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WO1996028967A1 - Mammiferes transgeniques autres que des primates, ou des serotypes de primates superieurs ont ete exprimes par un transfert de genes etrangers et procede pour creer ceux-ci - Google Patents

Mammiferes transgeniques autres que des primates, ou des serotypes de primates superieurs ont ete exprimes par un transfert de genes etrangers et procede pour creer ceux-ci Download PDF

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Publication number
WO1996028967A1
WO1996028967A1 PCT/JP1996/000703 JP9600703W WO9628967A1 WO 1996028967 A1 WO1996028967 A1 WO 1996028967A1 JP 9600703 W JP9600703 W JP 9600703W WO 9628967 A1 WO9628967 A1 WO 9628967A1
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WIPO (PCT)
Prior art keywords
primate
gene
mammal
type
antigen
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PCT/JP1996/000703
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English (en)
Japanese (ja)
Inventor
Chihiro Koike
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Chihiro Koike
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from PCT/JP1995/000488 external-priority patent/WO1996028966A1/fr
Application filed by Chihiro Koike filed Critical Chihiro Koike
Priority to AU49562/96A priority Critical patent/AU4956296A/en
Priority to PCT/JP1996/000703 priority patent/WO1996028967A1/fr
Priority to JP52828796A priority patent/JP3809189B2/ja
Publication of WO1996028967A1 publication Critical patent/WO1996028967A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A01K2267/025—Animal producing cells or organs for transplantation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases

Definitions

  • the present invention relates to a transgenic non-S long-sized mammal having cells, tissues or organs suitable for transplantation into a higher primate by introducing an exogenous gene and a method for producing the animal.
  • the present invention relates to a transgenic animal in which a mammalian glycan antigen is converted into a form unique to a higher primate, and its state is genetically stabilized, and a method for producing the same.
  • kumio includes organs such as bold and liver, as well as skin, blood vessels, and cells.
  • artificial materials such as artificial vessels and artificial blood vessels are used. This includes self tissues, and even tissues and cells of the same or different animal species.
  • Livestock on the other hand, are non-primate mammals, with well-established breeding methods, readily available, and with few ethical issues, making them a promising alternative donor, the so-called donor.
  • tissues isolated from such mammals are known to cause severe rejection in humans, particularly severe rejection that occurs within 24 hours (hereinafter referred to as “super! This is also called a reaction.
  • Such hyperacute rejection occurs in the endothelial glaze within the donor's tissue when the replenished donor tissue vessels are anastomosed with the recipient vessels to flush the blood.
  • This hyperacute rejection reaction in combination with a species in which a human has a natural antibody, is one of the two activation pathways of the human trapping system, one of the classical pathways triggered by the antigen-antibody reaction. It is considered to be the main reaction.
  • This antigen-antibody reaction is thought to be due to the following causes.
  • vascular cells and hemocyte cells containing vascular endothelial cell membranes of non-S-long mammals such as pigs (hereinafter, also simply referred to as vascular cells and hemocyte cells) is high. Because it is different from that of higher primates such as G.
  • non-primate mammals such as septa have their own carbohydrate antigens (hereinafter referred to as G-type antigens or G-type S) that higher primates do not have on their vascular / hemocyte cell membrane surfaces. Because it is.
  • G-type antigens hereinafter referred to as G-type S
  • the anti-G natural antibody in the human blood and the G-type antigen on the vascular endothelial cell membrane in human blood are Antigen-antibody reaction is triggered, and the resulting antigen-antibody complex activates the complement system, causing porcine vascular endothelial cells to rupture, leading to the destruction of the replenished donor tissue, resulting in hyperacute rejection It is.
  • G-type antigen in the donor tissue it is possible to suppress the expression of G-type antigen in the donor tissue by introducing antisense DNA to a gene involved in the expression of G-type antigen in the donor or by completely suppressing the expression of this gene.
  • the GS1 antigen precursor is subjected to SS in the donor male. Since this precursor is also a so-called Bombay-type antigen, it may still cause hyperacute rejection by natural antibodies present in humans.
  • ABH-type substances are expressed in higher primates according to the present invention. Only in rodents to which chimpanzees, orangutans, baboons, Japanese, etc. belong, and in other primates and non-primate mammals, expressed in gastrointestinal muco-knee receptor cells etc. But not expressed in vascular and blood cells.
  • non-primate mammals have a G-type substance in vascular and blood cells that is a substance in which galactose binds to a precursor of ABH-type substance (N-acetyllactosamine) in a 113 structure. is there.
  • the H-type substance also referred to as H-type antigen
  • the H-type antigen is different from the HT precursor (N-acetyllactosamine) in terms of GDP-fucose:) SD-galactoside.
  • G-type substances are the same precursors as the aforementioned H-type substances, N- For cetyl lactosamine, UDP galactose: ⁇ SD-galactosyl-1.4-N-acetyl-darcosaminide 3-galactosyltransferase (hereinafter referred to simply as G-choice or (1.3) galactosyltransferase). It is generated by acting.
  • the difference in serotype between vasculature and blood cells in humans and pigs is due to the difference in enzymes acting on precursors, in other words, the enzyme GT exists in pigs and the like.
