TRANSGENIC MICE CONTAINING TRP GENE DISRUPTIONS

Abstract: The present invention relates to transgenic animals, compositions and methods relating to the characterization of gene function.

Description: TRANSGENIC MICE CONTAINING TRP GENE DISRUPTIONS The present application claims benefit of U.S. Provisional Application 60/161,488, filed October 26, 1999, the entire contents of which are incoφorated herein by reference. Field of the Invention The present invention relates to transgenic animals, compositions and methods relating to the characterization of gene function. Background of the Invention Many polymoφhic trinucleotide repeats have been identified in the human genome. These mutations are produced by heritable, unstable DNA and are termed "dynamic mutations" because of changes in the number of repeat units inherited from generation to generation (Koshy, et al, Brain Pathol, 7:927-42 (1997)). Although these repeats are highly polymoφhic, their number usually does not exceed 40 repeats in normal individuals (Online Mendelian Inheritance in Man, OMIM (TM). Johns Hopkins University, Baltimore, MD. MIM Number: 603279: jlewis .7/14/1999; World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim; Koshy, et al (1997)). In contrast, abnormally expanded trinucleotide repeats have been found to cause disease (OMIM 603279). Expansions causing disease typically contain more than 40 trinucleotide repeats and tracts of 200 or more repeats have been reported (OMIM 603279; Slegtenhorst- Eegdeman, et al, Endocrinology, 139:156-62 (1998)). Four types of trinucleotide repeat expansions have been identified: (1) long cytosine-guanine-guanine (CGG) repeats in the two fragile X syndromes (FRAXA and FRAXE), (2) long cytosine-thymine-guanine (CTG) repeat expansions in myotonic dystrophy, (3) long guanine-adenine-adenine repeat expansions in Friedreich's ataxia and (4) short cytosine-adenine-guanine repeat expansions (CAG) which are implicated in neurodegenerative disorders. (Koshy, et al. (1997)). At least 12 diseases, classified into Type 1 and Type 2 disorders, are caused by trinucleotide expansion mutation, most with neuropsychiatric features (Margolis, et al, Hum Genet., 100:114-122 (1997)). Type 1 disorders are caused by a (CAG) n expansion in an open reading frame, resulting in an expanded glutamine repeat. Type 1 disorders include spinocerebellar ataxia type 1 (SCA1, Orr, et al, Nat Genet, 4:221-6 (1993); SCA2 (Imbert, et al, Nat Genet, 14:285-91 (1996); Pulst, et al, Nat Genet, 14:269-76 (1996); Sanpei, et al, Nat Genet, 14:277-84 (1996)); Machado-Joseph disease (MJD or SCA3, Kawaguchi, et al, Nat Genet, 8:221-8 (1994)); SCA6 (Zhuchenko, et al, Nat Genet, 15:62-9 (1997)); dentatutorubral pallidoluysioan atrophy (DRPLA, Koide, et al, Nat Genet, 6:9-13 (1994)); Huntington's disease (HD, Huntington's Disease Collaborative Research Group, Cell, 72:971-83 (1993)); and spinal I and bulbar muscular atropy (SBMA, La Spada, Nature, 352:77-9 (1991)). Type 2 disorders can be caused by expansions in 5' untranslated (Jacobsen's syndrome, Jones, et al, Nature, 376:145- 9 (1995); fragile X syndrome, Fu, et al, Science, 1992 255:1256-8 (1992)), 3' untranslated (myotonic dystrophy, Brook, et al, Cell, 68:799-808 (1992); Philips, et al, Science, 280:737-41 (1998)) and intronic regions (Fredreich's ataxia, Campuzano, et al, Science, 271: 1423-7 (1996)). The mechanism and timing of the expansion events are poorly understood, however (Bates, et al, Hum Mol Genet., 6:1633-7 (1997)). Diseases that are caused by trinucleotide repeat expansions exhibit a phenomenon called anticipation that cannot be explained by conventional Mendelian genetics (Koshy, et al. (1997)). Anticipation is defined as an increase in the severity of disease with an earlier age of onset of symptoms in successive generations. Anticipation is often influenced by the sex of the transmitting parent, and for most CAG repeat disorders, the disease is more severe when paternally transmitted. The severity and the age of onset of the disease have been correlated with the size of the repeats (Koshy, et al (1997)). Longer expansions result in earlier onset and more severe clinical manifestations. The phenomenon of anticipation has led to the suspicion that instability in the expanded repeat underlies a given disorder (OMIM 603279). The proteins harbouring expanded trinucleotide repeat tracts are unrelated and are widely expressed, with extensively overlapping expression patterns (Bates, et al (1997)). Most are novel with the exception of the androgen receptor and the voltage gated alpha 1A calcium channel, which are mutated in spinal and bulbar muscular atrophy and spinocerebellar ataxia type 6. It is intriguing that CAG repeat proteins