result

Mutations in CD163 were generated using CRISPR/Cas9 technology as described in Example 1 above. Several founder animals were produced from zygote injections and somatic cell nuclear transfer. Some of these founder animals were mated to produce offspring for study. A single founder male was mated to a female with two genotypes. The founder male (67-1) had an 11 bp deletion in exon 7 in one allele and a 2 bp addition in exon 7 of the other allele (and a 377 bp deletion in the preceding intron) and was predicted to be a non-responsive animal ( CD163 ^-/- ). One founder female (65-1) had a 7 bp addition in exon 7 in one allele and the corresponding uncharacterized allele and was therefore predicted to be heterozygous for the knockout ( CD163 ^-/? ). The second progenitor female genotype (3 cloned animals) contained an uncharacterized allele and an allele with a 129 bp deletion in exon 7. This deletion is predicted to result in a 43 amino acid deletion in domain 5. Matings between these animals resulted in piglets that inherited the uncharacterized allele from the boars and either the 43 amino acid deletion or the uncharacterized allele from the sows. In addition to the wild-type piglets that served as positive controls for virus challenge, this produced four additional genotypes (Table 8).

Table 12. Genotypes tested for resistance to PRRSV challenge (NVSL and KS06 strains).

At weaning, genetically edited piglets and wild-type age-matched piglets were transported to Kansas State University for PRRSV challenge. PRRSV challenge was performed as previously described (Prather et al., 2013). Piglets were transferred to the challenge facility at 3 weeks of age and maintained in a single group. All experiments were initiated after approval by the institutional animal use and biosafety committees. After acclimation, pigs were challenged with the PRRSV isolate, NVSL 97-7895 (Ladinig et al., 2015) and seeded on MARC-145 cells (Kim et al., 1993). Pigs were challenged with approximately 10 ⁵ TCID ₅₀ of virus. Half of the inoculum was delivered intramuscularly and the remainder intranasally. All infected pigs were maintained in a single group, allowing for continuous exposure to virus from infected pen mates. Blood samples were collected at various days up to 35 days post-infection and at the end of the study on day 35. Pigs were necropsied and tissues fixed in 10% buffered formalin, embedded in paraffin, and processed for histopathology. PRRSV-associated clinical signs recorded during the course of infection included dyspnea, anorexia, lethargy, and fever. Results for clinical signs over the study period are summarized in Figure 9 . As expected, wild type ( CD163 +/+ ) pigs showed early signs of PRRSV infection, which peaked between days 5 and 14 and persisted in the group for the remainder of the study. The percentage of febrile pigs peaked around day 10. In contrast, nonresponsive ( CD163 -/- ) piglets showed no signs of clinical signs over the entire study period. Respiratory signs during acute PRRSV infection are reflected in significant histopathologic changes in the lungs (Table 9 ). Infection of wild-type pigs resulted in histopathology consistent with PRRS, including interstitial edema and mononuclear cell infiltration (Fig. 10). In contrast, nonresponsive ( CD163 -/-) pigs showed no signs of lung changes. The sample sizes for the various genotypes were small; nevertheless, the mean scores were 3.85 (n=7) for wild-type, 1.75 (n=4) for nonspecific A, 1.33 (n=3) for nonspecific B, and 0 (n=3) for nonresponsive (CD163 -/-).

Table 13. Lung evaluation using microscopy

The maximum clinical signs correlated with the level of PRRSV in the blood. Measurement of viral nucleic acid was performed by isolation of total RNA from serum followed by amplification of PRRSV RNA using a commercial reverse transcriptase real-time PRRSV PCR test (Tetracore, Rockville, MD). A standard curve was generated by preparing serial dilutions of the PRRSV RNA control provided in the RT-PCR kit, and the results were normalized as the number of templates per 50 μl PCR reaction. PRRSV isolation followed the course of PRRSV viremia in wild-type CD163 +/+ pigs (Fig. 11). Viremia was evident on day 4, peaked on day 11, and then declined through the end of the study. In contrast, viral RNA was not detected in CD163 ^-/- pigs at any time during the study. Consistent with viremia, antibody production by non-responsive and uncharacterized allele pigs was detectable by day 14 and increased by day 28. Antibody production was absent in non-responsive animals (Fig. 12). Together, these data demonstrate that wild-type pigs develop clinical signs consistent with PRRS and support PRRSV replication. In contrast, knockout pigs did not develop viremia or clinical signs, even when vaccinated and continuously exposed to infected penmates.

At the end of the study, porcine alveolar macrophages were removed by lung lavage and stained for surface expression of SIGLEC1 (CD169, clone 3B11/11) and CD163 (clone 2A10/11) as previously described (Prather et al., 2013). Relatively high levels of CD163 expression were detected in CD163 +/+ wild-type animals (Figure 13). In contrast, CD163 -/- pigs showed only background levels of anti-CD163 staining, thus confirming the knockout phenotype. Expression levels for another macrophage marker, CD169, were similar for both wild-type and knockout pigs (Figure 14). Other macrophage surface markers, including MHC II and CD172, were identical for both genotypes (data not shown).

Although the sample size was small, wild-type pigs tended to gain less weight over the course of the experiment (mean daily gain 0.81 kg ± 0.33, n = 7) than pigs of the other three genotypes (noncharacterized A 1.32 kg ± 0.17, n = 4; noncharacterized B 1.20 kg ± 0.16, n = 3; nonresponders 1.21 kg ± 0.16, n = 3).

In a second attempt, six wild-type, six △43 amino acid, and six pigs carrying the uncharacterized allele (B) were challenged as described above, except that piglets were inoculated with KS06-72109. Similar to the NVSL data, wild-type and uncharacterized B piglets developed viremia. However, KS06 did not induce viremia in the △43 amino acid pigs (Fig. 15; Table 7).

Results and Conclusions

The most clinically relevant disease in the swine industry is PRRS. Although vaccination programs have been successful in preventing or ameliorating most swine pathogens, PRRSV has proven to be rather challenge-resistant. Here, CD163 is identified as the entry vector for this virus strain. The founder male pigs were produced by injecting CRISPR/Cas9 into zygotes (Whitworth et al., 2014) and are therefore transgenic. Additionally, one of the alleles from the sow (also produced using CRISPR/Cas9) does not contain a transgene. Thus, piglet #40 has a 7 bp addition to one allele and an 11 bp deletion to the other allele, but no transgene. This virus-resistant allele of CD163 represents minimal genome editing considering that the pig genome is approximately 2.8 billion bp (Groenen et al., 2012). If similarly produced animals were introduced into the food supply, significant economic losses could be prevented.

References

Boddicker, N.J., Bjorkquist, A., Rowland, R.R., Lunney, J.K., Reecy, J.M., Dekkers, J.C., 2014. Genome-wide association and genomic prediction for host response to porcine reproductive and respiratory syndrome virus infection. Genetics, selection, evolution: GSE 46, 18.

Etzerodt, A., Kjolby, M., Nielsen, M.J., Maniecki, M., Svendsen, P., Moestrup, S.K., 2013. Plasma clearance of hemoglobin and haptoglobin in mice and effect of CD163 gene targeting disruption. Antioxidants & redox signaling 18, 2254-2263.

Etzerodt, A., Moestrup, S.K., 2013. CD163 and inflammation: biological, diagnostic, and therapeutic aspects. Antioxidants & redox signaling 18, 2352-2363.

Graversen, J.H., Svendsen, P., Dagnaes-Hansen, F., Dal, J., Anton, G., Etzerodt, A., Petersen, M.D., Christensen, P.A., Moller, H.J., Moestrup, S.K., 2012. Targeting the hemoglobin scavenger receptor CD163 in macrophages highly increases the anti-inflammatory potency of dexamethasone. Molecular therapy: the journal of the American Society of Gene Therapy 20, 1550-1558.

Groenen, M.A., Archibald, A.L., Uenishi, H., Tuggle, C.K., Takeuchi, Y., Rothschild, M.F., Rogel-Gaillard, C., Park, C., Milan, D., Megens, H.J., Li, S. ., Larkin, D.M., Kim, H., Frantz, L.A., Caccamo, M., Ahn, H., Aken, B.L., Anselmo, A., Anthon, C., Auvil, L., Badaoui, B., Beattie, C.W., Bendixen, C., Berman, D., Blecha, F., Blomberg, J. , Bolund, L., Bosse, M., Botti, S., Bujie, Z., Bystrom, M., Capitanu, B., Carvalho-Silva, D., Chardon, P., Chen, C., Cheng, R., Choi, S.H., Chow, W., Clark, R.C., Clee, C., Crooijmans, R.P., Dawson, H.D., Dehais, P., De Sapio, F., Dibbits, B., Drou, N., Du, Z.Q., Eversole, K., Fadista, J., Fairley; S., Faraut, T., Faulkner, G.J., Fowler, K.E., Fredholm, M., Fritz, E., Gilbert, J.G., Giuffra, E., Gorodkin, J., Griffin, D.K., Harrow, J.L., Hayward; A., Howe, K., Hu, Z.L., Humphray, S.J., Hunt, T., Hornshoj; H., Jeon; J.T., Jern, P., Jones, M., Jurka, J., Kanamori, H., Kapetanovic, R., Kim, J., Kim, J.H., Kim, K.W., Kim, T.H., Larson, G., Lee , K., Lee, K.T., Leggett, R., Lewin, H.A., Li, Y., Liu, W., Loveland, J.E., Lu; Y., Lunney, J.K., Ma, J., Madsen, O., Mann, K., Matthews, L., McLaren, S., Morozumi, T., Murtaugh, M.P., Narayan, J., Nguyen, D.T.; Ni, P., Oh, S.J., Onteru, S., Panitz, F., Park, E.W., Park, H.S., Pascal, G.; Paudel, Y.; Perez-Enciso, M., Ramirez-Gonzalez, R., Reecy, J.M., Rodriguez-Zas, S., Rohrer, G.A., Rund, L., Sang, Y., Schachtschneider, K., Schraiber, J.G., Schwartz; J., Scobie, L., Scott, C., Searle, S., Servin, B., Southey, B.R.; Sperber, G., Stadler, P., Sweedler, J.V., Tafer, H., Thomsen, B., Wali, R., Wang, J., Wang, J., White, S., Xu, X., Yerle. , M., Zhang, G., Zhang, J., Zhang, J., Zhao, S., Rogers, J., Churcher, C., Schook, L.B., 2012. Analyzes of pigs genomes provide insight into porcine demography and evolution. Nature 491, 393-398.

Halbur, P.G., Paul, P.S., Frey, M.L., Landgraf, J., Eernisse, K., Meng, X.J., Lum, M.A., Andrews, J.J., Rathje, J.A., 1995. Comparison of the pathogenicity of two US porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus. Veterinary pathology 32, 648-660.

Holtkamp, D.J., Kliebenstein, J.B., Neumann, E.J., Zimmerman, J.J., Rotto, H.F., Yoder, T.K., Wang, C., Yeske, P.E., Mowrer, C.L., Haley, C.A., 2013. Assessment of the economic impact of porcine reprodutive and respiratory syndrome virus on United States pork producers. Journal of Swine Health and Production 21, 72-84.

Keffaber, K.K., 1989. Reproductive failure of unknown etiology. American Association of Swine Practitioners Newsletter 1, 1-9.

Kim, H.S., Kwang, J., Yoon, I.J., Joo, H.S., Frey, M.L., 1993. Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line. Arch Virol 133, 477-483.

Ladinig, A., Detmer, S.E., Clarke, K., Ashley, C., Rowland, R.R., Lunney, J.K., Harding, J.C., 2015. Pathogenicity of three type 2 porcine reproductive and respiratory syndrome virus strains in experimentally inoculated pregnant gilts. . Virus Res 203, 24-35.

Merck, The Merck Veterinary Manual. http://www.merckmanuals.com/vet/appendixes/reference_guides/normal_rectal_temperature_ranges.html.

Miao, Y.L., Sun, Q.Y., Zhang, Environmental and molecular mutagenesis 50, 666-671.

Patience, J.F., Thacker, P.A., 1989. Swine Nutrition Guide, in: Center, P.S. (Ed.), University of Saskatchewan, Saskatoon, CA, pp. 149-171.

Prather, R.S., Rowland, R.R., Ewen, C., Trible, B., Kerrigan, M., Bawa, B., Teson, J.M., Mao, J., Lee, K., Samuel, M.S., Whitworth, K.M.; Murphy, C.N., Egen, T., Green, J.A., 2013. An intact sialoadhesin. (Sn/SIGLEC1/CD169) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus. J Virol 87, 9538-9546.

Rowland, R.R., Lunney, J., Dekkers, J., 2012. Control of porcine reproductive and respiratory syndrome (PRRS) through genetic improvements in disease resistance and tolerance. Frontiers in genetics 3, 260.

Schaer, C.A., Schoedon, G., Imhof, A., Kurrer, M.O., Schaer, D.J., 2006a. Constitutive endocytosis of CD163 mediates hemoglobin-heme uptake and determines the noninflammatory and protective transcriptional response of macrophages to hemoglobin. Circulation research 99, 943-950.

Schaer, D.J., Schaer, C.A., Buehler, P.W., Boykins, R.A., Schoedon, G., Alayash, A.I., Schaffner, A., 2006b. CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin. Blood 107, 373-380.

Schaer, D.J., Schaer, C.A., Schoedon, G., Imhof, A., Kurrer, M.O., 2006c. Hemophagocytic macrophages constitute a major compartment of heme oxygenase expression in sepsis. European journal of haematology 77, 432-436.

Trible, B.R., Suddith, A.W., Kerrigan, M.A., Cino-Ozuna, A.G., Hesse, R.A., Rowland, R.R., 2012. Recognition of the different structural forms of the capsid protein determines the outcome following infection with porcine circovirus type 2. J Virol 86, 13508-13514.

Van Breedam, W., Delputte, P.L., Van Gorp, H., Misinzo, G., Vanderheijden, N., Duan, X., Nauwynck, H.J., 2010. Porcine reproductive and respiratory syndrome virus entry into the porcine macrophage. J Gen Virol 91, 1659-1667.

Van Gorp, H., Delputte, P.L., Nauwynck, H.J., 2010. Scavenger receptor CD163, a Jack-of-all-trades and potential target for cell-directed therapy. Mol Immunol 47, 1650-1660.

Wensvoort, G., Terpstra, C., Pol, J.M.A., ter Laak, E.A., Bloemrad, M., de Kluyer, E.P., Kragten, C., van Buiten, L., den Besten, A., Wagenaar, F. , Broekhuijsen, J.M., Moonen, P.L.J.M., Zetstra, T., de Boer; E.A., Tibben, H.J., de Jong, M.F., van't Veld, P., Groenland, G.J.R., van Gennep, J.A., Voets, M.T.H., Verheijden, J.H.M., Braamskamp, J., 1991. Mystery swine disease in The Netherlands: the isolation of Lelystad virus. Veterinary Quarterly 13, 121-130.

Whitworth, KM, Lee, K., Benne, JA, Beaton, BP, Spate, LD, Murphy, SL, Samuel, MS, Mao, J., O'Gorman, C., Walters, EM, Murphy, CN, Driver. , J., Mileham, A., McLaren, D., Wells, KD, Prather, RS, 2014. Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro -derived oocytes and embryos. Biol Reprod 91, 78.

Winckler, C., Willen, S., 2001. The reliability and repeatability of a lameness scoring system for use as an indicator of welfare in dairy cattle. Acta Agricultura Scandinavica Section A. Animal Science, 103-107.

The embodiments disclosed herein are provided by way of example and are not intended to limit the scope of the invention.

In light of the above, it will be seen that several objects of the present invention are achieved and other advantageous results are achieved.

Since various changes could be made in the above products and methods without departing from the scope of the present invention, all matters contained in the above description and illustrated in the attached drawing[s] should be construed in an illustrative rather than a limiting sense.

