MXPA00003442A - Mammals lacking expression of osteoprotegerin - Google Patents

Abstract

Disclosed is a mammal in which expression of the gene encoding Osteoprotegerin is suppressd. Also disclosed is a nucleic acid construct useful in preparing such a mammal, and a cell line containing such construct.

Description

MSMEs that lack OSTEOPROTEGERIN EXPRESSION BACKGROUND Field of the invention This invention relates to a mammal in which the production of the protein encoded by the endogenous osteoprotegerin gene (OPG) has been completely deleted.

Description of the related state of the art - # et al. Am. J. Path. 145. 827-836 (1994)), and c- "i" src "/ _ and c-fos_" mice (Soriano et al., Cell 64: 693-702 (1991), Wang et al., Nature 360: 741- 745 (1992), Grigoriadis et al., Science 266: 443-448 (1994), Johnson et al., Cell 71: 20 577-586 (1992)) all exhibit osteopetrosis accompanied * • * due to odd tooth eruption and delayed growth. Defects in these genetic mutants are generally associated with decreased bone resorption J. attributable to decreased numbers of osteclasts or Inactive osteoclasts (Yoshida et al., Supra, Wiktor-Jedrzejczak et al., Supra; Graves et al., Supra; Grigoriadis et al., Supra; Boyce et al., J. Clin. Invest. 90: 1622-1627 (1992 ), Lowe et al., Proc. Nati, Acad. Sci.

USA = > J0: 4485-4489 (1993)). In general, the long bones of the characterized osteopetrotic mouse models are shortened in length and the mouse exhibits moderately severe cranial and facial abnormalities. Osteopetrosis in 5 OPG founding transgenic animals was severe in high exprestors, still occurring without shortening of long bones or odd eruption of teeth (Simonet et al., Supra).

It has been established that pharmacological doses of OPG result in an increased bone density. However, it is a necessity to understand the physiological role of OPG in the development and maintenance of bone mass and other metabolic processes. In particular, it is a need to determine if OPG is a physiological regulator of bone mass, and if other factors can compensate to maintain normal bone mass in its absence. In addition, an animal model in which the density of bone is diminished would be valuable to project therapeutic novelties for bone loss diseases.

Accordingly, it is an object of this invention to provide a mammal in which the gene encoding OPG has been deleted. This and other such objects will be readily apparent to one of ordinary skill in the state of the art.

! »^ V BRIEF DESCRIPTION OF THE INVENTION. - i The inv. is related to a mammal in which the expression of the gene encoding OPG is suppressed. It also provides a nucleic acid construct useful in the preparation of such a mammal, and a cell line containing such a construct.

In one embodiment, the present invention provides a mammal. _ comprising the gene encoding OPG, wherein an allele of the gene has been interrupted: In another embodiment, this invention provides a mammal comprising the gene encoding OPG, wherein both alleles of the gene have been interrupted. In still another embodiment, this invention provides a mammal comprising an interrupted OPG mutation, wherein the disruption results in a null mutation of the gene encoding OPG.

Preferably, the mammal is a non-human mammal. More preferably, the mammal is a rodent. Optional, the rodent is a mouse.

In still another embodiment, this invention provides a nucleic acid molecule comprising an OPG knockout construct. Optionally this construct can be inserted into an expression and / or amplification vector, and the vector can be useful for the transformation of a prokaryotic or eukaryotic cell, or an embryo.

In a further embodiment, the present invention provides a cell line of murine origin RW4 comprising an OPG knockout construct.

The suppression of OPG expression results in a phenotype of decreased bone density and increased bone resorption. The OPG blocked mammals described herein provide a method for projection of compounds that modulate bone resorption, and can help identify drugs that treat bone diseases such as osteoporosis.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 represents the preparation of an OPG knockout construction. The restriction sites are indicated as follows: "RI" is echo Pl; "X" is Xmnl; , P "is PstI, the exons are indicated with black boxes, and the introns as thin white lines. (A) represents a fragment of a native OPG gene containing exons 2-5, part of intron 1 and introns 2-4. (B) represents the knockdown construct in which the thymidine kinase (MC1-TK) cartridge and the neomycin cartridge (Neo) have been ligated into the OPG gene (C) Represents the structure of an allele objectified following homologous recombination to the locus OPG The small open box represents 1 ..cr used to project recombinant ES cell clones.

Figure 2 depicts Southern Blot analysis of wild-type digested EcoRI and genomic is an EcoRI fragment of. ~ * and the objectified allele is an EcoRI fragment of 3.2. kb.

Figure 3 illustrates Southern blot analysis of PstI of digested wild type DNA gene (+/-), and heterozygous knockout mice (+/-) - The wild type allele is us fragment c PstI of 2.3. kb and objectified allele is a fragment of 3.0 kb Figure 4A illustrates whole-body X-rays of mice not] u-i < _-OPG d2 2 months of wild type (+/-), and homicigóticos \ - / -).

Figure 4B shows the X-rays of the femurs of the ratc: .-- -OPG - / -, + / + and +/-. The strongest phenotype joins in # 1-38. The vertical is thinner, the place of growth is not visible. mice +/- are not different from mice + / +.