  • the enzyme GT does not exist in humans and the like and the enzyme purple FT exists.
  • the presence or absence of ABH-type substances and enzymes in such various tissues is considered to be based on evolution.
  • the present invention provides a non-primate mammal, such as a pig, which is widely bred as a domestic animal and is easily obtainable, by a new method which is not used in the past.
  • the aim is to mitigate rejection and apply non-primate mammals to transplantation into higher primates.
  • the present inventor has proposed that in non-primate mammals such as bushus, gene transfer engineering and developmental engineering, for example, the production of higher-grade glucotransferases such as F-chote.
  • non-primate mammals such as bushus, gene transfer engineering and developmental engineering
  • F-chote higher-grade glucotransferases
  • GT vascular and blood cell
  • the conventional, natural antibodies Attention has been focused on the suppression of the capturer activation pathway after the binding of a protein to an antigen.
  • the present invention can be said to be a more essential solution to the conventional method in that the binding itself between the natural anti-G antibody and the G-type antigen in higher primates such as humans is suppressed.
  • a first aspect of the present invention relates to a non-human mammal having a DNA sequence containing a DNA sequence encoding FT of a higher primate, wherein the DNA construct has been transformed to express an H-type antigen.
  • Transgenic animal In the animal into which this DNA construct has been introduced, FT is expressed, and this FT acts on N-acetyl lactosamine to generate an H-type antigen. Express. For this reason, even if a non-S long mammal has a GT gene, when FT consumes N-acetyl lactosamine, which is a substrate of GT, the non-S long mammal's GT and N-acetyl lactosamine may be intermingled. bond or is inhibited, these foreign DNA beta expression is suppressed G group antigens, be held in a heterozygous state, but may be held at Jo homo-, held in homozygous Jo virtual It is a preferred idea to do so.
  • higher primates refer to narrow monkeys belonging to humans, chimpanzees, orangutans, baboons, Japanese birds, etc. among primates.
  • the non-higher primates are the broad-nosed primates and the protozoa among the primates.
  • Non-primate mammals or non-primate mammals are mammals that are not primates.
  • the second invention comprises a first DNA construct comprising a DNA sequence encoding higher order FT and a second DNA comprising a DNA sequence which is antisense to the GT gene of a non-primate mammal.
  • a non-primate mammal trait characterized in that it has been transformed to express a higher length H-type antigen while reducing expression of a non-primate mammal's G-type antigen. Convertible animal.
  • exogenous DNAs may be maintained in a heterologous state or a homologous state, but it is preferred that they be maintained in a homozygous state.
  • the present invention relates to a method wherein a DNA construct containing a DNA sequence encoding FT is inserted by homologous recombination into a part of the GT gene of a non-primate mammal, thereby expressing a non-primate mammalian G-type antigen.
  • a non-S long mammal transformed animal characterized by being transformed to express a primate H-type antigen.
  • a mutation is introduced into the GT gene of a non-S long mammal by this DNA construct, so that GT is not expressed and G-type antigen is not expressed.
  • FT is expressed, and H-type antigen is expressed by the FT.
  • DNA constructs that cause homologous recombination in part of the GTif gene are:
  • exogenous DNAs may be retained in a hetero state or in a homo state, but are preferably retained in a homo state ffi.
  • a DNA construct containing a DNA sequence encoding FT is inserted into a part of the GT gene of a non-primate mammal by homologous recombination to express a G-type antigen of a non-S long mammal.
  • This is a transplant material obtained from a non-S long mammal transformed animal that has been transformed to express a higher primate H-type antigen without any modification.
  • the cell itself obtained from the transgenic animal, a tissue containing the cell as a component, 3 ⁇ 4, or other materials can be widely used as a material for transplantation into a higher primate.
  • a preferable aspect of the transgenic animal in these inventions is that the non-primate mammal is bush. If the transformed animal is a pig, this can provide material suitable for transplantation into higher primate mammals.
  • a fifth invention is to express a non-primate mammalian G-type antigen by inserting a DNA construct containing a DNA sequence encoding FT into a part of the GT gene of the non-primate mammal by homologous recombination. This is a method for producing a non-primate mammal transformed animal that expresses the H-type antigen of a higher primate.
  • the sixth invention is directed to a method for avoiding rejection reaction between a natural antibody of a higher primate and an antigen of a non-primate mammal in a space for transplanting a tissue of a non-S long mammal into a higher primate.
  • a DNA construct containing a DNA sequence encoding FT is inserted by homologous recombination into a part of the GT gene of a non-primate mammal.
  • H-type of a higher primate without expressing the G-type antigen of the non-primate mammal Creating a non-S long mammal transformed to express the antigen. Transplanting the non-primate mammal progeny obtained in this step into a higher primate.
  • non-primate mammal tissues express the HSS antigen but not the GSS [antigen]. Therefore, when this kumio is transplanted to the higher primate, it is not rejected by the anti-G natural antibody of the higher primate.
  • FIG. 2 is a process diagram of the construction operation of the plasmid pMAM / FT having the FT gene of Example I.
  • FIG. 3 is a schematic view of the fragment for exclusive use of the FT gene of Example I.
  • FIG. 4 is a process (2) of constructing and operating pREP8 / AS / GT of the second embodiment. You.