Sequence table

<110> Curators of the University of Missouri Prather, Randall Wells, Kevin Whitworth, Kristin <120> PATHOGEN-RESISTANT ANIMALS HAVING MODIFIED CD163 GENES <130> UMO 16001.USP <160> 117 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> DNA <213> Sus scrofa <400> 1 ggaaacccag gctggttgga ggg 23 <210> 2 <211> 23 <212> DNA <213> Sus scrofa <400> 2 ggaactacag tgcggcactg tgg 23 <210> 3 <211> 23 <212> DNA <213> Sus scrofa <400> 3 cagtagcacc ccgccctgac ggg 23 <210> 4 <211> 23 <212> DNA <213> Sus scrofa <400> 4 tgtagccaca gcagggacgt cgg 23 <210> 5 <211> 23 <212> DNA <213> Sus scrofa <400> 5 ccagcctcgc ccagcgacat ggg 23 <210> 6 <211> 23 <212> DNA <213> Sus scrofa <400> 6 ctttcattta tctgaactca ggg 23 <210> 7 <211> 23 <212> DNA <213> Sus scrofa <400> 7 ttatctgaac tcagggtccc cgg 23 <210> 8 <211> 23 <212> DNA <213> Sus scrofa <400> 8 cagctgcagc atatatttaa ggg 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 9 ctcctcgccc ttgctcacca tgg 23 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 10 gaccaggatg ggcaccaccc cgg 23 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 11 ctctccctca ctctaaccta ctt 23 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 12 tatttctctc acatggccag tc 22 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 13 ctctccctca ctctaaccta ctt 23 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 14 gactggccat gtgagagaaa ta 22 <210> 15 <211> 27767 <212> DNA <213> Sus scrofa <400> 15 atacaagtgc cttttacaga caatctgcac aagttatttg ttagacatat ttgattatag 60 aattaatatt aaaaggggtt ataacaatca agcattgata atttaattat gtttgcctat 120 tttactttag ttttttgaca taactgtgta actattgcga tttttttat cctaatgtaa 180 ttagttcaaa acaaagtgca gaaatttaaa atattcaatt caacaacagt atataagtca 240 atattccccc cttaaatttt tacaaatctt tagggagtgt ttctcaattt ctcaatttct 300 ttggttgttt catgtcccat atggaagaaa acatgggtgt gaaagggaag cttactcttt 360 tgattacttc ccttttctgg ttgactccac ctccattatg aagcctttct gtatttttgt 420 ggaagtgaaa tgatttttag aattcttagt ggttctcttc ttcaggagaa catttctagg 480 taataataca agaagattta aatggcataa aaccttggaa tggacaaact cagaatggtg 540 ctacatgaaa actctggatc tgcaggtaaa atcttctcat ttattctata tttacctttt 600 aaatagagtg tagcaatatt ccgacagtca atcaatctga tttaatagtg attggcatct 660 ggagaagaag taacagggaa aaaggcaata agctttataa ggggaacttt tatcttccat 720 agactcaaaa ttgaagacgt gactagaaga ttgctagatt tggcatcagt tttgtaaaat 780 tgctgaggtg aaattaagta agggatgaaa attaactaaa ttgtgttgag tatgaaacta 840 gtagttgtta gaaaagatag aacatgaagg aatgaatatt gattgaaagt tgatgaccta 900 gaggacattt agactaacac ctctgagtgt caaagtctaa tttatgattt acatcgatgc 960 gttaaactca tttaacattc ttactttttt cccctcaagc atttaagctg aagtataaca 1020 tttcacatga aagcctggat tataaatgca cagttcagtg acctatctca gaggagtgac 1080 tgccatagca ttttttttgt ctttttgcct tcagagccac agcaacgcgg gatccgaagc 1140 cgcgtctgcg acccacca cagctcacgg caatgccgga tctttaaccc actgagcgag 1200 gccggggatc gaacccgcag tctcatggtt cctagtagga ttcgttaacc actgcgccac 1260 gacgggaact cctaccatag catttttact tttaagttac tgttggttta gagtaagaag 1320 gagaaatgag agtgatggag cgtttgctat atttggagac aaggtcctat attggaggtt 1380 ctcaaatata aattttgtcg ctttttcctc caatgtattg ttcaactact atttagcagg 1440 ccactgtgcc aggtactggt gaaactggtg aacatgatag atgtaattca ttccctcatg 1500 gaactttcca tctaacaatg tggatcaggt aggcttggag atgagaatgc cagtggttga 1560 ctatgactct gtggctgaag ggagagctac tcacttcgta gtttcatcaa tgtctttttg 1620 gttttccagg ttttaagccc tgctcttgca attcttttcc cttctccaac tttcttctaa 1680 tttctcaccc ctaggatgcc tataaacatg agtattttca aagctacttc actgaggtta 1740 tatgatcctg gtgtgaattt ttcctgcctg acttgccatt tagaaggaag tgtttcctgg 1800 aatttccatt gtggcttggt ggttaaagac cctgcattgt ctctgtgagg atgtgggttc 1860 aatctctggc ctcattcagt gagtgggtta aggatctggt gtcgctgcaa gctgtggcta 1920 agatcccaca ttgccatgtc tgtggtgtag actggcacct ggagctctga tttgaccaca 1980 atcttaggaa cttcagatgt ggccataaaa aggaaaaaaaa agttaggaag ggttttctgt 2040 cttgtttgga ccttcgttaa tctcaaacct ttggaaccat ctctcctcca aaacctcctt 2100 tgggtaagac tgtatgtttg ccctctctct tcttttcgca gactttagaa gatgttctgc 2160 ccatttaagt tccttcactt tggctgtagt cgctgttctc agtgcctgct tggtcactag 2220 ttctcttggt gagtactttg acaaatttac ttgtaaccga gcccaactgt gacaagaaac 2280 actgaaaagc aaataattgc tcctgaagtc tagatagcat ctaaaaacat gcttcatggt 2340 ttcaaggatc atatattgaa accccaggga tcctctagag tcgacctgca gcatgcaggg 2400 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 2460 gggggggggg gggggggggg gggggggggg gggggggggg gtgcataagg aaagactatc 2520 tcaacgtctt attcctcagc ttacattaga tttgaaactc tagtcaccta aaatgcaaat 2580 ctcatttact taccatcaga gatattaatg acctatagaa ttcagcataa ataaagtttc 2640 atgtatggat attagcttat ggttctagtc actgctaatt gaaacctgtg atattgctgt 2700 ttgttttgac tcctatgaaa taacattctc ccattgtacc atggatgggt ccagaaacat 2760 ttctcaaatc ctggcttgaa aaaataaata agtaatctaa agaataataa ttctctactt 2820 gctctttgaa tcttgaccaa ttgctgcatt tacctattgt tacaggagga aaagacaagg 2880 agctgaggct aacgggtggt gaaaacaagt gctctggaag agtggaggtg aaagtgcagg 2940 aggagtgggg aactgtgtgt aataatggct gggacatgga tgtggtctct gttgtttgta 3000 ggcagctggg atgtccaact gctatcaaag ccactggatg ggctaatttt agtgcaggtt 3060 ctggacgcat ttggatggat catgtttctt gtcgagggaa tgagtcagct ctctgggact 3120 gcaaacatga tggatgggga aagcataact gtactcacca acaggatgct ggagtaacct 3180 gctcaggtaa gacatacaca aataagtcaa gcctatacat gaaatgcttt gtgggaaaaa 3240 atgtatagat gagttaaaaa caaaaaggaa ccagttttct ataagtcatc tagtccatgt 3300 ataaaattac ccaatccatt actaaaagac cacttctggt attttacaca tgacaaagcc 3360 catattaaaa aaaaaaaatt cagaagagat tctgaatgct ataataaatg agcaagtgac 3420 tagcttcaat tttatattag gtcattctac cttctacttc tacatgaaaa tatcataatg 3480 tctaagttaa ttccttgtcc cctttcccaa taaagcactg ctttcatgca ctggcctatg 3540 aatcatgaac tttttgccct ttaactgatg atcaacttac caaatcaaga aataaatatt 3600 cttagcactg atcctttttt gttgttgttg gaggaagaat gttttgcaaa gtagaattgc 3660 ttttttctgt ttaacagtgc tattcatttc atttacatgg tcgttttaat ttataaaaca 3720 tttcataagt ttcacctcat atgcccttac aataactcag gaagttatat gttagacctt 3780 tctgctgaca aatcccagag tcatgtttct gacccagttc agattccttg gcttcccatt 3840 tctctttgct catgtcattg acctttatgc agccctctta cctcccacct ttctattaca 3900 gaccatctcc tccataggac tggtgttaga aagtactaat ctctacccag gcattgtggt 3960 gcaatgtggg cagcacaggc tggtatctag aaaaatgctg aagtgaattc cagctcagct 4020 gctcgttaat actatcgttt taagtaagct gttcaatcct ttgaaattca ctttctgagc 4080 actcagtgat ataataaatg tagagctact ggtacactgt ctggtatgta ataggtgtta 4140 ccaattaacc ttagtttcct catgggtcac tggttctcat tacctagaca actcatttct 4200 ctttcttcct ctttctcttt ctccattctc ctcctccttc ttcctcttct tcttgtctgt 4260 tattgttata tcattttgct gagaaagtta agaaataaca actctaacct ctacatcgac 4320 cacctagagc aaagttaaaa ataataataa accttgccag actcttacta taattgttgc 4380 tgtctataga gttgactgtt taagttaaga catcagtata tatttttaat ttttgtgttt 4440 tttttttcat acttttacat gaggatcctt tatataagga tgagttaaac aaacttgatt 4500 tttgaagttt atacccctga ggctcaactg cataataata gaaagggatc catagcctct 4560 caaggactta actagtttca tgagttttca gaatctgaat ttctgagatt ctccacccca 4620 attaaagctc aagcctcaga acatatatcc ttctcttggt aaattctatt cttatcacat 4680 gcgtaataat aaaaaagaga gatgttggag acagattttt ttcctcacat tctgtctcta 4740 ctgttttcta ggtgtttgat tctgtgttat ttaacctcag tttgcttatc tgtgaagtag 4800 ggattatggt aataacatat aatgctttat gttgtaaaga ctaaagaaga tagcatatgt 4860 aacacatttg gaacagggaa tgcatatttt gattgtgagc tcttattatt attaccattc 4920 agccctaata aaaatcttgg taagtggaag gctttggatt tcagaacttt taaaatctaa 4980 ttactttttc aaaaaagaac ttcttagggt tttttttttt taaccacaaa gtgtttctat 5040 tttttaggtg tcccaaaatt tcgttccaaa tatctttttc tcagatattt tagtcctcat 5100 agaacaccta gggatagtgg atagagaaaa ttttctttat taaaaagctg ttctttgcta 5160 aaaattgtag caggtacttt tgggaggggg gaaaactttg attcagaaac tgctaagaca 5220 tggagtgttt tgactaattt ttcctcaatt tttaatgttt tttataccat agggtacttt 5280 tgcaaactat tatgcatact tatatatttt tacttttttc ctgtctttta acttccaaat 5340 tcaacttcag acaattattc atgcactaaa ctgtttgtag taagaaagat taaaattaaa 5400 aaattaacca ttcaacaaat gactggtttg ccatttttac tactttgttg tatgaacaat 5460 ttttttttct acaaatgaat actttgagtc tgatttatcc attcctacat aaaagttttt 5520 actatatctt agtattggaa ggaaacaaaa caaaacacaa tgtaaatttt aatctataaa 5580 ttttgggggg gtaaatatac atagatgaaa gtcttaacca ttaattagag tcaaaagatt 5640 aaaattctcc aatatgtgaa cttaggctgc atccaaaatg aagcatcatt tttaaggaca 5700 gcatcaaaag tgaccagagg aattttactt tctttctttt tttttttttt gaattttagt 5760 ttctaaactc acttctgaat aaatacaact tctaaattct cgtcttttct ctactctaga 5820 tggatctgat ttagagatga ggctggtgaa tggaggaaac cggtgcttag gaagaataga 5880 agtcaaattt caaggaacgt ggggaacagt gtgtgatgat cacttcaaca taaatcatgc 5940 ttctgtggtt tgtaaacaac ttgaatgtgg aagtgctgtc agtttctctg gttcagctaa 6000 ttttggagaa ggttctggac caatctggtt tgatgatctt gtatgcaatg gaaatgagtc 6060 agctctctgg aactgcaaac atgaaggatg gggaaagcac aattgcgatc atgctgagga 6120 tgctggagtg atttgcttaa gtaaggactg acctgggttt gttctgttct ccatgagagg 6180 gcaaaaaaag gggagtaaaa gtcttaaaag ctcaaactgt taaaaacata atgatgattg 6240 cttcttttat catcttatta ttatctaatt tcaggtcgaa attctagtac ctgtgcagtt 6300 ttttacctta actgaaatta agataaatag gatagggagg aaggatgagc agtgacattt 6360 aggtccaagt catgaggtta gaaggaaatg ttcagagaat agcccattcc ctcagccctc 6420 aaagaaagaa agaaagaaaa agaaaaaaaaa aaagaaagct taactagaaa attttgttct 6480 ctggatgttt tagaggcaaa ccatcccttt atcattccta cctacaaagc cttctcttaa 6540 tcacattacc caccctttcc tactatagtc aggggggggg gggggggggg gggggggggg 6600 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 6660 gggggggggg gtgaaaaaag aaccaaacaa tttcaacaaa aaaccaaaca attccaacaa 6720 aattggtcca ataagcaaac ctcttagataa atttcagtgc cctggatgtt ttgttaggaa 6780 ctcttcctac aatgcgtgct ttccattctg aaaagtccta tctacttgcc tgatccactt 6840 ctccttccat cctaaacgat tttcagtggt agtatattac tgttgtctct gtctctactt 6900 atatatcttc cccttttcac tcactcctct caggtacagc tcttcagttt gcccttattc 6960 ttgtttcctt gtcaatgact tgttttgtgt ccctcttaca gatggagcag acctgaaact 7020 gagagtggta gatggagtca ctgaatgttc aggaagattg gaagtgaaat tccaaggaga 7080 atggggaaca atctgtgatg atggctggga tagtgatgat gccgctgtgg catgtaagca 7140 actgggaggt ccaactgctg tcactgccat tgtcgagtta acgccagtga gggactggac 7200 acattggctc acacacatac agccatgaca cgatctgctc tatggtccga tgattaaagg 7260 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 7320 gggggggggg gggggggggg gggggggggg ggggggggag aagagctggt ggacatttct 7380 ggaaaggaac caaaacccgg aagggccttg ttcttcagga tttgggatgg attggggagg 7440 gagaaaattg tttctaatat ttcttggtgg gaattctttt acagttgtga caaatctttc 7500 acatattctt catttgagta gtttggaggg ttgtctgact gttttctata ataaatgtcc 7560 caagtgctat gaggtaccac atttcaaatt ctaattctac ctgaagctcc aaaaagacaa 7620 aatgttatag gtcttttctt tatatctaat ttgcttatgg tttttagcca ttgacaattt 7680 ttttttctta actcttgaaa ctataaccct atttctaacc aaattcatgt tctatactgg 7740 ctcttcaaaa acccaggaga tgggaaagcc agaatctcca gtgtttcagc ttctgggaag 7800 gagcaagttt ttaaaaatac cctctgggag ctaaattcca catgtatcta tggcctaagt 7860 gtatgtttat tttgcagatg gatcagatct ggaactgaga cttaaaggtg gaggcagcca 7920 ctgtgctggg acagtggagg tggaaattca gaaactggta ggaaaagtgt gtgatagaag 7980 ctggggactg aaagaagctg atgtggtttg caggcagctg ggatgtggat ctgcactcaa 8040 aacatcatat caagtttatt ccaaaaccaa ggcaacaaac acatggctgt ttgtaagcag 8100 ctgtaatgga aatgaaactt ctctttggga ctgcaagaat tggcagtggg gtggacttag 8160 ttgtgatcac tatgacgaag ccaaaattac ctgctcaggt aagaatttca atcaatgtgt 8220 taggaaattg cattctactt tcttttacat gtagctgtcc agttttccca gcaccacttg 8280 ttgaagagac tgtcttttct tcatcatata gtcctacatc ctttgtcata aattaattga 8340 ccataggtgt gtgggtttat atctgggctc tctattctgt tcctttgatc tatatgtctg 8400 tttttatgcc agcaccatgc tgttttgatt actatagctt tgtagtatca tctgaagtca 8460 ggaaacatga ttcctccagc tttgttcttc tttctcaaga ttgttttgtc tattcagagt 8520 ttatgttccc atgcagattt aatttttaaa tttatttaat ttttattttt tatttttaat 8580 ttaaattaat ttaaattttt tatttcccaa cgtacagcca agggggccag ggtaaccttt 8640 acatgtatac attaaaaatt tcaggttttt cccccaccca tttctttctg ttggcaagta 8700 aatttttgaa caaagtttcc caatgctttt taaggggaat tcccttgggg gggggggggg 8760 gggggggggg gggggggggg gg gggggggg gggggggggg gggggggggg gggggggggg 8820 gggggggggg gggggggggg gggggggggg gggggggggg agacgaaatt gactatattt 8880 tctttgttgg gaatctttta cagttgtgac aaatctttca catattcttc atttgagtag 8940 tttggagggt tgtctgactg ttttctataa taaatgtccc aagtgctatg aggtaccaca 9000 tttcaaattc taattctacc tgaagctcca aaaagacaaa atgttatagg tcttttcttt 9060 atatctaatt tgcttatggt ttttagccat tgacaatttt tttttcttaa ctcttgaaac 9120 tataacccta tttctaacca aattcatgtt ctatactggc tcttcaaaaa cccaggagat 9180 gggaaagcca gaatctccag tgtttcagct tctgggaagg agcaagtttt taaaaatacc 9240 ctctgggagc taaattccac atgtatctat ggcctaagtg tatgtttat ttgcagatgg 9300 atcagatctg gaactgagac ttaaaggtgg aggcagccac tgtgctggga cagtggaggt 9360 ggaaattcag aaactggtag gaaaagtgtg tgatagaagc tggggactga aagaagctga 9420 tgtggtttgc aggcagctgg gatgtggatc tgcactcaaa acatcatatc aagtttattc 9480 caaaaccaag gcaacaaaca catggctgtt tgtaagcagc tgtaatggaa atgaaacttc 9540 tctttgggac tgcaagaatt ggcagtgggg tggacttagt tgtgatcact atgacgaaac 9600 caaaattacc tgctcaggta agaatttcaa tcaatgtgtt aggaaaaattg cattctactt 9660 tcttttacat gtagctgtcc agttttccca gcaccacttg ttgaaaaaac tgtctttttc 9720 ttcatcatat agtcctacat cccttggcca taaattaatt gaccataagg ggtgtgggtt 9780 taatatccgg ggctcctcaa ttcgggtccc ttggatccta aaagccggtt ttataacccg 9840 acacatggcc