Figure 5B illustrates bone morphology in OPG - / -; € mouse against a normal control (# 45), H and E, 500 microns bar of such normal morphology observed; Wet B - OPG - / - (# 38), Hy E, size bar microns, severe mechanical osteoporosis of the articular surface; C - control vertebra n "> _ (# 45), H and E, 500 microns of size bar, observe mopo-normal; D - vertebra OPG - / - (# 38), H and E, 500 microns of size bar, observe severe osteoporosis and degenerative damage of the intervertebral disc; E - normal control humerus (# 45), H and E, 100 micron bar size, observe normal morphology; F - humerus OPG - / - (# 38), H and E, 100 microns of size bar, observe severe osteoporosis, increased porosity in the cortical bone; g - normal control vertebra (# 45), TRAP stain, 25 microns bar size, observe normal number of osteoclasts (marrow); H - vertebra OPG - / - (# 38), spot TRAP, 25 microns bar size, observe slightly increased osteoclast number (marrow).

DETAILED DESCRIPTION OF THE INVENTION The term "knocked-out" refers to parsial reduction or co-expression of the expression of al méñós "un pó pó * có" ñ dé ün polypeptide encoded by an endogenous gene (such as OPG) of a single cell, selected cells, or the total d3 cells of a mammal. The mammal may be a "heterozygous knockout", where an allele of the endogenous gene has been disrupted. Alternatively, the mammal may be a "homozygous knockout" where both alleles of the endogenous gene have been disrupted.

The term "knockdown construct" refers to a nucleotide sequence that is designed to decrease or suppress the expression of a polypeptide encoded by an endogenous gene in one or more mammalian cells. The nucleotide sequence used as the knockout construct is typically comprised of (1) DNA from some portion of the endogenous gene (one or more exon sequences, introns sequences, and / or promoter sequences) to be deleted and (2) a sequence marker used to detect the presence of a knockout construct in the cell. The knockout construct is inserted into a cell that contains the endogenous gene to be knocked out. The knockout construct can then be integrated into one or more alleles of the endogenous OPG gene, and such integration of the OPG knockout construct can interrupt or prevent transcription of the full-length endogenous OPG gene. The integration of knockout constructs into cellular chromosomal DNA is typically accomplished by means of homologous recombination (ie, the regions of the OPG knockout constructs that are homologous or complementary to endogenous OPG DNA sequences can hybridize to each other when the construction noqueadora is inserted inside the cell; these regions can then recombine so that the knockout construct is incorporated into corresponding positions of the endogenous DNA).

Typically, the knockout construct is inserted into an undifferentiated cell belonging to an embryonic cell lineage (ES cell). ES cells are usually derived from an embryo or blastocyst of the same species in which the embryo develops within which it can be introduced, as discussed above.

The phrases "gene alteration", "gene alteration", "suppressing expression", and "gene deletion" refer to insertions of an OPG knockout nucleotide sequence (usually containing one or more exons) and / or the promoter region of this gene in order to decrease and prevent the expression of the full-length OPG molecule in the cell. The insertion is usually carried out by homologous recombination. By way of example, a "nucleotide knockout" sequence can be prepared by inserting a nucleotide sequence comprising an antibiotic resistance gene into a portion of an isolated nucleotide sequence that encodes OPG that is to be altered. The knockout construct is then inserted into an embryonic lineage cell ("ES cell"), the construct can integrate into the genomic DNA of at least one OPG allele, thus, many progeny of the cell will not express longer OPG at least in some cells, or will express this at a decreased level and / or in a truncated form, since at least part of the endogenous coding region of OPG is now altered by an antibiotic resistance gene.

The term "marker sequence" refers to a nucleotide sequence that is (1) used as part of a longer construction nucleotide sequence (ie, the "knockout construct") to interrupt the expression of OPG, and (2) used as a means to identify those cells that have the OPG knockout construction incorporated into the chromosomal DNA. The marker sequence can be any sequence that serves these purposes, although typically it will be a sequence encoding a protein that confers a detectable trait in the cell, such as a penicillin resistance gene or a testable enzyme not naturally found in the cell. The marker sequence will also typically contain a homologous or heterologous promoter that regulates its expression.

The term "rodent" and "rodents" refers to all members of the phylogenetic order Rodentia including any and all procjenie of all future generations derived from it.

The term "murine" refers to any and all members of the muridae family, including without limitation, rats and mice. The term "progeny" refers to any and all future generations derived or descended from a particular mammal, i.e., a mammal containing one or more knockout constructs inserted into its genomic DNA, if the mammal is heterozygous. u homozygous for the knockout construction. The progeny of any successive generation is included here such that the progeny, the generations Fl, F2, F3 and so on indefinitely containing knockout constructions are included in this definition.

Included within the scope of this invention is a mammal in which one or both OPG alleles, as well as one or both alleles of another gene (s) have been knocked out. Such a mammal can be generated by repeating the procedures reported herein for generation of an OPG knockout mammal but using another gene, or by breeding two mammals, one with one or both knockout OPG alleles, and one with one or both alleles of a second knockout gene, for another, and projection for that offspring that has the double knockout genotype (either a double heterozygous knockout genotype or a homozygous double, or a variation thereof).