  • FIG. 5 is a schematic diagram of an AS / GT3 ⁇ 4 gene introduction fragment of Example 2.
  • FIG. 6 is a process diagram of a construction operation of PREP 9 / GT3-4 of the third embodiment.
  • FIG. 7 shows pREP9 / GT3-room SspI nom DraI of Example 3.
  • This figure illustrates the process of assembling I and the process of manufacturing the gene fragment GT 3-/ FT / p 01 yA.
  • FIG. 8 is a process diagram of a construction operation of the pGT / FT of the embodiment S.
  • FIG. 9 is a graph showing the results of 5 , Cr release atsee.
  • FIG. 10 is a diagram showing a method for obtaining fragment A from exons 2 to 3 of G-choedogene.
  • Figure 11 shows a method for obtaining fragment B from exons 4 to 6 of the GT3 ⁇ 4 gene.
  • FIG. 12 is a diagram showing a method for obtaining a plasmid D containing cDNA obtained from exons 1 and 2 of the FT gene.
  • FIG. 1S is a diagram illustrating a method for obtaining a plasmid E from a fragment B and a plasmid D.
  • FIG. 1S is a diagram illustrating a method for obtaining a plasmid E from a fragment B and a plasmid D.
  • Figure 14 shows how to construct from fragment A and plasmid E. 96/28967 N
  • FIG. 1 A first figure.
  • FIG. 15 is a diagram showing a mutant gene when a GT gene and a construct undergo orthotopic recombination.
  • FIG. 16A is a diagram in which the sequence in FT is detected from FT by Southern blotting
  • 16B is a diagram in which the homologous portion of the FT gene on the upstream side is detected by PCR.
  • Fig. 16C shows that 16C confirmed by PCR that homologous recombination occurred in the downstream part of the construct F-finger.
  • 16D shows FT transmission by Northern plotting.
  • Fig. 16 shows that mRNA of the offspring was detected.
  • Fig. 16E shows that D9-29 cells do not express H antigen by D-cytometry using lectin UE A1.
  • Fig. 16F is a diagram showing that the detection of the H antigen of L922 cells into which the FTS gene was introduced by the lectin UE A1 was detected by flow cytometry.
  • 17A is a diagram in which the FT gene was detected by PCR
  • 17B is a PC indicating that homologous recombination was performed in the homologous portion of the construct upstream of FT.
  • R is a diagram
  • 17C is a PCR diagram showing that homologous recombination was performed in the homologous portion downstream of the FT of the construct
  • 17D is an mRNA of the FTS gene.
  • 17E is a flow cytometry diagram showing that L929 cells do not express H antigen using lectin UEA1
  • FIG. FIG. 3 is a diagram showing, by flow cytometry, the detection of H antigen of L929 cells introduced with FT gene into the lectin UEA1.
  • non-primate mammals such as pigs
  • it is an S gene or its expression substance that only higher primates such as humans have and that non-S primate mammals such as septa do not.
  • To express the protein it is necessary to create transgenic animals.
  • blood group matching is one of the basic principles in human-to-human transplantation, but the basic substance of the so-called blood group, or ABO classification, is an H-type substance.
  • the previous substance N Is formed by the action of FT on acetylsilactamine.
  • non-chordate mammals such as pigeons have GT instead of FT, thereby forming a different substance (G-type substance) from human.
  • an exogenous gene fragment containing an FT gene encoding glycosyltransferase FT, which produces an H-type substance from its precursor is introduced into a fertilized egg such as a pig, and this gene fragment is transferred to the chromosome.
  • a fertilized egg such as a pig
  • this gene fragment is transferred to the chromosome.
  • Non-II long mammals expressing H-type K have similar acute serotypes in their blood and blood serotypes, and hyperacute rejection when transplanting the tissues of these animals into higher primates. The response can be mitigated, making these animal tissues suitable for transplantation into higher primates.
  • the enzyme FT consumes the precursor ⁇ , it is expected that the binding of the endogenous enzyme GT to the precursor will be inhibited and the production of type G substances will be suppressed.
  • the FT3 ⁇ 4 gene can be stably transmitted to progeny after it has been incorporated into the chromosome.
  • the present invention inactivates the expression of glycosyltransferases endogenous in non-primate mammals such as pigs, for example, the gene transfer of the plant transferase GT, which produces a G-type substance from its precursor.
  • a foreign gene fragment containing the antisense DNA of the GT G gene is introduced into the fertilized egg.
  • this gene fragment is integrated on the human chromosome, if this antisense DNA is transcribed in each cell, the transcript will be paired with the endogenous GT transcript and the GT It is expected that translation of offspring will be suppressed.
  • the enzyme F is actively expressed, the expression of the enzyme GT is suppressed, and H-type substances are produced, while production of G-type substances is reduced. Evening can be obtained.
  • foreign gene fragments such as antisense DNA can be stably transmitted to progeny after being integrated into the chromosome.
  • the foreign gene Once the foreign gene has been integrated into the chromosome, it can be homogenized by mating with the foreign gene, and the foreign gene that expresses the transferase of higher primates can be obtained.
  • higher primate sugars can be used.