tgtttttgac taaataaaac ctttggaaaa caatcccgaa ggtcgaggaa 9900 catggaatcc ccccaacaaa ggaccttctt tccccaaaaa tgcggctcag ccaactcaaa 9960 aagattttat gaatcacaaa ccgcacatta tcttcctaaa attactattc ctatgtttta 10020 atttgcaaag tcattccgat atagttggcg cagagtaact catttagata tccaccccac 10080 cagttcctca ctcaagtaag gggggggggg gggggggggg gggggggggg gggggggggg 10140 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggc 10200 ccccatgtga gattttgtgt gtcctttaag agtggagtct ctatttccca ctgctctctg 10260 gttctcccca aagtaagccc tgctggcttt caaaacttct gggagcttgc cttcttggta 10320 taggactcct gggctaggga gtctaatgtt tggcttagac cccttactgc ttgggaagaa 10380 tctctgcaac tgtaatgaat tatcttccta tttgtgggtt gctgaggata tggtcttaac 10440 tgttctgtgt tctacccctc ctatccatct tgttgtggtt ccttctttat atctttagtt 10500 gtagaaaagt ttttcttatc aacagttgct ctgtaaattg taacttgggt gtacacctag 10560 taggaggtga gctcagggtc ttcctactct gccatcttgg ccatgtcctc taaacatttt 10620 ggtgtatttc actgcaacct ttttaaaaat ctcaaaagtg agctgtgatt ggctagtctt 10680 gtggataatc tctagcattt gatgctaatc atatttatac aaatactttg ttgaaaagtg 10740 atgccttttt aactattatt aaaaaacgta ttgacataac tattgctatt atactgaaaa 10800 gaaagacctt agagaaaata gcataagagc aaaaccatta aacatggaga catctagtca 10860 tagggtggaa attttatgtg gtccatatcc cctaaccagt ggctttacac caggcacatc 10920 ctaactaaga tctgctccca agtgtcttcc ctgatgcttt aaattgtgtt acatggaaac 10980 tatcctttga tgaagaaatg caacctttta aaatacaaca ttgaaacttt tgtgctttaa 11040 ttttgctttt caacattttt tctttttaaa agaagaaatt tatttgtttt tttaaatttt 11100 aatggccacg gcatatggaa gttctcaggc cagggataga attcaagcca caggtgcgac 11160 ccatgccaca actgctgcaa caccagatcc tttaacccac tgcaccaggc cagggattga 11220 agccttgcct tactgacaat ctgagccact tcagtcagat aaagaaattt cttcattaag 11280 cagagtattc acatggttta aacttcaaaa tattaaagtg taaactcttt ccccaccact 11340 gtccccagct caccaactct acttaccaca gacaactgat gtggttaggg tatttaaata 11400 gtaaatccaa gaaaatataa acaaatccgt atatataggt ttcaccccat tttattatcc 11460 taatgttgca tatcatataa actatactgt cccttgggta ttcacttagt aaaatatttt 11520 gatcataatt tcctatcagt atttaaagag ctttctgaaa ttatttctgt ataacatttc 11580 ttttctcatc ggtagggggg gggggggggg gggggggggg gggggggggg gggggggggg 11640 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg ggggaatggg 11700 aagaaaaaac caccatggtt aatttttttt atccctctac acccgggaaa attacccttg 11760 gggccacact tttctataga aaggggatta tttaaaaggg tctgaaaaag aatttttttt 11820 tcgaaagggg aaatatttgg cctaacttag tcacataagc catgttctct ggcaagttag 11880 gtaacataca tttttgtcat tgggggcaac aaaaacaatt ttccttttgg accttttggg 11940 actccgcatt ggttagggaa ggggaagtat attggaattc ggaaaattcc ttccaaatta 12000 aaaaaggttt gttatttca tattaaccta tttcatatta attagcatga attccagcgc 12060 cattaaaagg gaaaacacct ggagtggtaa gaaaaaagtt tttttttctc tttttttttt 12120 ttttttttta atggccacat ctgtggcatg tgaagttccc aggctagggg tcgaatagga 12180 gctacagctg ccagcttgca ccacagccac aacaatgcca gagccaagcc tcatctgcga 12240 cctataccac aactcatggc aatgctggtt ccttaacccc ctgagtgagg cctggggtca 12300 aacccacatc ctcatggata ctaaccggct ttgttaccgc tgagccatga gggaaactcc 12360 ctttttctca ttgaaaataa gtcaaataga taagcagctt aaggctgttt gggtgattct 12420 gtggtccagt aattatcaaa tcctactgga caagaataga gaatgtgcaa atgagggaac 12480 gtgttggtga gatcaggctc tgcccactga gctatcctct gtcatgggcc ctgtgctgtt 12540 ctcagagctg tacttcctag ggcattgttc tcatttcaat tctgagttca gtgtggagag 12600 tatacgtgtg tgggggctgc acgcttttca caacccactt tctgctgata ctgatttagg 12660 gatccttgga ttgctttaca gttgagtcat cattaactag tgtcacttgc cttcaaagtc 12720 agcaaaataa ttgtctccaa actagtaggc ttctagtgta tttgctttaa tccaatgcca 12780 tgtgaaagta acatggtcaa agaataagtt atataccttg acctaccctg tgaccaggct 12840 cttcctctta atttattgac cactgcctta aggtcatttg aaaccatggg tttgggagga 12900 aggcaaggcc taaatcccgt ctttgttgga aggctcactg tccttgtctt tagagcatca 12960 ttttttttta aactggggta cagtttattt acagtgttgt gtcaatttct gctgtacagc 13020 acagtgaccc agtcatacac atacatacat tctttttctc atactatctt caattttat 13080 ttctgctaag tctgccattt tatcatcacc tcagtttgaa ggacaggata tttagagttt 13140 gttttttttt tccccccaat cctgcaattt ctaaattata agactctcaa ttagccgtat 13200 ataacagctg caggcacagg atgtctccct cacaaaattg gtatttttcc ttccatttct 13260 tcttgcagtt tggctatttc ttgtctgagt tcatctctct ttttaagtgt taaaaagggc 13320 aaggaggatt catgctatgt caacattatg attttttctt ttctatactt gataagagta 13380 tacttttccc aaatgtcatc caacttttca gcatcagttt ggacatggtt ttcttttcaa 13440 ggtggtattt ctctaatgtc acttgaataa caagactcgt tagttctcca ggctacaata 13500 tcctagtctg agtatattct gcatgttaat tctattcagc cacatccata atttaggttt 13560 tattcctgga acacctcact tttttttttt tttttggtct ttttatagcc ataaccatgg 13620 catatggagg ttcccaggct aggggtctaa tctgagcttt agccactggc ccatgccaca 13680 gccacagcca tgccacatct gagccacatc tgtgaccttt tccacagctc acagaaacac 13740 cagatcccta acccactgag tgaggccagg ggtcaaacct gtaacccttc catggttcct 13800 agtcagattc gttcctctgt accacgatgg gaattcctaa tacctcactt atgataacac 13860 attctgaatt atttaggatt ctattatact gcatgtaata gaaatcccaa atagcaaaat 13920 ttgcaactta aggcaggttc ctgtctttac aaaatcatgt tttcctttgc tatatgtgca 13980 ctttgctttc ctctgtgaat tccctttttt gttatatttc tatagctttt ggaaacactt 14040 ttacttattt gggggggcct agatttttaa ccctctcctt gtttttctag aaatagagtt 14100 tataatttta tttcttcatt tacttgatac tttcaagaga ttcccaggaa aaaaattatg 14160 gaaatactgt ctctgtgcct gccaagttca aactaagaat tgtataatct gttttaattc 14220 ttaagcattt atagatgaca aggctttgtg tctgataggg gccagcgaac tcagtaaaga 14280 gggaagatga gaaagataat ggcaagaatt tatccctgaa gtgtagtttt gacaaaccag 14340 tcacaaagag gtctaagaaa ttttggtcac aaagttgttt tgaatcccag gcattttatt 14400 tgcaatgatt gcatatgttc tggaaaaggac atctgaacct aagaaatagt tcatttgcat 14460 tgtgttatat tttactaagg tctgagaaat aatcttgaga tgagaatgaa ctctacttct 14520 tcagagtctg gaaggaataa attatgaaaa tgtattaatg cttctttaaa ccatattgta 14580 tatttatcta ttactaaaaca aaaagaagta gctctattta tttattatt tatttattta 14640 tttatgtctt ttgtctcttt agggccacac ctgtggcata tggaggttcc caggctagag 14700 gtccaattgg agatgtagca gccagcctat gccagagcca ccgcaacacg ggatctgagc 14760 cacgtctgtg acttacacca cagctcacag caacgcctga tcctcaaccc actgagcgag 14820 gccagggatc gaacccatgt cctcatggat gctagttggg ttcgttaact gctgagccat 14880 gatgggaact ccaaattaat tatttcttat atttgttctt catatattca tttctataga 14940 aagaaataaa tacagattca gttaatgatg gcaggtaaaa gcttaactta ttaatcaaag 15000 gagttaatcc aggcacaaaa attcaattca tggctctctg ttaaaattta ggtataggtt 15060 tagcaggaag aaaaggttag tagatgcaga ctattacatt tagaatggat ggacaatgaa 15120 gtcctactat acagcacagg gaactatatc caatctcttg ggatagaata tgatggaaga 15180 caaaatcaga acaagagagt atatatatat gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 15240 gtgtgtgtgt gtgtgactgg gtcaccctgc ggcacagcag aaattggcag aacattgtaa 15300 atcaactata ctttaatagg aaaaatactt ttaagggcta aatttccaat attctaacca 15360 tgtacacaga gtaaatgtca taaggatgcc agtctgtgta gagattgatg tgttactagc 15420 agattcatga aataaaggct gaggatgtag tccccaagtc acttctgagt ggaagaattt 15480 ctcctttgtc ctggactcaa atattttagg ataaaggaaa aaagaagata tttatagaag 15540 ggacttgttt tcaagtactt gacaaaattt caccattaaa gagaaatttg tgggagttcc 15600 catcgtggct cagtggaaac aaatccaact aggaaccatg aggttgtggg tttgatccct 15660 ggcctcactc agtgggttaa ggatccggtg ttgccgtgag ctgtggtgta ggttgcagac 15720 acggttctga tcctgcgttg ctgtggctgt ggctgtggtg taggccagca gcaaacagct 15780 ctgattagac ccctagcctg gaaacctcca tatgccacag gtgcagccct aaaaagacaa 15840 aaaaagagaa aagacaaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaag 15900 aacccccaga ggtatttatt tgtttttgcc ttttttcact gactgttctt tgtttgtttg 15960 tttgagactg atctagaaga ctagagatta caagaaatat ggatttggct cactctaaga 16020 aactgctttc attccaaggt ttgggtctat ccaaaagtgg aatagaatca tatgaatact 16080 agtttatgag tatttagtga gaggaatttc aagctcaaat aatgattcag caagattaaa 16140 ttaaggaggg aattttcctt gtggctgagt gggttaagga cccaatgttg tctctgtgag 16200 gatgtaggtt ccatcctggg ctttgctcat taggttaagg atctggcatt gctgcagctc 16260 agacccagtg ctgccctggt tgtggcttag gccaaagctg cagctccaat tcaatctctg 16320 gcctgggaac ctccatgtgc tacaaggtgc ggccttaaaa ggaaaaaaaa aaaattaaat 16380 caaggactca agagtctttc attatttgtg ttgtggaagc tatatttgtt ttaaagtctt 16440 agttgtgttt agaaagcaag atgttcttca actcaaattt gggagggaac ttgtttcata 16500 catttttaat ggataagtgg caaaattttc atgctgaggt gatctatagt gttgtaatgc 16560 agaatatagt cagatcttga acattttagg aagttggtga gggccaattg tgtatctgtg 16620 ccatgctgat aagaatgtca agggatcaca agaattcgtg ttatttgaca gcagtcatct 16680 ttaaaaggca tttgagaaag tccaatttca aatgcatttc ctttctttaa aagataaatt 16740 gaagaaaata agtctttatt tcccaagtaa attgaattgc ctctcagtct gttaaaagaa 16800 actcttacct tgatgattgc gctcttaacc tggcaaagat tgtctttaaa atctgagctc 16860 catgtcttct gctttatttc tggtgtgcct ttgactccag attacagtaa atggaggact 16920 gagtataggg ctaaaaagta gagagaatgg atgcatatta tctgtggtct ccaatgtgat 16980 gaatgaagta ggcaaatact caaaggaaag agaaagcatg ctccaagaat tatgggttcc 17040 agaaggcaaa gtcccagaat tgtctccagg gaaggacagg gaggtctaga atcggctaag 17100 cccactgtag gcagaaaaac caagaggcat gaatggcttc cctttctcac ttttcactct 17160 ctggcttact cctatcatga aggaaaaatat tggaatcata ttctccctca ccgaaatgct 17220 atttttcagc ccacaggaaa cccaggctgg ttggagggga cattccctgc tctggtcgtg 17280 ttgaagtaca acatggagac acgtggggca ccgtctgtga ttctgacttc tctctggagg 17340 cggccagcgt gctgtgcagg gaactacagt gcggcactgt ggtttccctc ctggggggag 17400 ctcactttgg agaaggaagt ggacagatct gggctgaaga attccagtgt gaggggcacg 17460 agtcccacct ttcactctgc ccagtagcac cccgccctga cgggacatgt agccacagca 17520 gggacgtcgg cgtagtctgc tcaagtgaga cccagggaat gtgttcactt tgttcccatg 17580 ccatgaagag ggtagggtta ggtagtcaca gacatctttt taaagccctg tctccttcca 17640 ggatacacac aaatccgctt ggtgaatggc aagaccccat gtgaaggaag agtggagctc 17700 aacattcttg ggtcctgggg gtccctctgc aactctcact gggacatgga agatgcccat 17760 gttttatgcc agcagcttaa atgtggagtt gccctttcta tcccgggagg agcacctttt 17820 gggaaaggaa gtgagcaggt ctggaggcac atgtttcact gcactgggac tgagaagcac 17880 atgggagatt gttccgtcac tgctctgggc gcatcactct gttcttcagg gcaagtggcc 17940 tctgtaatct gctcaggtaa gagaataagg gcagccagtg atgagccact catgacggtg 18000 ccttaagagt gggtgtacct aggagttccc attgtggctc agtggtaaca aactcgactg 18060 gtatccatga gggtatgggt ttgatccctg gccttgctca atgggttaag gatccagcat 18120 tgctgtgagc tgtggtatag gttgcagact ctgctcaggt cccatgttgc tgtgattgtg 18180 gtgtaggctg actgctgcag cttcaatttg acccctagcc cgggaatttc cataggccac 18240 acgtgcagca ctaaggaagg aaaaaaagaa aaaaaaaaaa aaagagtggg tgtgcctata 18300 gtgaagaaca gatgtaaaag ggaagtgaaa gggattcccc cattctgagg gattgtgaga 18360 agtgtgccag aatattaact tcatttgact tgttacaggg aaagtaaact tgactttcac 18420 ggacctccta gttacctggt gcttactata tgtcttctca gagtacctga ttcattccca 18480 gcctggttga cccatccccc tatctctatg gctatgttta tccagagcac atctatctaa 18540 cactccagct gatcttcctg acacagctgt ggcaaccctg gatcctttaa ccaactgtgc 18600 caggctggag atcaaaccta agcctctgca gcaacccaag ctgctgcagt cagattttta 18660 accccctgtg ccactgtggg tatctccgat attttgtatc ttctgtgact gagtggtttg 18720 ctgtttgcag ggaaccagag tcagacacta tccccgtgca attcatcatc ctcggaccca 18780 tcaagctcta ttatttcaga agaaaatggt gttgcctgca taggtgagaa tcagtgacca 18840 acctatgaaa atgatctcaa tcctctgaaa tgcattttat tcatgtttta tttcctcttt 18900 gcagggagtg gtcaacttcg cctggtcgat ggaggtggtc gttgtgctgg gagagtagag 18960 gtctatcatg agggctcctg gggcaccatc tgtgatgaca gctgggacct gaatgatgcc 19020 catgtggtgt gcaaacagct gagctgtgga tgggccatta atgccactgg ttctgctcat 19080 tttggggaag gaacagggcc catttggctg gatgagataa actgtaatgg aaaagaatct 19140 catatttggc aatgccactc acatggttgg gggcggcaca attgcaggca taaggaggat 19200 gcaggagtca tctgctcggg taagttctgc acatcacttc gggttacagt gatttaagaa 19260 acaactaagg tggggcaaag ggtagtgagg catatccatc agagcaaatt ccttgaaata 19320 cggactcaga gggaaccatt gtgagattga ggttcccaga ggtgtggatt taatgaatta 19380 gtgttacctc atgtacaagg tagtatacta ccagaaagat aaaaattcag aagcgagttt 19440 gcagcaaaac tcatagggag aacttctttt ataaataata tgaagctgga tatttagtgc 19500 accacctgat gaccacttta ttaataaata aagagttcct gttgtggcgc agcggaaatg 19560 aatccgacaa ataatcatga gtttgcgggt ttgatccctg acctcgctca gtgggttggg 19620 gatctggtgt tgccatgagc tgtggtgtag gtcgcagatg ctgcttggat cctgctttgc 19680 tgtggctgtg gtataggctt gtggctacag ctccgatttg accgctagcc tgggaacctc 19740 catatgctgc gggggtggcc ctcaaaagaa aaataaataa ataagtaaat aaataagtag 19800 tttaaaaagg acaagaagaa atatatttgg tgttatattc tacagagaca aagataatca 19860 ccatgcccga ttgatttttc aaggcatata aatgagacgt catgggagca aaaatggtca 19920 taatacaatg cccttgtttt gtgtacatgg taagatttta gaaagcattg tgaggtaaaa 19980 aagtgtactc agttataata tattggggaa aacagtacta tgagaagtaa aaaaatctac 20040 atgccggaag ttattttttt aatgtctctt ttagagtcgc acatgcggca tatggaggtt 20100 cccaggctag gggtcgaatc agagctatag ccactggctt atggcacagc cacaacaaca 20160 ctagatctga gccacatcag cgacctatac tatagctcat ggcaatgcca gatccttaac 20220 ctactgagcc aagccatggg tcaaatccag gtcctcacgg atcctaggca aattcatttc 20280 tgctgagcca cgaagggaac tcctcagaag tgattttgat gttactttct tttcatgaca 20340 aatctggtaa agtacataca catagaaact gaagtgtcag aaagggaaat atttcatttt 20400 aaggtaatgt atacaaaaca gtggttttac catctgagta tctcgctaaa ttttaactat 20460 caaggacaat tgccaaaaaa aaaaaaaaaa gagagagaga gagaacagaa tagggttatg 20520 aagctaaaat cacagggtta tgaagctaaa atcacagtaa tttagggaga aaaaaatcca 20580 aagcatgtaa ttgataaaag gttctgagcc tttgtttgag atttagaatt caacttagaa 20640 ataccggtgg tattttaaag cagtccataa gtataaaatc caaggctaaa aaaccagaag 20700 gtatttgtag aacaaatata