Also included within the scope of this invention is a mammal in which 1) one or both OPG alleles have been knocked out, and optionally one or both alleles of another gene (s), and 2) one or more transgenes have been inserted ( ie, sequence of exogenous ADβ (s) encoding a polypeptide (s) that may or may not be occurring naturally in the mammal).

Knockout technology 1. Isolation of the OPG Gene An OPG knockdown construct is typically prepared by isolating a portion of the OPG nucleotide sequence from cDNA or genomic DNA (usually encoding at least one exon and one intron), and inserting a marker sequence into the OPG sequence. The OPG gene or gene fragment to be used in the preparation of this construct can be obtained in a variety of ways. Generally, the OPG DNA molecule will be at least about 1 kilobase (kb) in length, and will preferably be 3-4 kb in length / thereby providing sufficient complementary sequence for recognition with chromosomal DNA (i.e., homologous recombination) when the knockout construct is introduced into the genomic DNA of the cell ES (discussed below).

A fragment of naturally occurring genomic OPG or cDNA molecule to be used in the preparation of the knockout construct can be obtained using methods well known in the art such as those described by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). Such methods include, for example, PCR amplification of a particular DNA sequence using oligonucleotide primers, or projection of genomic library of cells and tissues containing the OPG gene with a cDNA probe encoding at least a portion thereof or an OPG gene. highly homologous in order to obtain at least a portion of the genomic sequence of OPG. Alternatively, if a cDNA sequence is to be used in a knockdown construct, the cDNA can be obtained by projection of a cDNA genomic library (preferably one prepared from tissues or expressing OPG, where the tissues or cells are derived therefrom). mammalian species or similar to those to be produced by the knocked out mammal *.) with oligonucleotide probes, homologous cDNA probes, or antibodies (where the library is cloned into an expression vector). If a promoter sequence is to be used in the knockdown construct, synthetic DNA probes or primers may be designed to project a genomic library or for amplification using PCR, respectively.

Where the DNA sequence of the OPOG is known, a DNA fragment encoding the desired portion of such a gene can be manufactured synthetically, using chemical synthesis methods such as those described by Engels et al., (Angew, Chem. Int. Ed. Engl., 28_: 716-734 ((1989)) .These methods include methods of synthesizing nucleic acid inter alia, phosphodiester, phosphoramidite, and H-phosphonate.Typically, the fragment of genomic DNA to be prepared will be several. Hundreds of base pairs in length Since the chemical synthesis methods stated here can be used to make nucleic acid sequences above about 100 base pairs, the native genomic AD can be synthesized in fragments of 100 pairs of bases which can then be linked together using standard AD ligation methods ?.

The fragment of AD? OPG genomic or cAD molecule? prepared for use in the knockout construction must be generated in sufficient quantity for genetic manipulation. Amplification can be driven by 1) placing the fragment within a suitable vector and transforming bacterial cells or other cells that can amplify the vector rapidly, 2) by PCR amplification, 3) by synthesis with an AD synthesizer, or 4) by other suitable methods. 2. Preparation of an OPG opener construction The fragment of AD? OPG genomic, cDNA molecule, or PCR fragment to be used in the manufacture of the OPG knockdown construct can be digested with one or more restriction enzymes selected for short to a location (s) such that a second molecule of AD? encoding a marker gene can be inserted into the position itself within the OPG genomic DNA fragment, cDNA molecule, or PCR fragment to be used in the construction. The proper position for the insertion of the marker gene is one that will serve to diminish or prevent the transcription and / or expression of the entire length of the endogenous OPG gene. This position will depend on several factors such as the restriction sites available in the sequence to be cut, whether an exon sequence or a promoter sequence, or both are to be interrupted, and whether there are many isoforms of OPG in the mammal (due to alternative splicing) and only one such isoform is to be interrupted. Preferably, the enzyme (s) selected to count the OPG genomic DNA, cDNA molecule, or PCR fragment will generate a longer arm and a shorter arm, where the shorter arm is at least about 300 base pairs ( bp). In some cases it will be desirable to currently erase a portion or even all of one or more introns or exons of this native genomic or cDNA molecule. In these cases the OPG genomic DNA, cDNA molecule, or PCR fragment can be cut with appropriate restriction endonucleases such that a fragment of one's own size and own location can be removed. The marker gene used in the knockdown construct can be any nucleic acid molecule that is detectable and / or assayable after it has been incorporated into the genomic DNA of the ES cell, and ultimately the knockout mammal, however, typically this is an antibiotic resistance gene or another gene whose expression or presence in the genome can be easily detected. Preferably, the marker gene encodes a polypeptide that does not occur naturally in the mammal. The marker gene is usually operably linked to its own promoter or to another strong promoter such as the thymidine kinase (TK) promoter or the phosphoglycerol kinase (PGK) promoter from any source that will be activated or can be easily activated in the cell within which this inserted; however, the marker gene does not need to have its own label attached, such that it can be transcribed using the promoter of the gene to be knocked out. In addition, the marker gene will normally have a polyA sequence attached to its firL_ 3 '; this sequence serves to determine the transcription of the marker gene. Preferred marker genes are any antibiotic resistance gene such as neo (the neomycin resistance gene) and beta-gal (beta-galactosidase).