are ubiquitously expressed in both peripheral and central nervous tissue but in each neurological disorder only a select population of nerve cells are targeted for degeneration as a consequence of the expanded repeat (Koshy, et al (1997)). The mechanism by which expansion leads to neuronal dysfunction and cell death is unknown (Bates, et al. (1997)). Current thinking is that the presence of a repeat tract confers a gain-of-function onto the involved gene, message or protein. For example, inappropriate interaction of the expanded CUG repeat region of myotonic dytrophy gene (MD) transcripts with CUG-binding proteins has been postulated to titrate-out proteins which normally comprise heterogeneous nuclear ribonucleoprotein particles (Bhagwati, et al, Biochim Biophys Acta, 1317:155-7 (1996); Philips, et al. (1998)). The creation of novel protein-protein interactions or aberrant protein folding, as well as alterations in flanking gene expression and chromatin structure have also been suggested as mechanisms by which trinucleotide expansion may cause disease (Thornton, et al, Nat. Genet., 16:407-9 (1997)). Mouse models for trinucleotide repeat disorders hold great potential and promise for uncovering the molecular basis of these diseases and developing therapeutic interventions. Transgenic mice recapitulate many features of human disease and hence are excellent model systems to study the progression of disease in vivo. Using such mice, it will be possible to model both the pathogenic mechanism and the trinucleotide repeat instability in the mouse (Bates, et al. (1997)). Summary of the Invention The present invention generally relates to transgenic animals, as well as to compositions and methods relating to the characterization of gene function, and more specifically the present invention relates to genes encoding trinucleotide repeat proteins (TRP) such as gene T243. The present invention provides a cell, preferably a stem cell and more preferably an embryonic stem (ES) cell, comprising a disruption in a target DNA sequence encoding a TRP. Preferably, the target DNA sequence is T243. In a preferred embodiment, the stem cell is a murine ES cell. According to one embodiment, the disruption is produced by obtaining sequences homologous to the target DNA sequence and inserting the sequences into a targeting construct. The targeting construct is then introduced into the stem cell to produce a homologous recombinant which results in a disruption in the target DNA sequence. In a more preferred embodiment, the targeting construct is generated using ligation- independent cloning to insert two different fragments of the homologous sequence into a vector having a second polynucleotide sequence, preferably a gene that encodes a positive selection marker such that the second polynucleotide sequence is positioned between the two different homologous sequence fragments in the construct. In one aspect of this embodiment, the homologous sequences may be obtained by: generating two primers complementary to the target; annealing the primers to complementary sequences in a mouse genomic DNA library containing the target region; and amplifying sequences homologous to the target region. The products of the amplification reaction, which have endpoints formed by the primers, are then isolated. Preferably, amplification is by PCR; more preferably, amplification is by long-range PCR. In another embodiment, the vector also includes a gene coding for a screening marker. In a further embodiment, the vector also includes recombinase sites flanking the positive selection marker. The present invention further provides a vertebrate animal, preferably a mouse, having a disruption in a gene encoding a TRP. In one embodiment, the present invention provides a knockout mouse having a non-functional allele for the gene that naturally encodes and expresses a functional TRP. Included within the present invention is a knockout mouse having two nonfunctional alleles for the gene that naturally encodes and expresses functional TRP, and therefore is unable to produce wild type TRP. Preferably, the mouse is produced by injecting or otherwise introducing a stem cell comprising a disrupted gene encoding a TRP, either one described herein, or one available in the art, into a blastocyst. The resulting blastocyst is then injected into a pseudopregnant mouse which subsequently gives birth to a chimeric mouse containing the disrupted gene encoding the TRP in its germ line. A person skilled in the art will recognize that the chimeric mouse can be bred to generate mice with both heterozygous and homozygous disruptions in the gene encoding the TRP. According to one embodiment, the disruption alters at least one of a TRP gene