ttttaataag ctctaccaag tcatccagaa gctattaaag 20760 aattactggt cactgacata gtgtacctgt tttcaaggcc attcttacat cagaataaag 20820 ggagagcacc ctctgaatct tcagaaaaga tgtgaaagtg ctaattctct atttcatccc 20880 agagttcatg tctctgagac tgatcagtga aaacagcaga gagacctgtg cagggcgcct 20940 ggaagttttt tacaacggag cttggggcag cgttggcaag aatagcatgt ctccagccac 21000 agtgggggtg gtatgcaggc agctaggctg tgcagacaga ggggacatca gccctgcatc 21060 ttcagacaag acagtgtcca ggcacatgtg ggtggacaat gttcagtgtc ctaaaggacc 21120 tgacacacta tggcagtgcc catcatctcc atggaagaag agactggcca gcccctcaga 21180 ggagacatgg atcacatgtg ccagtgagta tccattcttt agcgccactg ttatcttctg 21240 atctacctaa gcagaagttt tataatctgt agttaatccc tattctacct ggatgatggg 21300 attcattctg tttaatttgg tgtgcaggta ttcagcatca gtgatcattt tcccaaagac 21360 catcatgctc tgatggtctt ctcaaaagtt ctaatcagtt gcttcctccg tgaacagttg 21420 aggagcagag aatatgtaat tcagaatttg actattgaat catcccattt ttctttcaca 21480 tagtcttttg ttgcactgaa tataaggaga gaagcagtca gaaagatcaa tcctgaatta 21540 tttctccatt ctacatctgt tttaaatttc aaaaaaaaaa attgttatag gtgatttaca 21600 atgtctgtca atttctgctc tacagcaaag tgacccagtt atttacatat acattctgtt 21660 tctcatattt ttaaaccagg agatttctat ctgcctggcg gtttgaggga atttaacatt 21720 atgcatttat gttaacttta ttcacctgat gttttctaag tcatactgag attcttatgc 21780 ccaggatgga atacacctgg tttgctggaa agacatgtgc tttcataaag acgaattttg 21840 gaaaaaatat aaaatttaaa aggcccatta aataagcaaa gttttaagag atttcaaaaa 21900 aaatttcatc tctctctttt cctctttgac ctcttgggca cgttcatctt ctcaaatatg 21960 atcttggtgt ttctgacttt tcagacaaaa taagacttca agaaggaaac actaattgtt 22020 ctggacgtgt ggagatctgg tacggaggtt cctggggcac tgtgtgtgac gactcctggg 22080 accttgaaga tgctcaggtg gtgtgccgac agctgggctg tggctcagct ttggaggcag 22140 gaaaagaggc cgcatttggc caggggactg ggcccatatg gctcaatgaa gtgaagtgca 22200 aggggaatga aacctccttg tgggattgtc ctgccagatc ctggggccac agtgactgtg 22260 gacacaagga ggatgctgct gtgacgtgtt caggtgaggg cagagagtct ggattgagct 22320 tggaagctct ggcagcaaag agagggtggg cggtgacctg cattgggtaa agattggaag 22380 gtccagccta aggatctggt ggtgggggga gacatgatgt ttcagtctga agaatgatga 22440 aaacctgtgt ggttacgcat gggccttcgc cgaggaaagg gacataacta ccatgtatcc 22500 tcctgcagag ggaggaagaa ctaggggatt ctagttttgt gtgggaagga gcagtttact 22560 tggctcagga ggcactaaag gctcagatag gaaacagaga tctgttccat tcttactccc 22620 agaactgatt ctcttctctt ttctcctaca gaaattgcaa agagccgaga atccctacat 22680 gccacaggta tatcaaaaag tttaagaaca tgggacccat tgtctgcatt ttgtggaatc 22740 cctcttatta agacattctg ggtcagaagt tctgaggatt tgacatttac ttcagctatc 22800 tgttatctta cccaagagag ggatggtaac taggaaccca ggtcttttag ctaagacatt 22860 atcacctctt gtgatgttta cttgttctca ggtcgctcat cttttgttgc acttgcaatc 22920 tttggggtca ttctgttggc ctgtctcatc gcattcctca tttggactca gaagcgaaga 22980 cagaggcagc ggctctcagg tctgaacaaa attacggtct ctctaatgtt tctatgggag 23040 aagaagcctc tctggataat aaaaaaaaaaaaattacattc aagtatcagt tggccagaaa 23100 gagggaacct agaagaggtt taagcagttt ctccgaaaca gggaacaaga attcagagaa 23160 gaaaaggcac attggctgta ctgatgatac ctgcactcgc tatgtatgtt taatggggga 23220 cagtagagaa ttgatagttt agaaggagta tgcttatatg gttctggatg aatcctgtat 23280 ccccccaaac atttattttc tcttactata tacttattac taatttaact cttctgtcaa 23340 gccatgtgct aggttctgaa gatggttcag acttggataa ccaagtgctt ttgttttcat 23400 ggaatttcca gtttagtgga agagataaat atgtaaaacaa ataaatgggg gggggggggg 23460 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23520 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23580 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23640 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23700 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23760 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23820 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23880 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23940 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 24000 gggggggggg gggggggggg gggggggggg gggggggggg ggggggggtt ggcgggcccc 24060 cctcgaggtc gacggtatcg ataagcttga tatcgaattc gtgagccaga ggacgagact 24120 agagatggat gatgactacg ttatgcttgc actgctgggg aaaagcacac atagggaggg 24180 aacgttttat tatgacccag tccctaacct atgacctctg ttatcagttt tctcaggagg 24240 agagaattct gtccatcaaa ttcaataccg ggagatgaat tcttgcctga aagcagatga 24300 aacggatatg ctaaatccct caggtccgtg ggttctttga ggggctgtag ccctggggtt 24360 cagatcagca gctgcagttg aggttgaggc atgctacttt gcatagcagt agaaagaaat 24420 ctcaactgta ataggaagct tgggatgcat atgaggaaga aaggcaagaa tgaactacaa 24480 attattcta gggaagataa aaattgcagt catggggaga cctctggctg agagggccgt 24540 gattatttct gacagaggga ttatggagta gaatatgatg gcttggacct tttttcacta 24600 aaacaagtca gtcttctcaa aggtagttta gcttttcata tatctttcac agtttcttcc 24660 attcccattt cctgccattt tcctttctct aacttttatt tattatattt tttcctaaaa 24720 gtttaaattt tctatatctt tatcccttca gaagccatcc ctagtcacag gactagtctc 24780 atttcccatt atgtaatgct tctttctctg tctgttgact tctatttaga accagtgcac 24840 taaatctgcc tttaggaaca tacctctgct aggttgcaag aaatatccca ttccccactc 24900 actctgtgaa gactcaatgc ttctcaatat tccttacctc ctgagaggga cttgcctcac 24960 ttctttaatc caagggactc gatttttgcc aaaactaagt caggaaaacc tacataagac 25020 ataggaaaga cttgctgtgc ttcttaaacc ccactgtttg ttttcctaat tgtgaacagt 25080 atttttaaag ttaacaagag agcttctaag gcacttgagg ggagatctga tttatttccc 25140 agtaattatt ttcttccttt cagaaaattc cactgaataa gatggtttta acggatgtgg 25200 gactaatttt tgtgtctaaa tctcttccta tttctggatg aaaaaaagga gaccactctg 25260 aagtacaatg aaaaggaaaa tgggaattat aacctggtga ggtgagtagg aagaatttat 25320 tcatcattgc tgaaaacagg tacattcctt ttgaaagttg agaactcctc tggtattaga 25380 aaaaaaaaaa gaacgtatat acacatatat ttccatgtct atgtttatgt ttgtaaatcc 25440 atattcagaa tatgcaacaa ctttttataa ctatgacttc agtccatctt ttagttacat 25500 atatattcta aacaacaact attgctaaga gaagctgggt aagtaaatgt gaataaatct 25560 tctaaagata ttacaggaag ttcctgctgc ggctcagtgg gttaaagact tgatgtcttt 25620 gtgaagatga gggctcgagc cctggcctca ctcagtgagt taaggatcta gcattgctgt 25680 aagctgcagc gtaggttgca gatagggctc agatccagtg ttgctgtggc tgtggcctca 25740 gttgcagctc tgattcaacc cttaggcgag gaacttccat atgcagcaaa tgtggccatt 25800 aaaaaaaaaa aaaacattat aggagtcatt tcataaaaga gataagacgt ttctatagtt 25860 atatagtgca tactctggta aagatagtat aggatactat aggaatatag aaagcttgcc 25920 tatgaaaatt tgggaagatt gtggaaaaga catctcaaaa tatggcatag aaaagaatca 25980 tatctttgag gaacagtaag tttttcattc aaaaccgtgt attgaacata cttgtggtga 26040 caagtggtgt cctgagtact aaaaattcag tgataaaaga tgctcttgac aaagacatgg 26100 ctgttgaata gaaggtctca ctgtcaatgt gtgggaatta tggacagcct atgtggacac 26160 agggaataga tgagactcta ggctggaagg ctgcattgag cccaataatg aatggtcctg 26220 tctgatatat ttcatgctca tattttattt tagggactat tggggaggtg gtgggttttg 26280 gaagattaag ctgaggcaag acacaatcag attgcctttt ataatttact ttcaggagga 26340 aagtctaact aaaaaaagaa ttcgatatca agcttatcga taccgtcgac ctcgaggagg 26400 cccgcctgcc cttttggggg gggggggggg gggggggggg gggggggggg gggggggggg 26460 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggcagca 26520 ccaattttat tattggcggg aataaagaga aaaatgtaat ttcaaagatt gctgttggaa 26580 atgaggggtg tggtagcttt tggagaaagc attctggaga cttctattaa tttttttttt 26640 ttaagtgctt caaagatcct ttgatccaac aattctactc ctaaaaattt cttccataca 26700 gataaagcca tttgtctgta tataacaaat agaagagaat tcctttttgc agccttgtta 26760 gtagtgcccc caaactggaa acaaagtgaa tatcagtcag tggggtagcg gctggaaaaaa 26820 ttttagtgca cccaaccaac aaagaaaaac catgcacaaa aattcaataa atatcatctc 26880 acttttgtgt tcatgttat gaatataatt aaacataatg tttacatcta taaaattatc 26940 atatgtatac atgtaaagaa acattaaaac atttttaaca gactgtaaac ttgaggactg 27000 tgaatgactt ttgattgata atctcaaaca tatggatact attctgatgt aataaataat 27060 gattaaattt tttccctaaa gagtaatcac tactgaatcg ttgcctcaga atcatatgga 27120 ggtgctttta aaaaaggcat ttctgcactg ttgttctctg gaatagaagt aattcttatg 27180 tacactgaag tttgaaaatc attgcattta agtgttctgt tcaggaaagt agtgtgcttt 27240 ttaatatttg tgagtgaatg agtaacacaa tacattatat cacattttaa tgtaattcta 27300 cacatgtgca tatgaagaga aaagtaacat ttttttctat ttatgtcttt agttcagcct 27360 ttaagatacc ttgatgaaga cctggactat tgaatgagca agaatctgcc tcttacactg 27420 aagattacaa tacagtcctc tgtctcctgg tattccaaag actgctgttg aatttctaaa 27480 aaatagattg gtgaatgtga ctactcaaag ttgtatgtaa gactttcaag ggcattaaat 27540 aaaaaagaat attgctgatt cttgttcttg attttctgaa tttctgaatc tcttattggg 27600 cttctaattt aaaaaaaaaat atctgggcgc ccgcagatat cgaactcttg ggcagtgtga 27660 ccaaacgaag acatatccaa tcaagcatgc aaatggacca gcccactgta ctagcacgct 27720gtggcagcca atctgaccga gaaagcagac aaccgcaggg agcaacg 27767 <210> 16 <211> 55 <212> DNA <213> Sus scrofa <400> 16 ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatc 55 <210> 17 <211> 54 <212> DNA <213> Sus scrofa <400> 17 ggtcgccacc atggccatga gcaagggcga ggagctgttc accggggtgg tgcc 54 <210> 18 <211> 48 <212> DNA <213> Sus scrofa <400> 18 ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggt 48 <210> 19 <211> 55 <212> DNA <213> Sus scrofa <400> 19 ggtcgccacc atggttgagc aagggcgagg agctgttcac cggggtggtg cccat 55 <210> 20 <211> 43 <212> DNA <213> Sus scrofa <400> 20 tgcagggaac tacagtgcgg cactgtggtt tccctcctgg ggg 43 <210> 21 <211> 60 <212> DNA <213> Sus scrofa <400> 21 tgcagggaac tacagtgcgg cactgtaaac cactactact gtggtttccc tcctgggggg 60 60 <210> 22 <211> 41 <212> DNA <213> Sus scrofa <400> 22 tgcagggaac tacagtgcgg ctgtggtttc cctcctgggg g 41 <210> 23 <211> 46 <212> DNA <213> Sus scrofa <400> 23 tgcagggaac tacagtgcgg aactactgtg gtttccctcc tggggg 46 <210> 24 <211> 31 <212> DNA <213> Sus scrofa <400> 24 gaaacccagg ctggttggag gggacattcc c 31 <210> 25 <211> 24 <212> DNA <213> Sus scrofa <400> 25 gaaacccagg ctgggggacat tccc 24 <210> 26 <211> 13 <212> DNA <213> Sus scrofa <400> 26 aggggacatt ccc 13 <210> 27 <211> 13 <212> DNA <213> Sus scrofa <400> 27 gaaacccat ccc 13 <210> 28 <211> 31 <212> DNA <213> Sus scrofa <400> 28 ggtcgccacc atggtgagca agggcgagga g 31 <210> 29 <211> 32 <212> DNA <213> Sus scrofa <400> 29 ggtcgccacc atggctgagc aagggcgagg ag 32 <210> 30 <211> 29 <212> DNA <213> Sus scrofa <400>30 ggtcgccacc atggtgagag ggcgaggag 29 <210> 31 <211> 32 <212> DNA <213> Sus scrofa <400> 31 ggtcgccacc atggttgagc aagggcgagg ag 32 <210> 32 <211> 48 <212> DNA <213> Sus scrofa <400> 32 ggtcgccacc atggtgagca agggcgagga gaacccaggc tggttgga 48 <210> 33 <211> 49 <212> DNA <213> Sus scrofa <400> 33 tgctgtgcag ggaactacag tgcggcactg tggtttccct cctgggggg 49 <210> 34 <211> 38 <212> DNA <213> Sus scrofa <400> 34 tgctgtgcag ggaactctgt ggtttccctc ctgggggg 38 <210> 35 <211> 22 <212> DNA <213> Sus scrofa <400> 35 ctgtggtttc cctcctgggg gg 22 <210> 36 <211> 23 <212> DNA <213> Sus scrofa <400> 36 actgtggttt ccctcctggg ggg 23 <210> 37 <211> 50 <212> DNA <213> Sus scrofa <400> 37 tgctgtgcag ggaactacag tgcggcaact gtggtttccc tcctgggggg 50 <210> 38 <211> 10 <212> DNA <213> Sus scrofa <400> 38 tcctgggggg 10 <210> 39 <211> 8 <212> DNA <213> Sus scrofa <400> 39 ctgggggg 8 <210> 40 <211> 52 <212> DNA <213> Sus scrofa <400> 40 agagagcaga gccagcgact cgcccagcga catggggtac ctgccgtttg tg 52 <210> 41 <211> 33 <212> DNA <213> Sus scrofa <400> 41 agagagcaga gccagcgact cgcccagcga gat 33 <210> 42 <211> 30 <212> DNA <213> Sus scrofa <400> 42 agagagcaga gccagcgact cgcccagcga 30 <210> 43 <211> 50 <212> DNA <213> Sus scrofa <400> 43 agagccagcc tcgcccagca ggggtaccat ggggtacctg ccgtttgtgt 50 <210> 44 <211> 53 <212> DNA <213> Sus scrofa <400> 44 agagagcaga gccagcgact cgcccagcga gcagtgggta cctgccgttt gtg 53 <210> 45 <211> 53 <212> DNA <213> Sus scrofa <400> 45 agagagcaga gccagcgact cgcccagcga tcagtgggta cctgccgttt gtg 53 <210> 46 <211> 53 <212> DNA <213> Sus scrofa <400> 46 agagagcaga gccagcgact cgcccagcga acatggggta cctgccgttt gtg 53 <210> 47 <211> 4990 <212> DNA <213> Sus scrofa <400> 47 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactacag tgcggcactg tggtttccct cctgggggga gctcactttg 3180 gagaaggaag tggacagatc tgggctgaag aattccagtg tgaggggcac gagtcccacc 3240 tttcactctg cccagtagca ccccgccctg acgggacatg tagccacagc agggacgtcg 3300 gcgtagtctg ctcaagtgag acccagggaa tgtgttcact ttgttcccat gccatgaaga 3360 gggtagggtt aggtagtcac agacatcttt ttaaagccct gtctccttcc aggatacaca 3420 caaatccgct tggtgaatgg caagacccca tgtgaaggaa gagtggagct caacattctt 3480 gggtcctggg ggtccctctg caactctcac tgggacatgg aagatgccca tgttttatgc 3540 cagcagctta aatgtggagt tgccctttct atcccgggag gagcaccttt tgggaaagga 3600 agtgagcagg tctggaggca catgtttcac tgcactggga ctgagaagca catgggagat 3660 tgttccgtca ctgctctggg cgcatcactc tgttcttcag ggcaagtggc ctctgtaatc 3720 tgctcaggta agagaataag ggcagccagt gatgagccac tcatgacggt gccttaagag 3780 tgggtgtacc taggagttcc cattgtggct cagtggtaac aaactcgact ggtatccatg 3840 agggtatggg tttgatccct ggccttgctc aatgggttaa ggatccagca ttgctgtgag 3900 ctgtggtata ggttgcagac tctgctcagg tcccatgttg ctgtgattgt ggtgtaggct 3960 gactgctgca gcttcaattt gacccctagc ccgggaattt ccataggcca cacgtgcagc 4020 actaaggaag gaaaaaaaaga aaaaaaaaaa aaaagagtgg gtgtgcctat agtgaagaac 4080 agatgtaaaa gggaagtgaa agggattccc ccattctgag ggattgtgag aagtgtgcca 4140 gaatattaac ttcatttgac ttgttacagg gaaagtaaac ttgactttca cggacctcct 4200 agttacctgg tgcttactat atgtcttctc agagtacctg attcattccc agcctggttg 4260 acccatcccc ctatctctat ggctatgttt atccagagca catctatcta acactccagc 4320 tgatcttcct gacacagctg tggcaaccct ggatccttta accaactgtg ccaggctgga 4380 gatcaaacct aagcctctgc agcaacccaa gctgctgcag