  • a non-primate mammalian system that expresses a transferase and exhibits a higher serotype serotype can be obtained.
  • humans can be particularly targeted as higher primates.
  • Higher S-long bran transferase refers to a glycosyltransferase that is found in higher S-longs and contributes to the production of antigens not found in non-S-long mammary vascular and blood cells.
  • a specific example is FT.
  • the human FT is [EC 2, 4.1.69].
  • a DNA construct containing a DNA sequence encoding a higher length bran transferase can have a promoter, a terminator and the like in addition to the DNA sequence of the gene for the glycosyltransferase.
  • the terminator associated with the Boli A addition signal of the initial gene of SV40 is one of the sequences suitable for addition.
  • the expression level of the transgene is considerably influenced by the B-A signal taken from which gene, the B-A addition from the initial SV40 gene is not I'm good. This signal is placed before the terminator.
  • To form a foreign gene that can express FT it is necessary to know at least the DNA sequence of FT. Human FT has already been reported (Proc. Tl. Acad. Sci. USA. 87 (1990), pp ⁇ 67 4-6678).
  • non-vertebrate mammal in particular, an animal to be subjected to xenotransplantation to the higher primate can be targeted.
  • stags, puppies, sheep, goats, etc. raised as domestic animals are suitable.
  • These livestock are easy to breed and can be supplied stably, so they are suitable for replenishment of transplanting males.
  • Glycosyltransferases in non-primate mammals are ⁇ transferases involved in the production of antigens found in the vascular and blood cells of non-primate mammals and not found in higher primates.
  • a specific example is the pig-to-mouse GT.
  • the mouse GT is specified in [EC 2.4.1.15.1].
  • DNA constructs that suppress the expression of enzymes include DNA constructs containing a DNA sequence that is antisense to the DNA sequence that encodes the glycosyltransferase.
  • a DNA construct containing a DNA sequence that disrupts DNA can be used. To form this DNA construct, it is necessary to know at least the non-primate glycosyltransferase gene sequence.
  • the mouse GT gene has already been reported (Proc. Natl. Acad. Sci. USA. (1989), pp. 8227-8231). Also, the GTA gene of buta was reported to K (Xe notransplantation).
  • a DNA fragment in which a part or all of the cDNA of GT gene is incorporated in the antisense direction is formed as a DNA construct.
  • homologous recombination for example, it is preferable to form a region having as many regions of homology with the GT gene as possible.
  • glycosyltransferase in non-primate mammals can be suppressed by a conventionally known method other than introduction of a foreign gene.
  • the DNA construct can be constructed not only for non-homologous recombination on the chromosome but also for homologous recombination.
  • the pig FT gene is recombined with the pig GTit gene as a target, the suppression of GT expression and the expression of FT can be simultaneously performed.
  • a means for expressing the glycosyltransferase at a high rate when the gene fragment is recombined into a chromosome can be used.
  • a foreign gene fragment is prepared using pMAM as a vector plasmid, high expression can be induced by administration of steroid.
  • non-primate mammal There are many methods for introducing an exogenous gene into a non-primate mammal, such as a method using a retrovirus and a method using microinjection, and any method can be adopted. From the viewpoint of obtaining a progeny transformation when the t it is desired to germline transformation, non-primate mammalian embryo ⁇ or ES cells, or, is preferably performed with respect to a fertilized egg.
  • ⁇ -transferase Due to the expression of ⁇ -transferase in higher primates, or at the same time, the expression of ⁇ ⁇ -transferase in non-prolonged mammals is suppressed, the animal has a blood serotype similar to that of higher primates. Therefore, it is possible to mitigate the acute rejection reaction when these animal tissues are transplanted into higher S primates, and these animal tissues are suitable for transplantation into higher primates. It will be. In particular, when expressed in vascular endothelial cells, hyperacute rejection is greatly reduced. In addition, the expression of sugar priming enzymes in higher primates in red blood cells makes the serotype of blood similar to higher primates, and the blood of these animals is suitable as a substitute for higher S primates.
  • the H-type substance is expressed and the G-type substance is simultaneously reduced in the vascular and blood cell cells of non-long-term mammals.
  • Tissues will be animals that are similar to blood serotypes or higher primates.
  • the G-type substance is further reduced, and the animal becomes an animal whose tissue and blood serotypes are very similar to those of higher vertebrates.
  • FT when FT is expressed on the surface of vascular endothelial cells, hyperacute rejection in xenogeneic organs or tissue transplantation is greatly reduced. Also, if expressed in erythrocytes, it will be more suitable for blood substitutes.
  • one or more of the GT G gene loci on the chromosome do not exist, and FT
  • at least one GT exists on the chromosome, so that an individual with FT or two GTs can be obtained instead.
  • Such an individual provides a transplant material suitable for transplantation of a human or the like as a donor, regardless of the recipient's ABO blood type.
  • such a transformed non-primate mammal has a tissue that can be transplanted into a higher primate such as a human, and thus becomes a source of a substitute for a tissue such as a human because it has blood.
  • these animals are stably bred, they will be able to provide necessary pheasants and the like when needed, and will be used as storage for transplanted tissues.