promoter, enhancer, or splice site such that the mouse does not express a functional TRP protein. In another embodiment, the disruption is an insertion, missense, frameshift or deletion mutation. The phenotype of such knockout mice can then be observed. One aspect of the invention is a knockout mouse having a phenotype that includes reduced weight relative to an average normal, wild type adult mouse. Typically, the weight of the knockout mouse is reduced at least about 15%. Another aspect is a knockout mouse with a phenotype that includes decreased length relative to an average normal, wild type adult mouse. Commonly, length is decreased at least about 10%. Yet another aspect of the invention is a knockout mouse having a phenotype that includes a decreased ratio of weight to length relative to a normal, wild type adult mouse. Generally, a decrease of at least about 20% is observed. In another embodiment of the invention, the knockout mouse has a phenotype including cartilage disease. Typically, abnormal cartilage is present and cartilage formation reduced. Another aspect of the invention is a mouse having a phenotype that includes bone disease. Typically, the bone disease includes abnormal bone and reduced bone formation. In one embodiment, the phenotype of the knockout mouse is characterized by chondrodysplasia. In yet another embodiment of the invention, the phenotype of the knockout mouse includes kidney disease. Commonly, kidney malformation is observed. In one embodiment, the phenotype of the knockout mouse includes renal dysplasia. The present invention also provides a method of identifying agents capable of affecting a phenotype of a knockout mouse. According to this method, a putative agent is administered to a knockout mouse. The response of the knockout mouse to the putative agent is then measured and compared to the response of a "normal" or wild type mouse. The invention further provides agents identified according to such methods. In a further embodiment of the invention, a knockout cell is provided in which a target DNA sequence encoding a TRP has been disrupted. According to one embodiment, the disruption inhibits production of wild type TRP. The cell or cell line can be derived from a knockout stem cell, tissue or animal. In a further embodiment, the cell is a stable cell culture. The invention also provides cell lines comprising nucleic acid sequences encoding TRPs. Such cell lines may be capable of expressing such sequences by virtue of operable linkage to a promoter functional in the cell line. Preferably, expression of the sequence encoding the TRP is under the control of an inducible promoter. The present invention further provides novel, previously uncharacterized nucleic acid sequences encoding TRPs. Also provided is a method of identifying agents that interact with a TRP including the steps of contacting the TRP with an agent and detecting an agent/TRP complex. The invention also provides methods for treating bone disease by administering to an appropriate subject an agent capable of affecting a phenotype of a knockout mouse to a subject. Appropriate subjects include, without limitation, mammals, including humans. In one embodiment, the bone disease is chondrodysplasia. The invention also provides methods for ameliorating the symptoms of bone disease, such as shortened bones, abnormal growth plates and reduced vertebrae. Among the agents which may be administered are T243 protein, a fragment thereof, as well as natural and synthetic analogs of T243. Also provided are methods for treating cartilage disease by administering to a subject an agent capable of affecting a phenotype of a knockout mouse. In one embodiment, the cartilage disease is chondrodysplasia. Methods are also provided for ameliorating the symptoms of cartilage disease including large, irregular cartilage islands, short chondrocyte columns and thin irregular cartilage. A method of treating kidney disease is also included within the scope of the invention. According to this method, an effective amount of an agent such as T243 protein, a T243 protein fragment, or a natural or synthetic analog of T243, is administered to a subject. In one embodiment, the kidney disease is renal dyplasia. The invention also includes methods for ameliorating symptoms associated with kidney disease such as small, abnormally formed kidneys. The present invention also provides a method for determining whether expansion of the trinucleotide repeat in a TRP produces a phenotypic change. According to this method, a knockout stem cell in which a positive selection marker, flanked by recombinase sites, is contacted with a synthetic nucleic acid. The synthetic nucleic acid includes trinucleotide repeats flanked by