After the OPG genomic DNA fragment, cDNA molecule, or PCR fragment has been digested with the appropriate restriction enzyme (s), the marker gene molecule can be ligated with the native genomic DNA or cDNA molecule using well known methods for the skilled artisan and described in Sambrook et al., supra. In some cases, it will be preferred to insert the reversing marker sequence or orientation in contra-sense cpn to the OPG nucleic acid sequence; this reverse insertion is preferred where the marker gene is operably linked to a particularly strong promoter. The ends of the DNA molecules to be ligated must be compatible; This can be achieved both by cote of all the fragments with those enzymes that generate compatible ends, as by blunting the ends prior to ligation. The blunting can be done using methods known in the state of the art, as for example by using the Klenow fragment (DNA polymerase I) to fill the sticky ends. After ligation, ligated constructs can be protected by digestion of the endonuclease by selective restriction to determine which constructs contain the marker sequence in the desired orientation.

The knockdown construction of ligated DNA can be transfected directly into embryonic stem cells (discussed below), or it may first be placed in an appropriate vector for its amplification prior to its insertion. Preferred vectors are those that are rapidly amplified in bacterial cells such as pBluescript II SK vector (Stratagene, San Diego, CA) or pGEM7 (Promega Corp., Madison, Wl). 3. Transfection of Embryonic Stem Cells The OPG knockout construct is typically transfected into stem cells derived from an embryo (embryonic stem cells, or "ES cells"). ES cells are undifferentiated cells that are capable of taking extrachromosomal DNA and incorporating it into its chromosomal DNA. Generally, the ES cells used to produce the knockout mammal will be of the same species as the knockout mammal to be generated. For example, mouse embryonic stem cells will usually be used for the generation of knockout mice.

The line of embryonic stem cells used is typically selected for its ability to integrate and form part of the germline of a developing embryo as well as to create the germline of transmission of the knockout construct. Thus, any ES cell line that is believed to have this capability is appropriate for use here. Preferred cell lines ES for generation of knockout mice are murine D3 and E14 cell lines (American Type Culture Collection, 12301 Parkiawn Drive, Rockville, MD 20852-1776 USA, catalog numbers CRL 1934 and CRL 1821, respectively), or RW4 (Genome Systems, Inc., 8620 Pennell Drive, St. Louis, Mi 63114 USA, Catalog number ESVJ-1182). Cells are cultured and prepared for DNA insertion using methods well known to the skilled artisan such as those mentioned by Robertson (in: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E, J. Robertson, ed. IRL Press, Washington, DC. (1987)), by Bradley et al. (Current Topics in Devel. Biol., 20: 357-371 (1986)) and by Hogan et al. (Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1986)).

The insertion (also called "transfection") of the knockdown construct in ES cells can be completed using a variety of methods well known in the art including, for example, electroporation, microinjection, and calcium phosphate treatment (see Lovell -Badge, in Robertson, ed., Supra). A preferred method of insertion is electroporation.

The DNA knockout molecules constructed OPG to be transfected into the cells can first be linearized if the knockout construct has previously been inserted into a circular vector. The linearization can be completed by digesting the DNA with an appropriate restriction endonuclease selected to cut only within the vector sequence and not within the knockout sequence constructed. The isolated OPG knockout DNA can be added to ES cells under conditions appropriate to the chosen insertion method. Where more than one construction will be introduced into the ES cells, the DNA molecules that code for each construction can be introduced simultaneously or sequentially. Optionally, homozygous OPG knockout ES cells can be generated by adding excess OPG knockout DNA to the cells, or by carrying out successive rounds of transfection in an attempt to achieve homologous recombination of the knockdown construct in both endogenous OPG alleles.

If the ES cells are going to be electroporated, the ES cells and the knockout DNA constructed are exposed to an electrical pulse using an electroporation machine and following the manufacturer's instructions for use. After electroporation, cells are typically allowed to recover under appropriate incubation conditions. The cells are then protected by the presence of the knockout construct.

The protection of the ES cells can be achieved using a variety of methods, but typically, one protects for the presence of the portion of the marker sequence of the knockout construct. Where the marker gene is an antibiotic resistance gene, the cells can be cultured in the presence of an otherwise lethal concentration of antibiotic. Those cells that survive presumably have integrated the knockout construction. If the marker gene is other than an antibiotic resistance gene, a Southern blot of the ES cell genomic DNA can be applied with a DNA sequence designed to hybridize only the marker sequence. If the marker gene is a gene encoding an enzyme whose activity can be detected (e.g., beta-galactosidase), the substrate of the enzyme can be added to the cells under appropriate conditions, and the enzymatic activity of the marker gene can be analyzed.