  • a method to provide animals and materials that can mitigate hyperacute rejection in the case of xenograft ra transplantation by expressing the FTS gene specific to higher S-length mammals in non-S-length mammals is an unprecedented new method. And provide a more essential solution for the reconciliation of hyperacute rejection in xenotransplantation.
  • a sugar chain antigen of a higher primate is produced in its tissue and blood, or simultaneously, the production of a sugar chain antigen of a non-primate mammal is suppressed.
  • hyperacute rejection can be mitigated when non-s long mammal tissues are transplanted into higher primates, which are allogeneic transplants, and can be replaced with conventional artificial blood vessels, artificial organs, or allogeneic transplants. It is possible to provide a material that can be used in place of a magnetic device, a magnet assembly, and the like.
  • transforming a non-mammal mammal according to the present invention does not cause any damage or abuse to these animals. It does not affect the survival, reproduction, etc. of the fish. Further, the present invention does not exert any adverse effects on humans or any other animals.
  • examples of the present invention will be described.
  • the exogenous s gene in the present invention the two types of ⁇ -transferases already described, namely the G Tit gene and the cDNA of the F T gene, can be exemplified. However, the following embodiment does not limit the IS area of the claim.
  • MCS multicloning site
  • pMAM a product of CL0NETECH, Lee.F. (1981) Nature 294: 2278
  • restriction enzymes Nhel and Xhol restriction enzymes Nhel and Xhol
  • LMP gel LMP gel
  • the purified FT gene cDNA was inserted into this vector using T4 DNA ligase.
  • the plasmid DNA was used to transform JM109 bacterial cells in a combinatorial concept, and the resulting transformants were selected on an Ambicillin plate.
  • Each individual colony was allowed to grow with an LB medium (composition: Bacto-tryprone 10 g (manufactured by DIFC0), Bacto-yeast extract 5 g (manufactured by DIFC0), and NaCl 10 g to make 1 L with water, the same applies hereinafter).
  • a mini-plasmid was prepared. The extracted plasmid DNA was digested with various restriction enzymes and subjected to electrophoresis to spot the size and position of the inserted fragment. As a result, the plasmid integrated in the correct direction was called pMAMZ FT (see 02).
  • E. coli JM109 / p was used to obtain a linear fragment for gene transfer.
  • Plasmid preparation was performed after mass-culturing the MAM / FT strain in LB medium. Further, the obtained plasmid was oval by CsC1 density gradient centrifugation. After digestion of 10 g of the coffin-produced brasmid with the restriction enzymes PVu] and BamHI, this fragment was purified and separated to obtain a fragment for FTS gene transfer. A schematic diagram of the construction of this fragment is shown in FIG.
  • RNA was extracted, and cDNA corresponding to total RNA was synthesized using reverse transcriptase. This cDNA was transformed into type III, and PCR was performed using the primers described above.
  • the cDNA fragment obtained by this PCR was subjected to 1% agarose gel electrophoresis, a band having a length of about llOObp was obtained.
  • the base sequence was read by automatic base sequence reading (1), and it had the base sequence of the GT gene as previously reported. . Therefore, the cDNA was purified by electrophoresis on an LMP gel, treated with restriction enzymes Nhel and Xhol, and then separated and distributed using a Wizard system.
  • Escherichia coli JM109 / pREP8ZAS / GT strain was mass-cultured in LB medium to prepare a linear fragment for gene introduction, and then plasmid was prepared. Further, the obtained plasmid was purified by CsC1 density gradient centrifugation. After digesting 10 g of the purified plasmid with the restriction enzymes XbaI and PstI, this fragment was purified and separated by LMP gel electrophoresis and the Wizard system, and then used as a fragment for AS / GT gene transfer. did. The construction scheme of this fragment is shown in FIG.
  • Plasmid containing plasmid for homologous recombination (pREP 9ZGTZFT) and preparation of gene fragment for transfection (PG-cho / FT)
  • a primer with a restriction enzyme Kpn I cleavage site (referred to as ⁇ / ⁇ ) as a sense primer in exon 3 and an antisense primer A primer (pZB) containing a restriction enzyme Ss ⁇ I cleavage site at the start codon site in exon 4 and a restriction enzyme HindIII cleavage site downstream thereof was synthesized.
  • the PCR method was performed using these primers p / A and p / B, and the obtained fragment was cut with restriction enzymes KpnI and HindIII, purified and separated, and this was separated into GT3- Called 4.
  • the expression vector (pREP 9 Unvitrogen) is cut with the restriction enzyme BamHI, the end is blunted with T4 DNA polymerase, self-ligated with T4 DNA ligase, and the restriction enzyme BamHI is used.
  • the expression vector from which the cleavage site was deleted was designated as pREP9 / amBamHI.
  • the expression vector was called pREP 9ZABamH I Nom Dr a III. This vector was digested with restriction enzymes KpnI and HindIII, and the plasmid into which the above GT3-4 was inserted was designated as PREP9ZGT3-4.
  • PCR method was performed using these primers, the resulting fragment was digested with restriction enzymes NheI and XhoI, and pREP9 / GT3-4 was digested with restriction enzymes NheI and XhoI. Insert the cut plasmid. This was called pREP 9ZGT 3-4ZFT.