recombinase target sites. In the presence of a recombinase which recognizes the recombinase target sites, recombination occurs between the recombinase sites in the synthetic nucleic acid and those flanking the positive selection marker by enzyme-assisted site-specific integration, thereby producing a transgenic stem cell. The phenotype of the resulting transgenic stem cell can then be compared with a normal, wild type stem cell, to determine whether trinucleotide expansion produces a phenotypic change. Preferably, the synthetic nucleic acid includes at least about 20 trinucleotide repeats. The enzyme-assisted site-specific integration can be, for example, a Cre recombinase-lox target system or an FLP recombinase-FRT target system. The invention also provides a vertebrate, preferably a mouse, having a trinucleotide expansion of a gene encoding a TRP. In one embodiment, the mouse is produced by introducing a transgenic stem cell containing an expanded TRP gene into a blastocyst. The resulting blastocyst is then implanted into a pseudopregnant mouse which subsequently gives birth to a chimeric mouse containing the expanded trinucleotide repeat gene in its germ line. The chimeric mouse can then be bred to generate mice with either heterozygous or homozygous disruption in the gene encoding the TRP. The present invention further provides novel, expanded TRP genes and the proteins encoded by these genes. Also provided is a method of identifying agents which interact with an expanded TRP including the steps of contacting the expanded TRP with an agent and detecting an agent/expanded TRP complex, thereby identifying agents which interact with the expanded TRP. The invention also provides cell lines comprising nucleic acid sequences encoding expanded TRPs that are capable of expressing such sequences through operable linkage to promoters functional in the cell lines. Preferably, expression of the sequence encoding the expanded TRP is under the control of an inducible promoter. As used herein, "gene targeting" is a type of homologous recombination that occurs when a fragment of genomic DNA is introduced into a mammalian cell and that fragment locates and recombines with endogenous homologous sequences. "Disruption" of a target gene occurs when a fragment of genomic DNA locates and recombines with an endogenous homologous sequence such that production of the normal wild type gene product is inhibited. Non-limiting examples of disruption include insertion, missense, frameshift and deletion mutations. Gene targeting can also alter a promoter, enhancer, or splice site of a target gene to cause disruption, and can also involve replacement of a promoter with an exogenous promoter such as an inducible promoter described below. As used herein, a "knockout mouse" is a mouse that contains within its genome a specific gene that has been disrupted or inactivated by the method of gene targeting. A knockout mouse includes both the heterozygote mouse (i.e., one defective allele and one wild-type allele) and the homozygous mutant (i.e., two defective alleles). Also included within the scope of the invention are hemizygous mice. It will be understood that certain genes, such as sex-linked genes in a male, are present in only one copy in the normal, wild type animal (i.e., are hemizygous in the normal wild type animal). A knockout mouse in which a gene which is normally hemizygous is disrupted will have a single defective allele of that gene. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides and/or their analogs. Nucleotides may have any three- dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes single-, double-stranded and triple helical molecules. "Oligonucleotide" refers to polynucleotides of between 5 and about 100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art. A "primer" refers to an oligonucleotide, usually single-stranded, that provides a 3'-hydroxyl end for the initiation of enzyme-mediated nucleic acid synthesis. The following are non-limiting embodiments of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimi dines are known in the art, and include, but are not limited to, aziridinycytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, inosine, N6- isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5-pentylnyluracil and 2,6-diaminopurine. The use of uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine. A "fragment" of a polynucleotide is a polynucleotide comprised of at least 9 contiguous nucleotides, including 10, 11, 12, 13, or 14 contiguous nucleotides, preferably at least 15 contiguous nucleotides and more preferably at least 45 nucleotides, also including at least 60 nucleotides, of coding or non-coding sequences. As used herein, "base pair," also