The knockout construct can be integrated into several locations in the ES cell genome, and can be integrated in a different location in each cell genome, due to the occurrence of random insertion events; the desired location of the insertion is within the endogenous genetic sequence OPG. Typically, less than about 1-10 percent of the ES cells that incorporate the knockout construction will in fact integrate the knockout construct at the desired location. To identify those cells with proper integration of the knockdown construct, the chromosomal DNA can be extracted from the cells using standard methods such as those described by Sambrook et al., Supra. To this DNA then a Southern blot can be applied with a probe or probes designed to hybridize the constructed knockout DNA digested with (a) particular restriction enzyme (s). Alternatively, or additionally, a genomic DNA sequence can be amplified by PCR with probes specifically designed to amplify that DNA sequence such that only those cells that contain the knockdown construct in the appropriate position will generate DNA fragments of the appropriate size. . 4. Incorporation / Implantation of Embryonic ES Cells After the appropriate ES cells containing the knockout construction in the proper location have been identified, the cells can be incorporated into an embryo. Incorporation can be achieved in a variety of ways. A preferred method of incorporation of ES cells is by microinjection into an embryo that is in the blastocyst development stage. For microinjection, about 10-30 cells are collected in a micropipette and injected into a blastocyst to integrate the ES cell into the developing blastocyst.

The appropriate stage of development for the blastocyst is dependent on the species, however for mice it is about 3.5 days. Blastocysts can be obtained by perfusing the uterus of pregnant females. Appropriate methods for accomplishing this are known to the skilled artisan, and are mentioned for example by Bradley (in Robertson, ed., Supra).

While any blastocyst of the appropriate age / stage of development is appropriate for use, the preferred blastocysts are males and have genes that code for a hair color or other phenotypic marker that is different from the hair color or another phenotypic marker encoded by the animals. ES cell genes. In this way, progeny can be easily protected from the presence of the knockout construct by observing the mosaic hair color of another phenotypic marker (indicating that the ES cell was incorporated into the developing embryo). Thus, for example, if the ES cell line transports the gene for white hair, the selected embryo will preferably carry genes for black or brown hair.

An alternative method of preparing an embryo containing ES cells that possess the knockdown construct is to generate "aggregation chimeras". A morula at the appropriate stage of development (about 2 days of age for mice) is isolated. The zona pellucida can be removed by treating the morula with a mild acid solution for about 30 seconds, thereby exposing the "group" of cells comprising the morula. Certain types of ES cells such as the Rl cell line for mice can then be cultured in conjunction with the morula cells, forming an embryo morula aggregation chimera and ES cells.

A refinement of the chimera aggregation embryo method can be used to generate an embryo comprised essentially of only those ES cells containing the knockdown construct. In this technique a very early-stage zygote (e.g., a zygote in the two-cell stage for mice) is given a mild electric shock. This shock serves to fuse the nucleus of the cells in the zygote and therefore generates a single nucleus that has doubled (or more) the DNA of a zygote that occurs naturally from the same stage of development. These zygotic cells are excluded from the embryo in appropriate development, and contribute only to the formation of accessory structures to the embryo such as the extra embryonal membrane. Therefore, when ES cells are cultured in conjunction with zygotic cells, the developing embryo is comprised exclusively of ES cells.

After the ES cells have been incorporated, the transfected aggregation chimera or embryo can be implanted into the uterus of a stepmother fa u? ta ci-ici duci. even any stepmother can be iJ.i-.iii._, < j .. < j., iü? Preferred individuals are particularly successful in their ability to procreate and reproduce well, and in their ability to care for their offspring. Such stepmothers are typically prepared by cross-linking with vasectomized males of the same species. The stage of pseudo pregnancy of the stepmother is important for successful implantation, and is dependent on the species. For mice, this stage is about 2-3 days of pseudo pregnancy.

. OPG Knockout Protection Progeny born to the stepmother can be initially protected from mosaic hair color or another phenotypic marker where the phenotype selection strategy (as the hair color, as described above) has been used. In addition, or as an alternative, the chromosomal DNA obtained from the tail tissue of the progeny can be protected from the presence of the knockdown construct using Southern blots and / or PCR as described above. Progeny that are positive for OPG knockout construction will typically be heterozygous, although some homozygous knockers may exist, and can typically be detected by visual quantification of the amount of probe that hybridizes to Southern blots.

• If homozygous knock-out mammals are desired, they can be prepared by crossing that heterozygous progeny that is believed to transport the knockout construct in its germline to one another; such crosslinks can generate homozygous knockout animals. If it is not clear if the progeny will have germline transmission, they can be crossed with an animal of their own offspring or another chain and the progeny protected from heterozygosity. Homozygotes can be identified by applying the Southern blot to equivalent amounts of genomic DNA from mammals that are the product of this cross, as well as mammals of the same species that are known heterozygotes, and wild-type mammals. Probes to protect the Southern blots from the presence of the knockout construct in the genomic DNA can be designated as mentioned above.

Other means of identification and characterization of the killing progeny are also available. For example, Northern blots can be used to probe mRNA obtained from various tissues of the progeny from the presence or absence of coding transcripts of either the deleted gene, the marker gene, or both. In addition, Western blots can be used to assess the level of expression of the deleted gene in various tissues of this progeny by applying the Western blot with an antibody against the protein encoded by the deleted gene, or an antibody against the product. of the marker gene, where this gene is expressed. Finally in situ analysis (such as fixing cells and labeling them with an antibody) and / or FACS analysis (cell sorting by activated fluorescence) of several cells of the progeny can be carried out using appropriate antibodies to look for the presence or absence of the product of the knockdown construction gene.