  • pREP 9ZGT 3-4ZFT When this plasmid PREP / GT3-4ZFT was cleaved with restriction enzymes SspI and DraIII, blunt ends were generated.
  • both ends were linked by sdf-ligation, and the FT cDNA expression initiation site was located at the GT expression initiation site.
  • This brassmid was designated as pREP9 / GT3-4 /? C / m3spI / ⁇ railI.
  • these restriction enzymes KpnHindl! SspI, Nhe], DralII, and XhoI were all fragments from exon 3 to exon 4 and the expression site of FTcDNA. It has already been found not to cut fragments.
  • An antisense primer (p / E) was synthesized containing the 3 'side of p01yA of PRE P9 and containing the restriction enzyme BamHI cleavage site, and a sense primer (P / A) And pREP 9 / GT3-4 / FT / AS sp I / AD ra
  • the fragment was purified by cutting with HI and separated, and the obtained fragment was designated as GT3-4 / FTZpolyA (see FIG. 7).
  • the expression vector pREP9 was digested with restriction enzymes KpnI and BamHI, and the above fragment GT3-4 / FT / p01yA was introduced. This is pREP 9ZGT3
  • the exon 4 to exon 6 part of the GT was digested with the restriction enzyme BamHI site for both the sense primer (pZF) and antisense primer (p / G), and extracted by PCR.
  • the fragment obtained by digestion with the restriction enzyme BamHI was inserted into a plasmid pREP9 / GT3—4noFTZpo1yA that had been opened with the restriction enzyme BamHI (BamHI was GT It has already been ascertained that 3-4, FT, po 1 y A and T 4-7 do not cut).
  • the resulting plasmid was designated as PREP 9ZGT3-4 / FTZpolyolAZGT4-6, or simply as pREP9 / GTZFT.
  • the plasmid was subjected to PCR using primers p / A and p / G.
  • the fragment obtained after purification was used as a fragment for introduction into a fertilized egg, and was referred to as GT3-4 / FT / p01yA / GT416, or simply pGTZFT.
  • the transgene fragment for introduction thus prepared has high homology with the gene of the GT chromosome and is expected to undergo homologous recombination.
  • the individual (F o) expresses both GT and FT because F-cho is expressed instead of GT.
  • a valence having only FT can be obtained according to Mendel's law. This is the transgenic animal that has no antigen specific to non-primate mammals but has an antigen specific to higher primates. Since the homology between mouse GT and Busu GT is high, the homologous recombination gene based on mouse GT can be applied to pigs.
  • the solution containing the two fragments for transfection is prepared by adding these transfection fragments to a buffer solution (0.25 mM EDTA, 5m Tris (pH 7.4)), and adding 1.0 gZml each.
  • a buffer solution (0.25 mM EDTA, 5m Tris (pH 7.4)
  • the 'fertilized egg was transplanted to the oviduct of a female adult pigs that estrus. Transplantation was performed by intratubal transplantation after laparotomy under general anesthesia. After tubal transplantation, the animals were bred to term until delivery, and pups were obtained.
  • the fertilized eggs into which the gene has been injected are preferably transplanted immediately.
  • cDNA was synthesized from total RNA by reverse transcriptase, and PCR was performed using the primer used for FT gene extraction described above. As a result, one of 27 animals was determined to be 13 ⁇ 4. That is, it was confirmed that the introduced FT gene was translated into mRNA and produced an FT enzyme protein. A similar search was performed on 15 control pigs into which no gene had been introduced, but all were negative.
  • Example 6 It was determined to be positive in Example 6, that is, it is considered to be producing FT protein Blood was collected from the pupa pupa, and after the heart, blood cells were separated and swine blood cells were stained by the FITC method using a fluorescent antibody. On the other hand, blood was also collected from control pigs (producers without transgenes) and stained similarly.
  • an anti-H-type antibody was used as a primary antibody, and mouse 7 globulin (F (ab) 2 ) labeled with a fluorescent dye was used as a secondary antibody.
  • F (ab) 2 mouse 7 globulin labeled with a fluorescent dye
  • the so-called blood type is converted, and as a result, the same H-type substance as the human type 0 is produced. It was shown that the antibody did not form an antigen-antibody reaction with the natural antibody, or that the formation of the antigen-antibody reaction was infrequent.
  • fragment A fragment A (fragnientA) is described below with reference to Fig. 10.
  • fragment A was cleaved with various restriction enzymes including the restriction enzyme Sphl, and it was found that there was 1 Sf of the restriction enzyme Sphl cleavage site at about 4.81 cbp upstream of exon 3. Then, the plasmid A was cleaved with restriction enzymes Spill and Sail to obtain a fragment of about Slcbp. After purification and separation, the fragment was blunt-ended using T4 DNA polymerase (Takara Shuzo). This was called fragment A (fr3 ⁇ 4ineiitA).
  • fragment B (irftgm e nt B)
  • Brasmid J> GEM-T Promega
  • Brasmid B piumid B
  • fragment B (plasmid B) was found to be restricted to the cloning site on the 5 'side of the plasmid pGEM-T (Promega). The fragment was cut with NcoI to obtain a fragment of about 3. bp. After this was isolated from the manufacturer, the above fragment was blunt-ended using T4 DNA polymerase (Takara Shuzo). To this fragment was added Sac linker (New Eflland Bioiab nc.), which was filled with nucleotides. This was called fragment B (fragmem B).