tcagattttt aaccccctgt 4440 gccactgtgg gtatctccga tattttgtat cttctgtgac tgagtggttt gctgtttgca 4500 gggaaccaga gtcagacact atccccgtgc aattcatcat cctcggaccc atcaagctct 4560 attatttcag aagaaaatgg tgttgcctgc ataggtgaga atcagtgacc aacctatgaa 4620 aatgatctca atcctctgaa atgcatttta ttcatgtttt atttcctctt tgcagggagt 4680 ggtcaacttc gcctggtcga tggaggtggt cgttgtgctg ggagagtaga ggtctatcat 4740 gagggctcct ggggcaccat ctgtgatgac agctgggacc tgaatgatgc ccatgtggtg 4800 tgcaaacagc tgagctgtgg atgggccatt aatgccactg gttctgctca ttttggggaa 4860 ggaacagggc ccatttggct ggatgagata aactgtaatg gaaaagaatc tcatatttgg 4920 caatgccact cacatggttg ggggcggcac aattgcaggc ataaggagga tgcaggagtc 4980 atctgctcgg 4990 <210> 48 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 48 caccggaaac ccaggctggt tgga 24 <210> 49 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 49 aaactccaac cagcctgggt ttcc 24 <210> 50 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 50 caccggaact acagtgcggc actg 24 <210> 51 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 51 aaaccagtgc cgcactgtag ttcc 24 <210> 52 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 52 caccgcagta gcaccccgcc ctgac 25 <210> 53 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 53 aaacgtcagg gcggggtgct actgc 25 <210> 54 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 54 caccgtgtag ccacagcagg gacgt 25 <210> 55 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 55 aaacacgtcc ctgctgtggc tacac 25 <210> 56 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 56 caccgccagc ctcgcccagc gacat 25 <210> 57 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 57 aaacatgtcg ctgggcgagg ctggc 25 <210> 58 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 58 caccgcagct gcagcatata tttaa 25 <210> 59 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 59 aaacttaaat atatgctgca gctgc 25 <210> 60 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400>60 caccgctttc atttatctga actca 25 <210> 61 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 61 aaactgagtt cagataaatg aaagc 25 <210> 62 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400>62 caccgttatc tgaactcagg gtccc 25 <210> 63 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 63 aaacgggacc ctgagttcag ataac 25 <210> 64 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400>64 caccgctcct cgcccttgct cacca 25 <210> 65 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400>65 aaactggtga gcaagggcga ggagc 25 <210> 66 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 66 caccggacca ggatgggcac caccc 25 <210> 67 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 67 aaacgggtgg tgcccatcct ggtcc 25 <210> 68 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 68 ttgttggaag gctcactgtc cttg 24 <210> 69 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 69 acaactaagg tggggcaaag 20 <210> 70 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400>70 ttgttggaag gctcactgtc cttg 24 <210> 71 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 71 ggagctcaac attcttgggt cct 23 <210> 72 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 72 ggcaaaattt tcatgctgag gtg 23 <210> 73 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 73 gcacatcact tcgggttaca gtg 23 <210> 74 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 74 cccaagtatc ttcagttctg cag 23 <210> 75 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400>75 tacaggtagg agagcctgtt ttg 23 <210> 76 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 76 cccaagtatc ttcagttctg cag 23 <210> 77 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 77 ctcaaaagga tgtaaaccct gga 23 <210> 78 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 78 tgttgatgtg gtttgtttgc cc 22 <210> 79 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 79 tacaggtagg agagcctgtt ttg 23 <210> 80 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400>80 ggaggtctag aatcggctaa gcc 23 <210> 81 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 81 ggctacatgt cccgtcaggg 20 <210> 82 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 82 gcaggccact aggcagatga a 21 <210> 83 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 83 gagctgacac ccaagaagtt cct 23 <210> 84 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 84 ggctctagag cctctgctaa cc 22 <210> 85 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 85 ggacttgaag aagtcgtgct gc 22 <210> 86 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 86 taatacgact cactataggg agaatggact ataaggacca cgac 44 <210> 87 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 87 gcgagctcta ggaattctta c 21 <210> 88 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 88 ttaatacgac tcactatagg ctcctcgccc ttgctcacca 40 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 89 aaaagcaccg actcggtgcc 20 <210> 90 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400>90 ttaatacgac tcactatagg aaacccaggc tggttgga 38 <210> 91 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 91 aaaagcaccg actcggtgcc 20 <210> 92 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 92 ttaatacgac tcactatagg aactacagtg cggcactg 38 <210> 93 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 93 aaaagcaccg actcggtgcc 20 <210> 94 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 94 ttaatacgac tcactatagg ccagcctcgc ccagcgacat 40 <210> 95 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 95 aaaagcaccg actcggtgcc 20 <210> 96 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 96 ttaatacgac tcactatagg cagctgcagc atatatttaa 40 <210> 97 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 97 aaaagcaccg actcggtgcc 20 <210> 98 <211> 3484 <212> DNA <213> Sus scrofa <400> 98 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggcacattc cctgctctgg tcgtgttgaa gtacaacatg 1560 gagacacgtg gggcaccgtc tgtgattctg acttctctct ggaggcggcc agcgtgctgt 1620 gcagggaact acagtgcggc actgtggttt ccctcctggg gggagctcac tttggagaag 1680 gaagtggaca gatctgggct gaagaattcc agtgtgaggg gcacgagtcc cacctttcac 1740 tctgcccagt agcaccccgc cctgacggga catgtagcca cagcagggac gtcggcgtag 1800 tctgctcaag tgagacccag ggaatgtgtt cactttgttc ccatgccatg aagagggtag 1860 ggttaggtag tcacagacat ctttttaaag ccctgtctcc ttccaggata cacacaaatc 1920 cgcttggtga atggcaagac cccatgtgaa ggaagagtgg agctcaacat tcttgggtcc 1980 tgggggtccc tctgcaactc tcactgggac atggaagatg cccatgtttt atgccagcag 2040 cttaaatgtg gagttgccct ttctatcccg ggaggagcac cttttgggaa aggaagtgag 2100 caggtctgga ggcacatgtt tcactgcact gggactgaga agcacatggg agattgttcc 2160 gtcactgctc tgggcgcatc actctgttct tcagggcaag tggcctctgt aatctgctca 2220 ggtaagagaa taagggcagc cagtgatgag ccactcatga cggtgcctta agagtgggtg 2280 tacctaggag ttcccattgt ggctcagtgg taacaaactc gactggtatc catgagggta 2340 tgggtttgat ccctggcctt gctcaatggg ttaaggatcc agcattgctg tgagctgtgg 2400 tataggttgc agactctgct caggtcccat gttgctgtga ttgtggtgta ggctgactgc 2460 tgcagcttca atttgacccc tagcccggga atttccatag gccacacgtg cagcactaag 2520 gaaggaaaaa aagaaaaaaa aaaaaaaaga gtgggtgtgc ctatagtgaa gaacagatgt 2580 aaaaggggaag tgaaaagggat tcccccattc tgagggattg tgagaagtgt gccagaatat 2640 taacttcatt tgacttgtta cagggaaagt aaacttgact ttcacgggacc tcctagttac 2700 ctggtgctta ctatatgtct tctcagagta cctgattcat tcccagcctg gttgacccat 2760 ccccctatct ctatggctat gtttatccag agcacatcta tctaacactc cagctgatct 2820 tcctgacaca gctgtggcaa ccctggatcc tttaaccaac tgtgccaggc tggagatcaa 2880 acctaagcct ctgcagcaac ccaagctgct gcagtcagat ttttaacccc ctgtgccact 2940 gtgggtatct ccgatatttt gtatcttctg tgactgagtg gtttgctgtt tgcagggaac 3000 cagagtcaga cactatcccc gtgcaattca tcatcctcgg acccatcaag ctctattatt 3060 tcagaagaaa atggtgttgc ctgcataggt gagaatcagt gaccaaccta tgaaaatgat 3120 ctcaatcctc tgaaatgcat tttattcatg ttttattcc tctttgcagg gagtggtcaa 3180 cttcgcctgg tcgatggagg tggtcgttgt gctgggagag tagaggtcta tcatgagggc 3240 tcctggggca ccatctgtga tgacagctgg gacctgaatg atgcccatgt ggtgtgcaaa 3300 cagctgagct gtggatgggc cattaatgcc actggttctg ctcattttgg ggaaggaaca 3360 gggcccattt ggctggatga gataaactgt aatggaaaaag aatctcatat ttggcaatgc 3420 cactcacatg gttgggggcg gcacaattgc aggcataagg aggatgcagg agtcatctgc 3480 tcgg 3484 <210> 99 <211> 4997 <212> DNA <213> Sus scrofa <400> 99 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactacag tgcggctact actactgtgg tttccctcct ggggggagct 3180 cactttggag aaggaagtgg acagatctgg gctgaagaat tccagtgtga ggggcacgag 3240 tcccaccttt cactctgccc agtagcaccc cgccctgacg ggacatgtag ccacagcagg 3300 gacgtcggcg tagtctgctc aagtgagacc cagggaatgt gttcactttg ttcccatgcc 3360 atgaagaggg tagggttagg tagtcacaga catcttttta aagccctgtc tccttccagg 3420 atacacacaa atccgcttgg tgaatggcaa gaccccatgt gaaggaagag tggagctcaa 3480 cattcttggg tcctgggggt ccctctgcaa ctctcactgg gacatggaag atgcccatgt 3540 tttatgccag cagcttaaat gtggagttgc cctttctatc ccgggaggag caccttttgg 3600 gaaaggaagt gagcaggtct ggaggcacat gtttcactgc actgggactg agaagcacat 3660 gggagatgt tccgtcactg ctctgggcgc atcactctgt tcttcagggc aagtggcctc 3720 tgtaatctgc tcaggtaaga gaataagggc agccagtgat gagccactca tgacggtgcc 3780 ttaagagtgg gtgtacctag gagttcccat tgtggctcag tggtaacaaa ctcgactggt 3840 atccatgagg gtatgggttt gatccctggc cttgctcaat gggttaagga tccagcattg 3900 ctgtgagctg tggtataggt tgcagactct gctcaggtcc catgttgctg tgattgtggt 3960 gtaggctgac tgctgcagct tcaatttgac ccctagcccg ggaatttcca taggccacac 4020 gtgcagcact aaggaaggaa aaaaagaaaa aaaaaaaaaaa agagtgggtg tgcctatagt 4080 gaagaacaga tgtaaaaggg aagtgaaagg gattccccca ttctgaggga ttgtgagaag 4140 tgtgccagaa tattaacttc atttgacttg ttacagggaa agtaaacttg actttcacgg 4200 acctcctagt tacctggtgc ttactatatg tcttctcaga gtacctgatt cattcccagc 4260 ctggttgacc catcccccta tctctatggc tatgtttatc cagagcacat ctatctaaca 4320 ctccagctga tcttcctgac acagctgtgg caaccctgga tcctttaacc aactgtgcca 4380 ggctggagat caaacctaag cctctgcagc aacccaagct gctgcagtca gatttttaac 4440 cccctgtgcc actgtgggta tctccgatat tttgtatctt ctgtgactga gtggtttgct 4500 gtttgcaggg aaccagagtc agacactatc cccgtgcaat tcatcatcct cggacccatc 4560 aagctctatt atttcagaag aaaatggtgt tgcctgcata ggtgagaatc agtgaccaac 4620 ctatgaaaat gatctcaatc ctctgaaatg cattttattc atgttttat tcctctttgc 4680 agggagtggt caacttcgcc tggtcgatgg aggtggtcgt tgtgctggga gagtagaggt 4740 ctatcatgag ggctcctggg gcaccatctg tgatgacagc tgggacctga atgatgccca 4800 tgtggtgtgc aaacagctga gctgtggatg ggccattaat gccactggtt ctgctcattt 4860 tggggaaagga acagggccca tttggctgga tgagataaac tgtaatggaa aagaatctca 4920 tatttggcaa tgccactcac atggttgggg gcggcacaat tgcaggcata aggaggatgc 4980 aggagtcatc tgctcgg 4997 <210> 100 <211> 3710 <212> DNA <213> Sus scrofa <400> 100 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaagggaa 2820 agggattccc ccattctgag ggattgtgag aagtgtgcca gaatattaac ttcatttgac 2880 ttgttacagg gaaagtaaac ttgactttca cggacctcct agttacctgg tgcttactat 2940 atgtcttctc agagtacctg attcattccc agcctggttg acccatcccc ctatctctat 3000 ggctatgttt atccagagca catctatcta acactccagc tgatcttcct gacacagctg 3060 tggcaaccct ggatccttta accaactgtg ccaggctgga gatcaaacct aagcctctgc 3120 agcaacccaa gctgctgcag tcagattttt aaccccctgt gccactgtgg gtatctccga 3180 tattttgtat cttctgtgac tgagtggttt gctgtttgca gggaaccaga gtcagacact 3240 atccccgtgc aattcatcat cctcggaccc atcaagctct attatttcag aagaaaatgg 3300 tgttgcctgc ataggtgaga atcagtgacc aacctatgaa aatgatctca atcctctgaa 3360 atgcatttta ttcatgtttt atttcctctt tgcagggagt ggtcaacttc gcctggtcga 3420 tggaggtggt cgttgtgctg ggagagtaga ggtctatcat gagggctcct ggggcaccat 3480 ctgtgatgac agctgggacc tgaatgatgc ccatgtggtg tgcaaacagc tgagctgtgg 3540 atgggccatt aatgccactg gttctgctca ttttggggaa ggaacagggc ccattggct 3600 ggatgagata aactgtaatg gaaaagaatc tcatatttgg caatgccact cacatggttg 3660 ggggcggcac aattgcaggc ataaggagga tgcaggagtc atctgctcgg 3710 <210> 101 <211> 3617 <212> DNA <213> Sus scrofa <400> 101 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gattgaaagg gattccccca ttctgaggga ttgtgagaag 2760 tgtgccagaa tattaacttc atttgacttg ttacagggaa agtaaacttg actttcacgg 2820 acctcctagt tacctggtgc ttactatatg tcttctcaga gtacctgatt cattcccagc 2880 ctggttgacc catcccccta tctctatggc tatgtttatc cagagcacat ctatctaaca 2940 ctccagctga tcttcctgac acagctgtgg caaccctgga tcctttaacc aactgtgcca 3000 ggctggagat caaacctaag cctctgcagc aacccaagct gctgcagtca gatttttaac 3060 cccctgtgcc actgtgggta tctccgatat tttgtatctt ctgtgactga gtggtttgct 3120 gtttgcaggg aaccagagtc agacactatc cccgtgcaat tcatcatcct cggacccatc 3180 aagctctatt atttcagaag aaaatggtgt tgcctgcata ggtgagaatc agtgaccaac 3240 ctatgaaaat gatctcaatc ctctgaaatg cattttatattc atgttttatt tcctctttgc 3300 agggagtggt caacttcgcc tggtcgatgg aggtggtcgt tgtgctggga gagtagaggt 3360 ctatcatgag ggctcctggg gcaccatctg tgatgacagc tgggacctga atgatgccca 3420 tgtggtgtgc aaacagctga gctgtggatg ggccattaat gccactggtt ctgctcattt 3480 tggggaaagga acagggccca tttggctgga tgagataaac tgtaatggaa aagaatctca 3540 tatttggcaa tgccactcac atggttgggg gcggcacaat tgcaggcata aggaggatgc 3600 aggagtcatc tgctcgg 3617 <210> 102 <211> 4979 <212> DNA <213> Sus scrofa <400> 102 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactctgt ggtttccctc ctggggggag ctcactttgg agaaggaagt 3180 ggacagatct gggctgaaga attccagtgt gaggggcacg agtcccacct ttcactctgc 3240 ccagtagcac cccgccctga cgggacatgt agccacagca gggacgtcgg cgtagtctgc 3300 tcaagtgaga cccagggaat gtgttcactt tgttcccatg ccatgaagag ggtagggtta 3360 