Both the heterozygous knockout mammals and the OPG homozygotes of this invention will have a variety of uses, since OPG has been implicated in the regulation of osteoclast differentiation and activation. One such use will be to use the mammal as in an in vivo protection system for drugs that affect bone resorption. It is known that osteoclast numbers increase in certain bone diseases, as well as in response to certain cytokines or hormones (i.e., interleukin-1 (IL-1) or parathyroid hormone (PTH)) that stimulate bone resorption. In addition, certain diseases such as osteoporosis generally result in an imbalance between bone resorption and bone formation. As such, the described mammals can be used to protect from drugs useful for the alteration of the number and / or activity of osteoclasts, i.e., drugs that either increase or inhibit these activities, depending on the disease under study.

The protection of such useful drugs typically involves the administration of the candidate drug over a range of doses to the mammal, and testing in several plinths of the drug itself (s) the density of the drug in the disease being evaluated. Such assays would include, for example, search for increased or decreased numbers of osteocytes, increased or decreased bone resorption, production and / or increased or decreased bone density, levels and / or increased or decreased activity of chemical messengers such as interleukins, and / or increased or decreased expression levels of a gene (s) in particular involved in the modulation of bone density.

For example, patients with osteoporosis often experience bone fractures. It would be desirable to block the bone resorption mediated by osteoclasts in such individuals. If a mammal of the present invention could be used to protect a variety of compounds, since a mammal of the present invention could be used to protect a variety of compounds, either alone or in combination, to determine whether partial or total inhibition of bone resorption mediated by osteoclasts results from the use of such a drug.

The same strategy could be applied to find compounds that would be useful in the suppression of bone resorption in patients with other bone loss diseases, such as hypercalcemia of malignancy or in patients with alterations in bone remodeling, such as Paget's disease.

In addition, a mammal of the present invention can be useful for the evaluation of the development and function of various components of the skeletal system, and for the study of the effects of mutations of a particular gene. For example, in a mammal that does not express OPG, one can analyze the effect of the lack of such expression on other components of the skeletal system.

Other uses of the described mammals and compounds may be readily apparent to someone skilled in the art.

The invention will be understood more fully by reference to the following examples. These examples are not to be construed in any way as limiting the scope of this invention.

EXAMPLES Example 1: Preparation of an OPG knockdown construct To obtain clones of murine OPG genomic DNA, a genomic library of Mouse 129 SVJ was protected in Lambda Fix II Vector (Stratagene, Inc., 11011 North Torrey Pines Road, La Jolla, CA 92037 , Catalog number 946309) with a radiolabeled DNA fragment corresponding to nucleotides 90-1296 of the murine OPG cDNA (Genbank accession No. U94331). Eleven clones were obtained when the library was protected at a severity of about 55 ° C in about 40 mM sodium phosphate, at pH 7.4. These clones were subsequently subdivided by their protection with cDNA fragments from the 5 'to 3' ends of the coding sequence. The 5 'clones were analyzed by Southern blot analysis after digestion.

The recovery of the cloned sequences and the amplification of plasmids was carried out using LambdaSorb Phage Adsorbent ™ (Promega, Inc., 2800 Woods Hollow Road, Madison, Wl 53711-5399 USA, catalog # A7051) according to the manufacturer's protocol. A clone (fragment 1) was then prepared as a fragment EcoRI and EcoRI was approximately 1.7 kb. A second clone was prepared as an EcoRI / fragment EcoRI (fragment 2) and was about 5.5 kb in length. Fragment 1 contained a portion of intron 1 and most of exon 2 (see Figure 1) and fragment 2 contained the 3 'portion of exon 2, intron 2, exon 3, and most of intron 3 (see Figure 1) . A third clone prepared as a 1.1 kb Xmnl / Xmnl fragment (fragment 3) was derived from a subfragment of fragment 1 (see Figure 1). Fragments 2 and 3, together with a neo-cartridge containing a PGK (phosphoglycerate kinase) promoter derived from the pKJ-1 vector (Tybutewicz et al., Cell, 65: 1153-1163 (1991); Adra et al., Gene, 60 : 65-74 (1987)) and a TK cartridge (thymidine kinase gene with a PGK promoter; Tybutewicz et al., Supra) were directionally cloned, using standard ligation techniques, within the pBluescript vector (Stratagene, La Jolla, CA ) to generate a knockdown construct containing, from 5 'to 3', genomic fragment 3 OPG, and TK (see Figure 1). Both the TK cartridge and the neo cartridge were linked in the opposite direction. To confirm the proper ligation, the cloning junctions were sequenced.