  • this fragment (fra ment ed. Incorporated into an expression vector pCR3 (Invitrogen, Inc.), was designated At the bra corner de C (plasmid C) t
  • the above expression vector The only restriction enzyme ⁇ 11 is present at nucleotide position 1113 of pCR3, that is, immediately downstream of the poiy A site of pCR3.
  • This restriction enzyme is a fragment C containing FT (frtgment C ) Is cut off. Therefore, plasmid C is restricted with restriction enzyme C. Cut, this before
  • Brasmid E (piasmidE) is described below with reference to FIG.
  • the plasmid E was cut with the restriction enzyme Sail, blunt-treated, and ligated with the fragment A (ir meiitA) using the DNA ligese (Takara Shuzo).
  • the above plasmid F was cut with restriction enzymes Sai I and Ail III. This resulted in a fragment approximately 10 kbp in length. This was manufactured as described above, and the portability was adjusted to 5.0 ⁇ 8 / ⁇ 1; as a ⁇ gene fragment for introducing g gene, it was referred to as ⁇ / ⁇ .
  • this construct has a mutant sequence inserted in a form that deletes the sequence portion containing the translation initiation code of the .GT gene.
  • this inserted sequence Contains all the translation parts (including the translation start code and translation stop code) that encode the FT Liao gene.
  • the indented sequence is the .FT translation part. 3 'is to include a terminator.
  • the god-introduced array is a translator of GT's ⁇ ⁇ ⁇ It is linked to the untranslated part on the 5 'side of the ⁇ code.
  • Fifth. The length of the part of the inserted sequence is reduced. In other words, the length of the sequence of the construct is almost the same as the length of ⁇ that causes homologous recombination in the existing gene of GT.
  • homologous recombination is relatively likely to occur. If the homologous recombination force occurs, the GT will be expressed due to the lack of the translation start code in the GT igi cloud, and the FT will follow The child is translated and expressed.
  • FCS blood culture medium
  • RPMI RPMI until about 1.0x10 s cells are observed, and this day is the first day.
  • change to a serum-free medium and after about 3 hours The following solutions were added to the culture dishes. That is, a mixed solution was prepared by adding 2 ⁇ l of the gene fragment GT / FT and 7 ⁇ l of the pMAM w (l. ( ⁇ G ⁇ i) to the 7 ⁇ l DMEM solution to the 7 ⁇ l DMEM solution. In the DMEM solution,
  • the medium was replaced with S at 1 O ml, and the culture was further cultured for 7 days.
  • the medium was replaced with 10 ml of the PRMI solution with 1095 FBS and 10 ml of G418.
  • the above-mentioned transduced cells were further cultured.
  • the total NA was extracted.
  • RT-PCR was performed using the above primers and F.
  • three out of the three cell groups were subjected to RT-PCR! It was positive (Fig. 16D).
  • the exogenous gene group and the non-transfected cell group were stained with a lectin (UEA-1; EY bolatones) labeled with 3 ⁇ 4IS, and the expression of the H antigen was determined by flow cytometry. Confirmed ( Figures 16E and F).
  • FIG. 16E indicates that the expression level of H antigen was measured using the lectin UEA-I in a mouse LS29 cell group into which the neomycin-resistant ifi gene was introduced.
  • FIG. 1 shows L929 I FT ⁇ UEA-I of I5F. The degree of H antigen expression was measured using the lectin UEA-I in the mouse L929 cell group into which the exogenous pathogen L-GT / FT was introduced. Indicates that
  • TTGTGCCAQC AGTTTTCTGA ATTT6AAAGA GTATTACTCT GGCTACTTCC 118G
  • CB nucleic acid

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Abstract

On peut atténuer le rejet hyperaigu qui survient dans les cas de transplantation de tissus d'un mammifère autre qu'un primate chez des primates supérieurs en transférant des gènes étrangers d'un primate supérieur, qui expriment une sucre-transférase, chez un mammifère autre qu'un primate, de manière à exprimer des antigènes à chaînes saccharidiques du primate supérieur.