ggtagtcaca gacatctttt taaagccctg tctccttcca ggatacacac aaatccgctt 3420 ggtgaatggc aagaccccat gtgaaggaag agtggagctc aacattcttg ggtcctgggg 3480 gtccctctgc aactctcact gggacatgga agatgcccat gttttatgcc agcagcttaa 3540 atgtggagtt gccctttcta tcccgggagg agcacctttt gggaaaggaa gtgagcaggt 3600 ctggaggcac atgtttcact gcactgggac tgagaagcac atgggagatt gttccgtcac 3660 tgctctgggc gcatcactct gttcttcagg gcaagtggcc tctgtaatct gctcaggtaa 3720 gagaataagg gcagccagtg atgagccact catgacggtg ccttaagagt gggtgtacct 3780 aggagttccc attgtggctc agtggtaaca aactcgactg gtatccatga gggtatgggt 3840 ttgatccctg gccttgctca atgggttaag gatccagcat tgctgtgagc tgtggtatag 3900 gttgcagact ctgctcaggt cccatgttgc tgtgattgtg gtgtaggctg actgctgcag 3960 cttcaatttg acccctagcc cgggaatttc cataggccac acgtgcagca ctaaggaagg 4020 aaaaaaagaa aaaaaaaaaa aaagagtggg tgtgcctata gtgaagaaca gatgtaaaag 4080 ggaagtgaaa gggattcccc cattctgagg gattgtgaga agtgtgccag aatattaact 4140 tcatttgact tgttacaggg aaagtaaact tgactttcac ggacctccta gttacctggt 4200 gcttactata tgtcttctca gagtacctga ttcattccca gcctggttga cccatccccc 4260 tatctctatg gctatgttta tccagagcac atctatctaa cactccagct gatcttcctg 4320 acacagctgt ggcaaccctg gatcctttaa ccaactgtgc caggctggag atcaaaccta 4380 agcctctgca gcaacccaag ctgctgcagt cagattttta accccctgtg ccactgtggg 4440 tatctccgat attttgtatc ttctgtgact gagtggtttg ctgtttgcag ggaaccagag 4500 tcagacacta tccccgtgca attcatcatc ctcggaccca tcaagctcta ttatttcaga 4560 agaaaatggt gttgcctgca taggtgagaa tcagtgacca acctatgaaa atgatctcaa 4620 tcctctgaaa tgcattttat tcatgtttta tttcctcttt gcagggagtg gtcaacttcg 4680 cctggtcgat ggaggtggtc gttgtgctgg gagagtagag gtctatcatg agggctcctg 4740 gggcaccatc tgtgatgaca gctgggacct gaatgatgcc catgtggtgt gcaaacagct 4800 gagctgtgga tgggccatta atgccactgg ttctgctcat tttggggaag gaacagggcc 4860 catttggctg gatgagataa actgtaatgg aaaagaatct catatttggc aatgccactc 4920 acatggttgg gggcggcaca attgcaggca taaggaggat gcaggagtca tctgctcgg 4979 <210> 103 <211> 4615 <212> DNA <213> Sus scrofa <400> 103 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aagaaggaaa 2580 atattggaat catattctcc ctcaccgaaa tgctattttt cagcccacag gaaacccagg 2640 ctggttggag gggacattcc ctgctctggt cgtgttgaag tacaacatgg agaacacgtgg 2700 ggcaccgtct gtgattctga cttctctctg gaggcggcca gcgtgctgtg cagggaacta 2760 cagtgcggca cagtgtggtt tccctcctgg ggggagctca ctttggagaa ggaagtggac 2820 agatctgggc tgaagaattc cagtgtgagg ggcacgagtc ccacctttca ctctgcccag 2880 tagcaccccg ccctgacggg acatgtagcc acagcaggga cgtcggcgta gtctgctcaa 2940 gtgagaccca gggaatgtgt tcactttgtt cccatgccat gaagagggta gggttaggta 3000 gtcacagaca tctttttaaa gccctgtctc cttccaggat acacacaaat ccgcttggtg 3060 aatggcaaga ccccatgtga aggaagagtg gagctcaaca ttcttgggtc ctgggggtcc 3120 ctctgcaact ctcactggga catggaagat gcccatgttt tatgccagca gcttaaatgt 3180 ggagttgccc tttctatccc gggaggagca ccttttggga aaggaagtga gcaggtctgg 3240 aggcacatgt ttcactgcac tgggactgag aagcacatgg gagattgttc cgtcactgct 3300 ctgggcgcat cactctgttc ttcagggcaa gtggcctctg taatctgctc aggtaagaga 3360 ataagggcag ccagtgatga gccactcatg acggtgcctt aagagtgggt gtacctagga 3420 gttcccattg tggctcagtg gtaacaaact cgactggtat ccatgagggt atgggtttga 3480 tccctggcct tgctcaatgg gttaaggatc cagcattgct gtgagctgtg gtataggttg 3540 cagactctgc tcaggtccca tgttgctgtg attgtggtgt aggctgactg ctgcagcttc 3600 aatttgaccc ctagcccggg aatttccata ggccacacgt gcagcactaa ggaaggaaaa 3660 aaagaaaaaa aaaaaaaaag agtgggtgtg cctatagtga agaacagatg taaaagggaa 3720 gtgaaaggga ttcccccatt ctgagggatt gtgagaagtg tgccagaata ttaacttcat 3780 ttgacttgtt acagggaaag taaacttgac tttcacggac ctcctagtta cctggtgctt 3840 actatatgtc ttctcagagt acctgattca ttcccagcct ggttgaccca tccccctatc 3900 tctatggcta tgtttatcca gagcacatct atctaacact ccagctgatc ttcctgacac 3960 agctgtggca accctggatc ctttaaccaa ctgtgccagg ctggagatca aacctaagcc 4020 tctgcagcaa cccaagctgc tgcagtcaga tttttaaccc cctgtgccac tgtgggtatc 4080 tccgatattt tgtatcttct gtgactgagt ggtttgctgt ttgcagggaa ccagagtcag 4140 acactatccc cgtgcaattc atcatcctcg gacccatcaa gctctattat ttcagaagaa 4200 aatggtgttg cctgcatagg tgagaatcag tgaccaacct atgaaaatga tctcaatcct 4260 ctgaaatgca ttttatcat gttttatttc ctctttgcag ggagtggtca acttcgcctg 4320 gtcgatggag gtggtcgttg tgctgggaga gtagaggtct atcatgaggg ctcctggggc 4380 acatctgtg atgacagctg ggacctgaat gatgcccatg tggtgtgcaa acagctgagc 4440 tgtggatggg ccattaatgc cactggttct gctcattttg gggaaggaac agggcccatt 4500 tggctggatg agataaactg taatggaaaa gaatctcata tttggcaatg ccactcacat 4560 ggttgggggc ggcacaattg caggcataag gaggatgcag gagtcatctg ctcgg 4615 <210> 104 <211> 4866 <212> DNA <213> Sus scrofa <400> 104 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttctgtggt ttccctcctg gggggagctc actttggaga 3060 aggaagtgga cagatctggg ctgaagaatt ccagtgtgag gggcacgagt cccacctttc 3120 actctgccca gtagcacccc gccctgacgg gacatgtagc cacagcaggg acgtcggcgt 3180 agtctgctca agtgagaccc agggaatgtg ttcactttgt tcccatgcca tgaagagggt 3240 agggttaggt agtcacagac atctttttaa agccctgtct ccttccagga tacacacaaa 3300 tccgcttggt gaatggcaag accccatgtg aaggaagagt ggagctcaac attcttgggt 3360 cctggggggtc cctctgcaac tctcactggg acatggaaga tgcccatgtt ttatgccagc 3420 agcttaaatg tggagttgcc ctttctatcc cgggaggagc accttttggg aaaggaagtg 3480 agcaggtctg gaggcacatg tttcactgca ctgggactga gaagcacatg ggagattgtt 3540 ccgtcactgc tctgggcgca tcactctgtt cttcagggca agtggcctct gtaatctgct 3600 caggtaagag aataagggca gccagtgatg agccactcat gacggtgcct taagagtggg 3660 tgtacctagg agttcccatt gtggctcagt ggtaacaaac tcgactggta tccatgaggg 3720 tatgggtttg atccctggcc ttgctcaatg ggttaaggat ccagcattgc tgtgagctgt 3780 ggtataggtt gcagactctg ctcaggtccc atgttgctgt gattgtggtg taggctgact 3840 gctgcagctt caatttgacc cctagcccgg gaatttccat aggccacacg tgcagcacta 3900 aggaaggaaa aaaagaaaaa aaaaaaaaaaa gagtgggtgt gcctatagtg aagaacagat 3960 gtaaaaggga agtgaaaggg attcccccat tctgagggat tgtgagaagt gtgccagaat 4020 attaacttca tttgacttgt tacagggaaa gtaaacttga ctttcacgga cctcctagtt 4080 acctggtgct tactatatgt cttctcagag tacctgattc attcccagcc tggttgaccc 4140 atccccctat ctctatggct atgtttatcc agagcacatc tatctaacac tccagctgat 4200 cttcctgaca cagctgtggc aaccctggat cctttaacca actgtgccag gctggagatc 4260 aaacctaagc ctctgcagca acccaagctg ctgcagtcag atttttaacc ccctgtgcca 4320 ctgtgggtat ctccgatatt ttgtatcttc tgtgactgag tggtttgctg tttgcaggga 4380 accagagtca gacactatcc ccgtgcaatt catcatcctc ggacccatca agctctatta 4440 tttcagaaga aaatggtgtt gcctgcatag gtgagaatca gtgaccaacc tatgaaaatg 4500 atctcaatcc tctgaaatgc attttatca tgttttatt cctctttgca gggagtggtc 4560 aacttcgcct ggtcgatgga ggtggtcgtt gtgctgggag agtagaggtc tatcatgagg 4620 gctcctgggg caccatctgt gatgacagct gggacctgaa tgatgcccat gtggtgtgca 4680 aacagctgag ctgtggatgg gccattaatg ccactggttc tgctcatttt ggggaaggaa 4740 cagggcccat ttggctggat gagataaact gtaatggaaa agaatctcat atttggcaat 4800 gccactcaca tggttggggg cggcacaatt gcaggcataa ggaggatgca ggagtcatct 4860 gctcgg 4866 <210> 105 <211> 4867 <212> DNA <213> Sus scrofa <400> 105 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttactgtgg tttccctcct ggggggagct cactttggag 3060 aaggaagtgg acagatctgg gctgaagaat tccagtgtga ggggcacgag tcccaccttt 3120 cactctgccc agtagcaccc cgccctgacg ggacatgtag ccacagcagg gacgtcggcg 3180 tagtctgctc aagtgagacc cagggaatgt gttcactttg ttcccatgcc atgaagaggg 3240 tagggttagg tagtcacaga catcttttta aagccctgtc tccttccagg atacacacaa 3300 atccgcttgg tgaatggcaa gaccccatgt gaaggaagag tggagctcaa cattcttggg 3360 tcctgggggt ccctctgcaa ctctcactgg gacatggaag atgcccatgt tttatgccag 3420 cagcttaaat gtggagttgc cctttctatc ccgggaggag caccttttgg gaaaggaagt 3480 gagcaggtct ggaggcacat gtttcactgc actgggactg agaagcacat gggagattgt 3540 tccgtcactg ctctgggcgc atcactctgt tcttcagggc aagtggcctc tgtaatctgc 3600 tcaggtaaga gaataagggc agccagtgat gagccactca tgacggtgcc ttaagagtgg 3660 gtgtacctag gagttcccat tgtggctcag tggtaacaaa ctcgactggt atccatgagg 3720 gtatgggttt gatccctggc cttgctcaat gggttaagga tccagcattg ctgtgagctg 3780 tggtataggt tgcagactct gctcaggtcc catgttgctg tgattgtggt gtaggctgac 3840 tgctgcagct tcaatttgac ccctagcccg ggaatttcca taggccacac gtgcagcact 3900 aaggaaggaa aaaaagaaaa aaaaaaaaaaa agagtgggtg tgcctatagt gaagaacaga 3960 tgtaaaaggg aagtgaaagg gattccccca ttctgaggga ttgtgagaag tgtgccagaa 4020 tattaacttc atttgacttg ttacagggaa agtaaacttg actttcacgg acctcctagt 4080 tacctggtgc ttactatatg tcttctcaga gtacctgatt cattcccagc ctggttgacc 4140 catcccccta tctctatggc tatgtttatc cagagcacat ctatctaaca ctccagctga 4200 tcttcctgac acagctgtgg caaccctgga tcctttaacc aactgtgcca ggctggagat 4260 caaacctaag cctctgcagc aacccaagct gctgcagtca gatttttaac cccctgtgcc 4320 actgtgggta tctccgatat tttgtatctt ctgtgactga gtggtttgct gtttgcaggg 4380 aaccagagtc agacactatc cccgtgcaat tcatcatcct cggacccatc aagctctatt 4440 atttcagaag aaaatggtgt tgcctgcata ggtgagaatc agtgaccaac ctatgaaaat 4500 gatctcaatc ctctgaaatg cattttatattc atgttttat tcctctttgc agggagtggt 4560 caacttcgcc tggtcgatgg aggtggtcgt tgtgctggga gagtagaggt ctatcatgag 4620 ggctcctggg gcaccatctg tgatgacagc tgggacctga atgatgccca tgtggtgtgc 4680 aaacagctga gctgtggatg ggccattaat gccactggtt ctgctcattt tggggaagga 4740 acagggccca tttggctgga tgagataaac tgtaatggaa aagaatctca tatttggcaa 4800 tgccactcac atggttgggg gcggcacaat tgcaggcata aggaggatgc aggagtcatc 4860 tgctcgg 4867 <210> 106 <211> 4991 <212> DNA <213> Sus scrofa <400> 106 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactacag tgcggcaact gtggtttccc tcctgggggg agctcacttt 3180 ggagaaggaa gtggacagat ctgggctgaa gaattccagt gtgaggggca cgagtcccac 3240 ctttcactct gcccagtagc accccgccct gacgggacat gtagccacag cagggacgtc 3300 ggcgtagtct gctcaagtga gacccaggga atgtgttcac tttgttccca tgccatgaag 3360 agggtagggt taggtagtca cagacatctt tttaaagccc tgtctccttc caggatacac 3420 acaaatccgc ttggtgaatg gcaagacccc atgtgaagga agagtggagc tcaacattct 3480 tgggtcctgg gggtccctct gcaactctca ctgggacatg gaagatgccc atgttttatg 3540 ccagcagctt aaatgtggag ttgccctttc tatcccggga ggagcacctt ttgggaaagg 3600 aagtgagcag gtctgggaggc acatgtttca ctgcactggg actgagaagc acatggggaga 3660 ttgttccgtc actgctctgg gcgcatcact ctgttcttca gggcaagtgg cctctgtaat 3720 ctgctcaggt aagagaataa gggcagccag tgatgagcca ctcatgacgg tgccttaaga 3780 gtgggtgtac ctaggagttc ccattgtggc tcagtggtaa caaactcgac tggtatccat 3840 gagggtatgg gtttgatccc tggccttgct caatgggtta aggatccagc attgctgtga 3900 gctgtggtat aggttgcaga ctctgctcag gtcccatgtt gctgtgattg tggtgtaggc 3960 tgactgctgc agcttcaatt tgacccctag cccgggaatt tccataggcc acacgtgcag 4020 cactaaggaa ggaaaaaaag aaaaaaaaaaa aaaaagagtg ggtgtgccta tagtgaagaa 4080 cagatgtaaa agggaagtga aagggattcc cccattctga gggattgtga gaagtgtgcc 4140 agaatattaa cttcatttga cttgttacag ggaaagtaaa cttgactttc acggacctcc 4200 tagttacctg gtgcttacta tatgtcttct cagagtacct gattcattcc cagcctggtt 4260 gacccatccc cctatctcta tggctatgtt tatccagagc acatctatct aacactccag 4320 ctgatcttcc tgacacagct gtggcaaccc tggatccttt aaccaactgt gccaggctgg 4380 agatcaaacc taagcctctg cagcaaccca agctgctgca gtcagatttt taaccccctg 4440 tgccactgtg ggtatctccg atattttgta tcttctgtga ctgagtggtt tgctgtttgc 4500 agggaaccag agtcagacac tatccccgtg caattcatca tcctcggacc catcaagctc 4560 tattatttca gaagaaaatg gtgttgcctg cataggtgag aatcagtgac caacctatga 4620 aaatgatctc aatcctctga aatgcatttt attcatgttt tatttcctct ttgcaggggag 4680 tggtcaactt cgcctggtcg atggaggtgg tcgttgtgct gggagagtag aggtctatca 4740 tgagggctcc tggggcacca tctgtgatga cagctgggac ctgaatgatg cccatgtggt 4800 gtgcaaacag ctgagctgtg gatgggccat taatgccact ggttctgctc attttgggga 4860 aggaacaggg cccatttggc tggatgagat aaactgtaat ggaaaagaat ctcatatttg 4920 gcaatgccac tcacatggtt gggggcggca caattgcagg cataaggagg atgcaggagt 4980 catctgctcg g 4991 <210> 107 <211> 4860 <212> DNA <213> Sus scrofa <400> 107 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggt cctgggggga gctcactttg gagaaggaag 3060 tggacagatc tgggctgaag aattccagtg tgaggggcac gagtcccacc tttcactctg 3120 cccagtagca ccccgccctg acgggacatg tagccacagc agggacgtcg gcgtagtctg 3180 ctcaagtgag acccagggaa tgtgttcact ttgttcccat gccatgaaga gggtagggtt 3240 aggtagtcac agacatcttt ttaaagccct gtctccttcc aggatacaca caaatccgct 3300 tggtgaatgg caagacccca tgtgaaggaa gagtggagct caacattctt gggtcctggg 3360 ggtccctctg caactctcac tgggacatgg aagatgccca tgttttatgc cagcagctta 3420 aatgtggagt tgccctttct atcccgggag gagcaccttt tgggaaagga agtgagcagg 3480 tctggaggca catgtttcac tgcactggga ctgagaagca catggggagat tgttccgtca 3540 ctgctctggg cgcatcactc tgttcttcag ggcaagtggc ctctgtaatc tgctcaggta 3600 agagaataag ggcagccagt gatgagccac tcatgacggt gccttaagag tgggtgtacc 3660 taggagttcc cattgtggct cagtggtaac aaactcgact ggtatccatg agggtatggg 3720 tttgatccct ggccttgctc aatgggttaa ggatccagca ttgctgtgag ctgtggtata 3780 ggttgcagac tctgctcagg tcccatgttg ctgtgattgt ggtgtaggct gactgctgca 3840 gcttcaattt gacccctagc ccgggaattt ccataggcca cacgtgcagc actaaggaag 3900 gaaaaaaaga aaaaaaaaaaa aaaagagtgg gtgtgcctat agtgaagaac agatgtaaaa 3960 gggaagtgaa agggattccc ccattctgag ggattgtgag aagtgtgcca gaatattaac 4020 ttcatttgac ttgttacagg gaaagtaaac ttgactttca cggacctcct agttacctgg 4080 tgcttactat atgtcttctc agagtacctg attcattccc agcctggttg acccatcccc 4140 ctatctctat ggctatgttt atccagagca catctatcta acactccagc tgatcttcct 4200 gacacagctg tggcaaccct ggatccttta accaactgtg ccaggctgga gatcaaacct 4260 aagcctctgc agcaacccaa gctgctgcag tcagattttt aaccccctgt gccactgtgg 4320 gtatctccga tattttgtat cttctgtgac