This vector, containing all components in the proper orientation, was linearized with Notl and then electroporated into the embryonic stem cells RW4 as follows: about 25 μg of linearized DNA were added to about 9 x 106 ES cells in one volume of about 900 μl of PBS. The cells were pulsed at about 0.23 kilovolts and about 500 μF, and each cell bottle was then placed in two 60 mm cell culture dishes with feeder cells. The dishes contained about 10 ml of DMEM medium (Glbco / BRL, Grand Island, NY), 15 percent fetal calf serum (Gibco / BRL, Grand Island, NY or the equivalent of Hyclone Labs, Logan, UT), and leukemia inhibitory factor (Fung-Leung et al., Cell, 65: 443-449 (1991)), 105 M of B-mercaptoethanol, 2 mM of L-glutamine, and 1 mM of sodium pyruvate. After two days in culture, the cells were selected in the presence of gangciclovir and G418 to enrich the cells which had undergone homologous recombination (Cappecchi, Science, 244: 1288 [1989]; Shahinian et al., Science, 261: 609 (1993)); the surviving cells were harvested, and subsequently cultured in a medium containing G418 but not gangciclovir. To confirm homologous recombination, the cells that grew in the presence of G418 were then protected by Southern blot analysis using the genomic DNA prepared from the cells and cut with EcoRI (see Figure 2).

Samples of RW4 cells that have undergone homologous recombination to incorporate the OPG knockout construct into their genomic DNA have been deposited with the American Type Culture Collection ("ATCC", 12301 Parkiawn Drive, Rockville MD 20852, USA) as access number CRL -12418, with a deposit date of October 9, 1997.

Example 2: Preparation of OPG Knockout Mice The RW4 cells containing the OPG knockout construct were inserted into fertilized embryos (blastocysts) that were approximately 3.5 days old, which were obtained from C57BL / 6 mice by perfusing the uterus of C57BL female mice. 6 that had been coupled with male mice. The insertion was completed by microinjection of about 15-30 cells into each blastocyst. The embryos were then implanted in female CD1 pseudopregnant mice at day 2.5 post-coitus for gestation. The chimerical progeny males of these stepmothers were protected from agouti hair color and were crossed with C57BL females or Swiss black females. The germline transmission of the knockout construction was determined by the hair color of the offspring Fl; Agouti pups were identified as heterozygous OPG knockouts. These offspring Fl were crossed with one another to generate homozygous F2. The homozygotes (OPG "_) were identified and distinguished from the heterozygous (OPG + ~) and wild type (0PG + +) mice by Southern blot analysis of genomic DNA cut with PstI and probed with the specific OPG EcoRI / Xmnl probe which was used to confirm homologous recombination (see Figure 3).

Example 3: Characterization of Knocked Out Mice OPG The following procedures were used for all the analyzes described below in which the qualitative and / or quantitative phenotypic analyzes of bone and other tissues of OPG knockout mice and controls were carried out. At 8-10 weeks of age, 3 homozygous OPG knockout mice (OPG- / ~), 5 heterozygous OPG knockout mice (OPG + _) and 4 control mice (OPG + / + 1 were autopsied (see Table 1) The radiograph was carried out before the general dissection. The serum of the mice was analyzed for clinical chemistry and complete hematology. The total body and the major organs were weighed and fixed in formalin. The tibias for pQCT measurements were set at 70% ETOH. The density of the bone in the proximal tibial metaphysis and in the tibial cortical stem of the wild type, hectozygotes, and OPG_ ~ mice was determined by quantitative CT search (pQCT) (Stratec, Germany). Two standard bone slices, 0.5 mm thick 1.5 mm proximal end and a 0.5 mm single cut 4 mm from the proximal end of the tibia were used to determine the density of the trabecular bone in the metaphysis and the mineral content and density of the cortical stem respectively. Another bone tissue was decalcified using a formic acid solution, and all sections were stained with H and E. The histochemistry of the enzyme was carried out to determine the expression of tartarate-resistant acid phosphatase (TRAP). Seven additional OPG mice- "between 8 and 16 weeks of age were bled for blood chemistries These mice were designated 1-51, 1-56 / 1-68, 1-74, 1-83, 4-78 and 4-79 .

TABLE 1 Knockout Mice Passed Through Necropsy The 1-26 OPG- ~ mouse was the dwarf of the litter, about half the size of a normal mouse. He became dying and died a little before the scheduled sacrifice, showed the signs of respiratory failure a little before dying. Blood for hematology and blood chemistry was collected immediately after death by cardiac puncture and a regular necropsy was performed.

The 1-38 OPG_ "mouse was placed in a cage with the mouse 1-27 OPG - / - in preparation for the procedures and died within the last hour prior to slaughter, no blood could be collected for its test. the autopsy was carried out in the usual manner and the organs submitted for histology.

The pathological evaluation of the wild-type OPG of 8-10 weeks of age, heterozygous knockout mice and homozygous knockout mice indicated that all three homozygous knockout mice (OPG_ ~) have severe osteoporosis. The OPG_ / ~ mice were X-rayed adjacent to the wild-type and / or heterozygous mice using the same X-ray film, to allow direct comparison of bone density and structure (see Figure 4a). The three mice OPG "varied markedly in size with 27 and 3 being similar in size to wild-type mice and 26 being profoundly dwarf." The thoracic vertebrae of mouse 38 were clearly deviated indicating spinal fracture as the cause of death.