PCT/JP1996/000703 1995-03-17 1996-03-18 Mammiferes transgeniques autres que des primates, ou des serotypes de primates superieurs ont ete exprimes par un transfert de genes etrangers et procede pour creer ceux-ci WO1996028967A1 (fr)

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AU49562/96A AU4956296A (en) 1995-03-17 1996-03-18 Transgenic non-primatal mammals wherein serotypes of higher primates have been expressed by foreign gene transfer and method of creating the same
PCT/JP1996/000703 WO1996028967A1 (fr) 1995-03-17 1996-03-18 Mammiferes transgeniques autres que des primates, ou des serotypes de primates superieurs ont ete exprimes par un transfert de genes etrangers et procede pour creer ceux-ci
JP52828796A JP3809189B2 (ja) 1995-03-17 1996-03-18 外来遺伝子の導入により高等霊長類の抗原型を発現した非霊長哺乳類の形質転換動物及びその作出方法

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PCT/JP1995/000488 WO1996028966A1 (fr) 1995-03-17 1995-03-17 Transformant de mammifere autre que primate et dont un serotype de primate superieur s'exprime par transduction d'un gene etranger, procede de fabrication de ce transformant
JPPCT/JP95/00488 1995-03-17
PCT/JP1996/000703 WO1996028967A1 (fr) 1995-03-17 1996-03-18 Mammiferes transgeniques autres que des primates, ou des serotypes de primates superieurs ont ete exprimes par un transfert de genes etrangers et procede pour creer ceux-ci

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EP0769902A4 (fr) * 1994-06-15 2001-02-28 Alexion Pharma Inc Procedes permettant de reduire le rejet suraigu d'heterogreffes
US6355859B1 (en) 1997-05-20 2002-03-12 Biotechnology Research And Development Corporation Interactions between genotype and diet in swine that prevent E. coli associated intestinal disease
JP2002529509A (ja) * 1998-11-13 2002-09-10 オシリス セラピューティクス，インコーポレイテッド ヒト間葉幹細胞の子宮内移植で胎児を処置し間葉幹細胞を移植し移植器官を調製する方法と調製された雑種器官
WO2002074948A2 (fr) * 2001-03-21 2002-09-26 Geron Corporation Tissu animal comportant des antigenes carbohydrates compatibles en vue d'une greffe humaine
US6965022B2 (en) 1997-05-20 2005-11-15 The United States Of America As Represented By The Secretary Of Agriculture Methods to identify swine genetically resistant to F18 E. coli associated diseases
US7126039B2 (en) 2001-03-21 2006-10-24 Geron Corporation Animal tissue with carbohydrate antigens compatible for human transplantation
US7560538B2 (en) 2003-11-05 2009-07-14 University Of Pittsburgh Porcine isogloboside 3 synthase protein, cDNA, genomic organization, and regulatory region
US7795493B2 (en) 2002-08-21 2010-09-14 Revivicor, Inc. Porcine animals lacking any expression of functional alpha 1, 3 galactosyltransferase
US8106251B2 (en) 2002-08-21 2012-01-31 Revivicor, Inc. Tissue products derived from porcine animals lacking any expression of functional alpha 1,3 galactosyltransferase

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PROC. NATL. ACAD. SCI. U.S.A., Vol. 86, (1989), ROBERT D. LARSEN et al., "Isolation of a cDNA Encoding a Murine UDPgalactose: Beta-D-galactosyl-1,4-N-acetyl-D-glucosaminide Alpha-1,3-galactosyltransferase: Expression Cloning by Gene Transfer", pages 8227-8231. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 87, (1990), ROBERT D. LARSEN et al., "Molecular Cloning, Sequence and Expression of a Human GDP-L-fucose: Beta-D-galactoside 2-alpha-L-fucosyltransferase cDNA that can Form the H Blood Group Antigen", pages 6674-6678. *
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0769902A4 (fr) * 1994-06-15 2001-02-28 Alexion Pharma Inc Procedes permettant de reduire le rejet suraigu d'heterogreffes
US6965022B2 (en) 1997-05-20 2005-11-15 The United States Of America As Represented By The Secretary Of Agriculture Methods to identify swine genetically resistant to F18 E. coli associated diseases
US6355859B1 (en) 1997-05-20 2002-03-12 Biotechnology Research And Development Corporation Interactions between genotype and diet in swine that prevent E. coli associated intestinal disease
JP2002529509A (ja) * 1998-11-13 2002-09-10 オシリス セラピューティクス，インコーポレイテッド ヒト間葉幹細胞の子宮内移植で胎児を処置し間葉幹細胞を移植し移植器官を調製する方法と調製された雑種器官
US7126039B2 (en) 2001-03-21 2006-10-24 Geron Corporation Animal tissue with carbohydrate antigens compatible for human transplantation
WO2002074948A3 (fr) * 2001-03-21 2002-11-14 Geron Corp Tissu animal comportant des antigenes carbohydrates compatibles en vue d'une greffe humaine
WO2002074948A2 (fr) * 2001-03-21 2002-09-26 Geron Corporation Tissu animal comportant des antigenes carbohydrates compatibles en vue d'une greffe humaine
US7795493B2 (en) 2002-08-21 2010-09-14 Revivicor, Inc. Porcine animals lacking any expression of functional alpha 1, 3 galactosyltransferase
US8106251B2 (en) 2002-08-21 2012-01-31 Revivicor, Inc. Tissue products derived from porcine animals lacking any expression of functional alpha 1,3 galactosyltransferase
US10130737B2 (en) 2002-08-21 2018-11-20 Revivicor, Inc. Tissue products derived from animals lacking any expression of functional alpha 1, 3 galactosyltransferase
US10912863B2 (en) 2002-08-21 2021-02-09 Revivicor, Inc. Tissue products derived from animals lacking any expression of functional alpha 1, 3 galactosyltransferase
US11172658B2 (en) 2002-08-21 2021-11-16 Revivicor, Inc. Porcine animals lacking expression of functional alpha 1, 3 galactosyltransferase
US7560538B2 (en) 2003-11-05 2009-07-14 University Of Pittsburgh Porcine isogloboside 3 synthase protein, cDNA, genomic organization, and regulatory region

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