tgagtggttt gctgtttgca gggaaccaga 4380 gtcagacact atccccgtgc aattcatcat cctcggaccc atcaagctct attatttcag 4440 aagaaaatgg tgttgcctgc ataggtgaga atcagtgacc aacctatgaa aatgatctca 4500 atcctctgaa atgcatttta ttcatgtttt atttcctctt tgcagggagt ggtcaacttc 4560 gcctggtcga tggaggtggt cgttgtgctg ggagagtaga ggtctatcat gagggctcct 4620 ggggcaccat ctgtgatgac agctgggacc tgaatgatgc ccatgtggtg tgcaaacagc 4680 tgagctgtgg atgggccatt aatgccactg gttctgctca ttttggggaa ggaacagggc 4740 ccatttggct ggatgagata aactgtaatg gaaaagaatc tcatatttgg caatgccact 4800 cacatggttg ggggcggcac aattgcaggc ataaggagga tgcaggagtc atctgctcgg 4860 4860 <210> 108 <211> 4858 <212> DNA <213> Sus scrofa <400> 108 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggc tggggggagc tcactttgga gaaggaagtg 3060 gacagatctg ggctgaagaa ttccagtgtg aggggcacga gtcccacctt tcactctgcc 3120 cagtagcacc ccgccctgac gggacatgta gccacagcag ggacgtcggc gtagtctgct 3180 caagtgagac ccagggaatg tgttcacttt gttcccatgc catgaagagg gtagggttag 3240 gtagtcacag acatcttttt aaagccctgt ctccttccag gatacacaca aatccgcttg 3300 gtgaatggca agacccccatg tgaaggaaga gtggagctca acattcttgg gtcctggggg 3360 tccctctgca actctcactg ggacatggaa gatgcccatg ttttatgcca gcagcttaaa 3420 tgtggagttg ccctttctat cccgggagga gcaccttttg ggaaaggaag tgagcaggtc 3480 tggaggcaca tgtttcactg cactgggact gagaagcaca tgggagattg ttccgtcact 3540 gctctgggcg catcactctg ttcttcaggg caagtggcct ctgtaatctg ctcaggtaag 3600 agaataaggg cagccagtga tgagccactc atgacggtgc cttaagagtg ggtgtaccta 3660 ggagttccca ttgtggctca gtggtaacaa actcgactgg tatccatgag ggtatgggtt 3720 tgatccctgg ccttgctcaa tgggttaagg atccagcatt gctgtgagct gtggtatagg 3780 ttgcagactc tgctcaggtc ccatgttgct gtgattgtgg tgtaggctga ctgctgcagc 3840 ttcaatttga cccctagccc gggaatttcc ataggccaca cgtgcagcac taaggaagga 3900 aaaaaagaaa aaaaaaaaaaa aagagtgggt gtgcctatag tgaagaacag atgtaaaagg 3960 gaagtgaaag ggattccccc attctgaggg attgtgagaa gtgtgccaga atattaactt 4020 catttgactt gttacaggga aagtaaactt gactttcacg gacctcctag ttacctggtg 4080 cttactatat gtcttctcag agtacctgat tcattcccag cctggttgac ccatccccct 4140 atctctatgg ctatgtttat ccagagcaca tctatctaac actccagctg atcttcctga 4200 cacagctgtg gcaaccctgg atcctttaac caactgtgcc aggctggaga tcaaacctaa 4260 gcctctgcag caacccaaagc tgctgcagtc agatttttaa ccccctgtgc cactgtgggt 4320 atctccgata ttttgtatct tctgtgactg agtggtttgc tgtttgcagg gaaccagagt 4380 cagacactat ccccgtgcaa ttcatcatcc tcggacccat caagctctat tatttcagaa 4440 gaaaatggtg ttgcctgcat aggtgagaat cagtgaccaa cctatgaaaa tgatctcaat 4500 cctctgaaat gcattttat catgttttat ttcctctttg cagggagtgg tcaacttcgc 4560 ctggtcgatg gaggtggtcg ttgtgctggg agagtagagg tctatcatga gggctcctgg 4620 ggcaccatct gtgatgacag ctgggacctg aatgatgccc atgtggtgtg caaacagctg 4680 agctgtggat gggccattaa tgccactggt tctgctcatt ttggggaagg aacagggccc 4740 atttggctgg atgagataaa ctgtaatgga aaagaatctc atatttggca atgccactca 4800 catggttggg ggcggcacaa ttgcaggcat aaggaggatg caggagtcat ctgctcgg 4858 <210> 109 <211> 3523 <212> DNA <213> Sus scrofa <400> 109 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gagctgtggt ataggttgca gactctgctc 2460 aggtcccatg ttgctgtgat tgtggtgtag gctgactgct gcagcttcaa tttgacccct 2520 agcccgggaa tttccatagg ccacacgtgc agcactaagg aaggaaaaaa agaaaaaaaaa 2580 aaaaaaagag tgggtgtgcc tatagtgaag aacagatgta aaagggaagt gaaagggatt 2640 cccccattct gagggattgt gagaagtgtg ccagaatatt aacttcattt gacttgttac 2700 agggaaagta aacttgactt tcacggacct cctagttacc tggtgcttac tatatgtctt 2760 ctcagagtac ctgattcatt cccagcctgg ttgacccatc cccctatctc tatggctatg 2820 tttatccaga gcacatctat ctaacactcc agctgatctt cctgacacag ctgtggcaac 2880 cctggatcct ttaaccaact gtgccaggct ggagatcaaa cctaagcctc tgcagcaacc 2940 caagctgctg cagtcagatt tttaaccccc tgtgccactg tgggtatctc cgatattttg 3000 tatcttctgt gactgagtgg tttgctgttt gcagggaacc agagtcagac actatccccg 3060 tgcaattcat catcctcgga cccatcaagc tctattattt cagaagaaaa tggtgttgcc 3120 tgcataggtg agaatcagtg accaacctat gaaaatgatc tcaatcctct gaaatgcatt 3180 ttattcatgt tttattcct ctttgcaggg agtggtcaac ttcgcctggt cgatggaggt 3240 ggtcgttgtg ctgggagagt agaggtctat catgagggct cctggggcac catctgtgat 3300 gacagctggg acctgaatga tgcccatgtg gtgtgcaaac agctgagctg tggatgggcc 3360 attaatgcca ctggttctgc tcattttggg gaaggaacag ggcccatttg gctggatgag 3420 ataaactgta atggaaaaaga atctcatatt tggcaatgcc actcacatgg ttgggggcgg 3480 cacaattgca ggcataagga ggatgcagga gtcatctgct cgg 3523 <210> 110 <211> 3603 <212> DNA <213> Sus scrofa <400> 110 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgttgt ggagaattcc acaagaattc gtgttattg acagcagtca tctttaaaag 540 gcatttgaga aagtccaatt tcaaatgcat ttcctttctt taaaagataa attgaagaaa 600 ataagtcttt atttcccaag taaattgaat tgcctctcag tctgttaaaa gaaactctta 660 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 720 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 780 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 840 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 900 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 960 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 1020 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 1080 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 1140 gagatgttgt ggagaattcc acaagaattc gtgttattg acagcagtca tctttaaaag 1200 gcatttgaga aagtccaatt tcaaatgcat ttcctttctt taaaagataa attgaagaaa 1260 ataagtcttt atttcccaag taaattgaat tgcctctcag tctgttaaaa gaaactctta 1320 ccttgatgat tgcgctctta acctggcaaa gattgtcttt aaaatctgag ctccatgtct 1380 tctgctttat ttctggtgtg cctttgactc cagattacag taaatggagg actgagtata 1440 gggctaaaaa gtagagagaa tggatgcata ttatctgtgg tctccaatgt gatgaatgaa 1500 gtaggcaaat actcaaagga aagagaaagc atgctccaag aattatgggt tccagaaggc 1560 aaagtcccag aattgtctcc agggaaggac agggaggtct agaatcggct aagcccactg 1620 taggcagaaa aaccaagagg catgaatggc ttccctttct cacttttcac tctctggctt 1680 actcctatca tgaaggaaaa tattggaatc atattctccc tcaccgaaat gctatttttc 1740 agcccacagg aaacccaggc tggttggagg ggacattccc tgctctcact ttggagaagg 1800 aagtggacag atctgggctg aagaattcca gtgtgagggg cacgagtccc acctttcact 1860 ctgcccagta gcaccccgcc ctgacgggac atgtagccac agcagggacg tcggcgtagt 1920 ctgctcaagt gagacccagg gaatgtgttc actttgttcc catgccatga agagggtagg 1980 gttaggtagt cacagacatc tttttaaagc cctgtctcct tccaggatac acacaaatcc 2040 gcttggtgaa tggcaagacc ccatgtgaag gaagagtgga gctcaacatt cttgggtcct 2100 gggggtccct ctgcaactct cactgggaca tggaagatgc ccatgtttta tgccagcagc 2160 ttaaatgtgg agttgccctt tctatcccgg gaggagcacc ttttgggaaa ggaagtgagc 2220 aggtctggag gcacatgttt cactgcactg ggactgagaa gcacatggga gattgttccg 2280 tcactgctct gggcgcatca ctctgttctt cagggcaagt ggcctctgta atctgctcag 2340 gtaagagaat aagggcagcc agtgatgagc cactcatgac ggtgccttaa gagtgggtgt 2400 acctaggagt tcccattgtg gctcagtggt aacaaactcg actggtatcc atgagggtat 2460 gggtttgatc cctggccttg ctcaatgggt taaggatcca gcattgctgt gagctgtggt 2520 ataggttgca gactctgctc aggtcccatg ttgctgtgat tgtggtgtag gctgactgct 2580 gcagcttcaa tttgacccct agcccgggaa tttccatagg ccacacgtgc agcactaagg 2640 aaggaaaaaaa agaaaaaaaaa aaaaaaagag tgggtgtgcc tatagtgaag aacagatgta 2700 aaagggaagt gaaagggatt cccccattct gagggattgt gagaagtgtg ccagaatatt 2760 aacttcattt gacttgttac agggaaagta aacttgactt tcacggacct cctagttacc 2820 tggtgcttac tatatgtctt ctcagagtac ctgattcatt cccagcctgg ttgacccatc 2880 cccctatctc tatggctatg tttatccaga gcacatctat ctaacactcc agctgatctt 2940 cctgacacag ctgtggcaac cctggatcct ttaaccaact gtgccaggct ggagatcaaa 3000 cctaagcctc tgcagcaacc caagctgctg cagtcagatt tttaaccccc tgtgccactg 3060 tgggtatctc cgatattttg tatcttctgt gactgagtgg tttgctgttt gcagggaacc 3120 agagtcagac actatccccg tgcaattcat catcctcgga cccatcaagc tctattattt 3180 cagaagaaaa tggtgttgcc tgcataggtg agaatcagtg accaacctat gaaaatgatc 3240 tcaatcctct gaaatgcatt ttattcatgt tttattcct ctttgcaggg agtggtcaac 3300 ttcgcctggt cgatggaggt ggtcgttgtg ctgggagagt agaggtctat catgagggct 3360 cctggggcac catctgtgat gacagctggg acctgaatga tgcccatgtg gtgtgcaaac 3420 agctgagctg tggatgggcc attaatgcca ctggttctgc tcattttggg gaaggaacag 3480 ggcccatttg gctggatgag ataaactgta atggaaaaga atctcatatt tggcaatgcc 3540 actcacatgg ttgggggcgg cacaattgca ggcataagga ggatgcagga gtcatctgct 3600 cgg 3603 <210> 111 <211> 4962 <212> DNA <213> Sus scrofa <400> 111 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactacag tgcgtcactt tggagaagga agtggacaga tctgggctga 3180 agaattccag tgtgaggggc acgagtccca cctttcactc tgcccagtag caccccgccc 3240 tgacgggaca tgtagccaca gcagggacgt cggcgtagtc tgctcaagtg agacccaggg 3300 aatgtgttca ctttgttccc atgccatgaa gagggtaggg ttaggtagtc acagacatct 3360 ttttaaagcc ctgtctcctt ccaggataca cacaaatccg cttggtgaat ggcaagaccc 3420 catgtgaagg aagagtggag ctcaacattc ttgggtcctg ggggtccctc tgcaactctc 3480 actgggacat ggaagatgcc catgttttat gccagcagct taaatgtgga gttgcccttt 3540 ctatcccggg aggagcacct tttgggaaag gaagtgagca ggtctggagg cacatgtttc 3600 actgcactgg gactgagaag cacatgggag attgttccgt cactgctctg ggcgcatcac 3660 tctgttcttc agggcaagtg gcctctgtaa tctgctcagg taagagaata agggcagcca 3720 gtgatgagcc actcatgacg gtgccttaag agtgggtgta cctaggagtt cccattgtgg 3780 ctcagtggta acaaactcga ctggtatcca tgagggtatg ggtttgatcc ctggccttgc 3840 tcaatgggtt aaggatccag cattgctgtg agctgtggta taggttgcag actctgctca 3900 ggtcccatgt tgctgtgatt gtggtgtagg ctgactgctg cagcttcaat ttgaccccta 3960 gcccgggaat ttccataggc cacacgtgca gcactaagga aggaaaaaaaa gaaaaaaaaa 4020 aaaaaagagt gggtgtgcct atagtgaaga acagatgtaa aagggaagtg aaagggattc 4080 ccccattctg agggattgtg agaagtgtgc cagaatatta acttcatttg acttgttaca 4140 gggaaagtaa acttgacttt cacggacctc ctagttacct ggtgcttact atatgtcttc 4200 tcagagtacc tgattcattc ccagcctggt tgacccatcc ccctatctct atggctatgt 4260 ttatccagag cacatctatc taacactcca gctgatcttc ctgacacagc tgtggcaacc 4320 ctggatcctt taaccaactg tgccaggctg gagatcaaac ctaagcctct gcagcaaccc 4380 aagctgctgc agtcagattt ttaaccccct gtgccactgt gggtatctcc gatattttgt 4440 atcttctgtg actgagtggt ttgctgtttg cagggaacca gagtcagaca ctatccccgt 4500 gcaattcatc atcctcggac ccatcaagct ctattatttc agaagaaaat ggtgttgcct 4560 gcataggtga gaatcagtga ccaacctatg aaaatgatct caatcctctg aaatgcattt 4620 tattcatgtt ttatttcctc tttgcaggga gtggtcaact tcgcctggtc gatggaggtg 4680 gtcgttgtgc tgggagagta gaggtctatc atgagggctc ctggggcacc atctgtgatg 4740 acagctggga cctgaatgat gcccatgtgg tgtgcaaaca gctgagctgt ggatgggcca 4800 ttaatgccac tggttctgct cattttgggg aaggaacagg gcccatttgg ctggatgaga 4860 taaactgtaa tggaaaaagaa tctcatattt ggcaatgcca ctcacatggt tgggggcggc 4920 acaattgcag gcataaggag gatgcaggag tcatctgctc gg 4962 <210> 112 <211> 3603 <212> DNA <213> Sus scrofa <400> 112 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactacag tgcgattcat catcctcgga cccatcaagc tctattattt 3180 cagaagaaaa tggtgttgcc tgcataggtg agaatcagtg accaacctat gaaaatgatc 3240 tcaatcctct gaaatgcatt ttattcatgt tttattcct ctttgcaggg agtggtcaac 3300 ttcgcctggt cgatggaggt ggtcgttgtg ctgggagagt agaggtctat catgagggct 3360 cctggggcac catctgtgat gacagctggg acctgaatga tgcccatgtg gtgtgcaaac 3420 agctgagctg tggatgggcc attaatgcca ctggttctgc tcattttggg gaaggaacag 3480 ggcccatttg gctggatgag ataaactgta atggaaaaga atctcatatt tggcaatgcc 3540 actcacatgg ttgggggcgg cacaattgca ggcataagga ggatgcagga gtcatctgct 3600 cgg 3603 <210> 113 <211> 3619 <212> DNA <213> Sus scrofa <400> 113 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcagccagcg 3120 tgctttgcag ggaaccagag tcagacacta tccccgtgca attcatcatc ctcggacccca 3180 tcaagctcta ttatttcaga agaaaatggt gttgcctgca taggtgagaa tcagtgacca 3240 acctatgaaa atgatctcaa tcctctgaaa tgcattttat tcatgtttta tttcctcttt 3300 gcagggagtg gtcaacttcg cctggtcgat ggaggtggtc gttgtgctgg gagagtagag 3360 gtctatcatg agggctcctg gggcaccatc tgtgatgaca gctgggacct gaatgatgcc 3420 catgtggtgt gcaaacagct gagctgtgga tgggccatta atgccactgg ttctgctcat 3480 tttggggaag gaacagggcc catttggctg gatgagataa actgtaatgg aaaagaatct 3540 catatttggc aatgccactc acatggttgg gggcggcaca attgcaggca taaggaggat 3600 gcaggagtca tctgctcgg 3619 <210> 114 <211> 3270 <212> DNA <213> Sus scrofa <400> 114 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atattattct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac ttgttacagg gaaagtaaac 2460 ttgactttca cggacctcct agttacctgg tgcttactat atgtcttctc agagtacctg 2520 attcattccc agcctggttg acccatcccc ctatctctat ggctatgttt atccagagca 2580 catctatcta acactccagc tgatcttcct gacacagctg tggcaaccct ggatccttta 2640 accaactgtg ccaggctgga gatcaaacct aagcctctgc agcaacccaa gctgctgcag 2700 tcagattttt aaccccctgt gccactgtgg gtatctccga tattttgtat cttctgtgac 2760 tgagtggttt gctgtttgca gggaaccaga gtcagacact atccccgtgc aattcatcat 2820 cctcggaccc atcaagctct attatttcag aagaaaatgg tgttgcctgc ataggtgaga 2880 atcagtgacc aacctatgaa aatgatctca atcctctgaa atgcatttta ttcatgtttt 2940 atttcctctt tgcagggagt ggtcaacttc gcctggtcga tggaggtggt cgttgtgctg 3000 ggagagtaga ggtctatcat gagggctcct ggggcaccat ctgtgatgac agctgggacc 3060 tgaatgatgc ccatgtggtg tgcaaacagc tgagctgtgg atgggccatt aatgccactg 3120 gttctgctca ttttggggaa ggaacagggc ccatttggct ggatgagata aactgtaatg 3180 gaaaagaatc tcatatttgg caatgccact cacatggttg ggggcggcac aattgcaggc 3240 ataaggagga tgcaggagtc atctgctcgg 3270 <210> 115 <211> 7 <212> DNA <213> Sus scrofa <400> 115 tactact 7 <210> 116 <211> 12 <212> DNA <213> Sus scrofa <400> 116 tgtggagaat tc 12 <210> 117 <211> 11 <212> DNA <213> Sus scrofa <400> 117 agccagcgtg c 11