There were several consistent differences between the radiological appearance of the bones in the knockout mice compared to those of wild type and heterozygous mice. The most distinctly different region was the distal femur (see Figure 4b). In the distal femoral metaphysis there was a marked reduction in density that was particularly severe in 38 and 26 and less severe in 27. In particular in clear radionuclide contour of the growth plate seen in the wild type and heterozygous mice was absent in all knockout mice. The bone cortex of the femur appeared thin at 38 and 26 and in all knockout mice the density of the bone cortex was decreased. There was also an apparent flattening of the normally rounded distal end of the femur possibly indicating partial collapse or compression of the distal femoral epiphysis. At the proximal end of the tibia there was a loss of the clearly defined growth plate. In the vertebrae there was also a reduction in bone density relative to wild type mice with a larger reduction in 26 and 38 and lower in 27.

The histological morphology of OPG knockout mice was also assessed. The lumbar vertebrae and the proximal metaphysical region of the humerus were deeply osteoporotic with almost complete absence of trabeculae (see Figure 5A, B, C, D). The cortical stems of the humerus in these bones showed increased cortical porosity with the presence of very active remodeling of the bone cortex as evidenced by several osteoclasts and osteoblasts (Figure 5E, F). Bone cortex in wild type mice had few cavities or vascular channels and showed little evidence of remodeling (Figure 5E) whereas in the OPG_ ~ mice extensive cortical bone porosity was present (Figure 5F).

In the proximal epiphysis of the OPG- ~ mice there was evidence of subchondral bone resorption and collapse of the binding surface with increased remodeling of the trabecular bone (Figure 5B).

The density of trabecular bone was markedly reduced in the metaphysical region (268.5 + 0.16 mg / cm3 versus 443.8 ± 27 mg / cm3 in the heterozygotes and 473.4 ± 30 mg / cm3 in the wild-type group). The mineral content of the bone (0.526 mg / cm3 versus 0.9 ± 0.11 mg / cm3 in the heterozygotes and 0.86 + 0.2 mg / cm3 in the wild type), and the cortical thickness was significantly reduced (to 0.23 ± 0.03 mm for 0.3 ± 0.04 mm in the wild type and 0.32 + 0.03 in the heterozygous group) and the cortical density was markedly reduced but not statistically significant in the tibial cortical stem.

The histomorphometry of the vertebrae and humerus revealed that the volume of trabecular bone in the proximal metaphysis of the humerus is significantly reduced (6.58 ± 1.9% vs. 21.66 ± 8.6% in the wild-type group), and the number of osteoclasts per mm. The perimeter of trabecular bone was significantly increased (4.88 + 1.19 versus 3.2 + 0.25 in the wild-type group). Similar changes in the volume of trabecular bone were recorded in the vertebrae, however the numbers of osteoclasts were similar in the 0PG ~ _ and the wild type mice (Figure 5G and H).

No parameter of hematology was different between the groups OPG + / + and OPG + / "The mouse 27 OPG" was not different from the previous groups, the dwarf # 26 had high hematocrit, white blood cell count, red blood cells , lymphocytes, monocytes and eosinophils due to terminal hemoconcentration. Blood was not available for analysis of # 38.

The majority of the chemical values were normal and similar across the groups. Dwarf # 26 had serum calcium and elevated serum cholesterol levels and severely decreased serum glucose. However, all OPG_ ~ mice, including the seven examined in the additional bleeding had high serum alkaline phosphatase (ALP) levels. In well analysis of the blood chemistry data of the sacrificed OPG knockers and the additional bleeds, the OPG_ / ~ mice had ALP values of 376.3 ± 36.6 IU / 1 versus 115.7 ± 55 IU / 1 in the OPG + "and 116.4 ± 36.6 IU / 1 in the OPG + ~ mice (p < 0.001).

While the present invention has been described in terms of the preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover such equivalent variations which come within the scope of the invention as claimed.

Having described the invention as above, the contents of the following are declared as property:

Claims (8)

CLAIMS í

1. A non-human mammal comprising the gene encoding OPG characterized in that an allele of the gene has been altered. _.

2. A non-human mammal comprising the gene encoding OPG characterized in that both alleles of the gene have been altered.

A non-human mammal comprising an altered OPG mutation characterized in that the alteration results in a null mutation of the gene encoding OPG.

4. The non-human mammal of any of Claims 1, 2 and 3 characterized in that it is a rodent.

5. The non-human mammal of Claim 4 characterized in that it is a mouse.

6. The non-human mammal of any of Claims 1, 2 and 3 characterized in that it has decreased bone density q increased bone resorption. A nucleic acid molecule characterized in that it comprises an OPG knockdown construct. A vector characterized in that it comprises the nucleic acid of claim 7. A line of murine RW4 embryonic stem cells characterized in that it comprises the nucleic acid molecule of claim 8. A method of selecting compounds that modulate bone resorption characterized in that it comprises introducing the compounds into the non-human mammal of any of Claims 1, 2 and 3 and determining the increase or decrease in bone resorption. - MAMMALS THAT LACK EXPRESSION OF OS EOPROTEGERINA SUMMARY OF THE INVENTION The expression of a mammal in which the expression of the gene encoding Osteoprotegerin is suppressed is revealed. Also disclosed is a useful nucleic acid construct in the preparation of such a mammal, and a cell line containing such a construct.