Claims (13)

A method for increasing the resistance of a porcine animal or a progeny or cell thereof to porcine reproductive and respiratory syndrome virus (PRRSV) compared to a wild type porcine animal or a progeny or cell thereof, comprising modifying at least one chromosomal sequence of a gene encoding CD163 protein in the porcine animal or a progeny or cell thereof, wherein the modification comprises:
Mutations in exon 7 of the gene encoding the CD163 protein,
Mutations in exon 8 of the gene encoding the CD163 protein,
A variant selected from the group consisting of a variant in an intron adjacent to exon 7 or exon 8 of a gene encoding CD163 protein, and a combination thereof, wherein the variant is
An 11 base pair deletion from nucleotide 3137 to nucleotide 3147 compared to reference sequence SEQ ID NO: 47;
A 377 base pair deletion from nucleotides 2573 to 2949, with a 2 base pair insertion between nucleotides 3149 and 3150 compared to reference sequence SEQ ID NO: 47 on the same allele;
A 1930 base pair deletion from nucleotide 488 to nucleotide 2417, wherein the deleted sequence is replaced by a 12 base pair insertion starting at nucleotide 488 and having a 129 base pair deletion from nucleotides 3044 to nucleotides 3172 relative to reference sequence SEQ ID NO: 47 on the same allele;
A 1467 base pair deletion from nucleotide 2,431 to nucleotide 3,897 compared to reference sequence SEQ ID NO: 47;
A 1280 base pair deletion from nucleotide 2818 to nucleotide 4097 compared to the reference sequence SEQ ID NO: 47; and
A method comprising selecting from a group consisting of combinations of these. delete A method in claim 1, wherein the porcine animal or its offspring or cell is heterozygous for a CD163 gene modification. A method in claim 1, wherein the porcine animal or its offspring or cell is homozygous for a CD163 gene mutation. A method in claim 1, wherein the modification is produced by the action of CRISPR, TALEN or ZFN nuclease. A method for producing pigs having increased resistance to porcine reproductive and respiratory syndrome virus (PRRSV) compared to wild-type pigs,
The step of enucleating the oocyte;
A step of fusing the above oocyte with a donor somatic cell, wherein the genome of the donor somatic cell is modified with at least one chromosomal sequence of a gene encoding CD163 protein.
Mutations in exon 7 of the gene encoding the CD163 protein,
Mutations in exon 8 of the gene encoding the CD163 protein,
A variant selected from the group consisting of a variant in an intron adjacent to exon 7 or exon 8 of a gene encoding CD163 protein, and a combination thereof, wherein the variant
An 11 base pair deletion from nucleotide 3137 to nucleotide 3147 compared to reference sequence SEQ ID NO: 47;
A 377 base pair deletion from nucleotides 2573 to 2949, with a 2 base pair insertion between nucleotides 3149 and 3150 compared to reference sequence SEQ ID NO: 47 on the same allele;
A 1930 base pair deletion from nucleotide 488 to nucleotide 2417, wherein the deleted sequence is replaced by a 12 base pair insertion starting at nucleotide 488 and having a 129 base pair deletion from nucleotides 3044 to nucleotides 3172 relative to reference sequence SEQ ID NO: 47 on the same allele;
A 1467 base pair deletion from nucleotide 2,431 to nucleotide 3,897 compared to reference sequence SEQ ID NO: 47;
A 1280 base pair deletion from nucleotide 2818 to nucleotide 4097 compared to the reference sequence SEQ ID NO: 47; and
A step of selecting from a group consisting of combinations of these;
A step of activating the above oocytes to produce embryos; and
The step of transplanting the above embryo into the reproductive tract of a surrogate pig animal
A method of producing a pig animal having increased resistance to porcine reproductive and respiratory syndrome virus (PRRSV) compared to a wild type pig animal, wherein the surrogate pig animal is in estrus but has not yet completed ovulation; and wherein pregnancy and term farrowing result in the pig animal. A method in claim 6, wherein the porcine animal is homozygous for the mutation. A method in claim 6, wherein the porcine animal is heterozygous for the mutation. A method in claim 6, wherein the donor somatic cells include fibroblasts. A method according to claim 6, wherein the mutation comprises an insertion, a deletion, a frameshift mutation, a nonsense mutation, a missense mutation, a point mutation, or a combination thereof. A method in claim 6, wherein the porcine animal comprises a modification that alters the expression or activity of CD163. A method in claim 6, wherein inactivation of the CD163 allele results in a CD163 protein that reduces or eliminates cellular uptake of porcine reproductive and respiratory syndrome virus (PRRSV). A method for producing a genetically modified pig animal comprising a mutation in both chromosomal sequences encoding CD163 protein, wherein the pig animal has increased resistance to porcine reproductive and respiratory syndrome virus (PRRSV) compared to a wild type pig animal,
A step of producing F1 offspring by breeding a genetically modified female pig produced by the method of claim 6 with a genetically modified male pig produced by the method of claim 6; and
A step of screening the above F1 offspring to identify genetically modified pig animals containing a mutation in both chromosomal sequences of the gene encoding CD163 protein.
, and the above modifications include
Mutations in exon 7 of the gene encoding the CD163 protein,
Mutations in exon 8 of the gene encoding the CD163 protein,
A variant selected from the group consisting of a variant in an intron adjacent to exon 7 or exon 8 of a gene encoding CD163 protein, and a combination thereof, wherein the variant is
An 11 base pair deletion from nucleotide 3137 to nucleotide 3147 compared to reference sequence SEQ ID NO: 47;
A 377 base pair deletion from nucleotides 2573 to 2949, with a 2 base pair insertion between nucleotides 3149 and 3150 compared to reference sequence SEQ ID NO: 47 on the same allele;
A 1930 base pair deletion from nucleotide 488 to nucleotide 2417, wherein the deleted sequence is replaced by a 12 base pair insertion starting at nucleotide 488 and having a 129 base pair deletion from nucleotides 3044 to nucleotides 3172 relative to reference sequence SEQ ID NO: 47 on the same allele;
A 1467 base pair deletion from nucleotide 2,431 to nucleotide 3,897 compared to reference sequence SEQ ID NO: 47;
A 1280 base pair deletion from nucleotide 2818 to nucleotide 4097 compared to the reference sequence SEQ ID NO: 47; and
A method comprising selecting from a group consisting of combinations of these.