designated "bp," refers to the complementary nucleic acid molecules. In DNA there are four "types" of bases: the purine base adenine (A) is hydrogen bonded with the pyrimidine base thymine (T), and the purine base guanine (G) with the pyrimidine base cytosine (C). Each hydrogen bonded base pair set is also known as a Watson- Crick base-pair. A thousand base pairs is often called a kilobase pair, or kb. A "base pair mismatch" refers to a location in a nucleic acid molecule in which the bases are not complementary Watson-Crick pairs. The phrase "does not include at least one type of base at any position" refers to a nucleotide sequence which does not have one of the four bases at any position. For example, a sequence lacking one nucleotide (i.e., lacking one type of base) could be made up of A, G, T base pairs and contain no C residues. As used herein, the term "construct" refers to an artificially assembled DNA segment to be transferred into a target tissue, cell line or animal, including human. Typically, the construct will include the gene or a sequence of particular interest, a marker gene and appropriate control sequences. The term "plasmid" refers to an autonomous, self-replicating extrachromosomal DNA molecule. In a preferred embodiment, the plasmid construct of the present invention contains a positive selection marker positioned between two flanking regions of the gene of interest. Optionally, the construct can also contain a screening marker, for example, green fluorescent protein (GFP). If present, the screening marker is positioned outside of and some distance away from the flanking regions. The term "polymerase chain reaction" or "PCR" refers to a method of amplifying a DNA base sequence using a heat-stable polymerase such as Taq polymerase, and two oligonucleotide primers; one complementary to the (+)-strand at one end of the sequence to be amplified and the other complementary to the (-)-strand at the other end. Because the newly synthesized DNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce exponential and highly specific amplification of the desired sequence. PCR also can be used to detect the existence of the defined sequence in a DNA sample. "Long-range" refers to PCR conditions which allow amplification of large nucleotides stretches, for example, greater than 1 kb. As used herein, the term "positive selection marker" refers to a gene encoding a product that enables only the cells that carry the gene to survive and/or grow under certain conditions. For example, plant and animal cells that express the introduced neomycin resistance (Neo r ) gene are resistant to the compound G418. Cells that do not carry the Neo r gene marker are killed by G418. Other positive selection markers will be known to those of skill in the art. "Positive-negative selection" refers to the process of selecting cells that carry a DNA insert integrated at a specific targeted location (positive selection) and also selecting against cells that carry a DNA insert integrated at a non-targeted chromosomal site (negative selection). Non- limiting examples of negative selection inserts include the gene encoding thymidine kinase (tk). Genes suitable for positive-negative selection are known in the art, see e.g., U.S. Patent 5,464,764. "Screening marker" or "reporter gene" refers to a gene that encodes a product that can readily be assayed. For example, reporter genes can be used to determine whether a particular DNA construct has been successfully introduced into a cell, organ or tissue. Non-limiting examples of screening markers include genes encoding for green fluorescent protein (GFP) or genes encoding for a modified fluorescent protein. "Negative screening marker" is not to be construed as negative selection marker; a negative selection marker typically kills cells that express it. The term "vector" refers to a DNA molecule that can carry inserted DNA and be peφetuated in a host cell. Vectors are also known as cloning vectors, cloning vehicles or vehicles. The term includes vectors that function primarily for insertion of a nucleic acid molecule into a cell, replication vectors that function primarily for the replication of nucleic acid, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions. In a preferred embodiment, the vector contains sites useful in the methods described herein, for example, the vectors "pDG2" or "pDG4" as described herein. A "host cell" includes an individual cell or cell culture which can be or has been a recipient for vector(s) or for incoφoration of nucleic acid molecules and/or proteins. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in moφhology or in total DNA complement) to the original parent due to natural, accidental, or deliberate mutation. A host cell includes cells transfected with the constructs of the present invention. The term "genomic library" refers to a collection of clones made from a set of randomly generated overlapping DNA fragments representing the genome of an organism. A "cDNA library" (complementary DNA library) is a collection of mRNA molecules present in a cell, tissue, or organism, turned into cDNA molecules with the enzyme reverse transcriptase, then inserted into vectors (other DNA molecules which can continue to replicate after addition of foreign DNA). Exemplary vectors for libraries include bacteriophage (also known as "phage"), which are viruses that infect bacteria, for example lambda phage. The library can then be probed for the specific cDNA (and thus mRNA) of interest. In one embodiment, library systems which combine the high efficiency of a phage vector system with the convenience of a plasmid system (for example, ZAP system from Stratagene, La Jolla, CA) are used in the practice of the present invention. The term "homologous recombination" refers to the exchange of DNA fragments between two DNA molecules or chromatids at the site of homologous nucleotide sequences, i.e., those sequences preferably having at least about 70 percent sequence identity, typically at least about 85 percent identity, and preferably at least about 90 percent identity, alternatively, at least about 95-98 percent identity. Homology and/or percent identity can be determined using a "BLASTN" algorithm, such as BLAST (Basic Local Alignment Search Tool) 2.0, available online at http://www.ncbi.nlm.nih. gov:80/BLAST/, (Basic, Advanced or PSI) and as described in any of Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410. (Medline); Gish, W. & States, D.J. (1993) "Identification of protein coding regions by database similarity search." Nature Genet.3:266-272. (Medline); Madden, T.L., Tatusov, R.L. & Zhang, J. (1996) "Applications of network BLAST server" Meth. Enzymol. 266:131-141. (Medline); Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25:3389-3402. (Medline); and Zhang, J. & Madden, T.L. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation." Genome Res. 7:649- 656. (Medline) It is understood that homologous sequences can accommodate insertions, deletions and substitutions in the nucleotide sequence. Thus, linear sequences of nucleotides can be essentially identical even if some of the nucleotide residues do not precisely correspond or align. As used herein the term "ligation-independent cloning" is used in the conventional sense to refer to incoφoration of a DNA molecule into a vector or chromosome without the use of kinases or ligases. Ligation-independent cloning techniques are described, for instance, in Aslanidis & de Jong, Nucleic Acids Res., 18:6069-74 and U.S. Patent Application Serial No. 07/847,298 (1991). As used herein, the term "target sequence" (alternatively referred to as "target gene sequence" or "target DNA sequence") refers to the nucleic acid molecule with any polynucleotide having a sequence in the general population that is not associated with any disease or discernible phenotype. It is noted that in the general population, wild-type genes may include multiple prevalent versions that contain alterations in sequence relative to each other and yet do not cause a discernible pathological effect. These variations are designated "polymoφhisms" or "allelic variations." In a preferred embodiment, the target DNA sequence comprises a portion of a particular gene or genetic locus in the individual's genomic DNA. Preferably, the target DNA sequence encodes a TRP, preferably having CTG trinucleotide repeats which encode leucine. According to one embodiment, the target DNA comprises part of a particular gene or genetic locus in which the function of the gene product is not known, for example, a gene identified using a partial cDNA sequence such as an EST. In a preferred embodiment, the target TRP gene is T243. Preferably, the target DNA sequence comprises SEQ ID NO:47 (murine) or SEQ ID NO:57 (human), or a naturally occurring allelic variation thereof. The term "exonuclease" refers to an enzyme that cleaves nucleotides sequentially from the free ends of a linear nucleic acid substrate. Exonucleases can be specific for double or single-stranded nucleotides and/or directionally specific, for instance, 3'-5' and/or 5'-3'. Some exonucleases exhibit other enzymatic activities, for example, T4 DNA polymerase is both a polymerase and an active 3'-5' exonuclease. Other exemplary exonucleases include exonuclease III which removes nucleotides one at a time from the 5'-end of duplex DNA which does not have a phosphorylated 3'-end, exonuclease VI which makes oligonucleotides by cleaving nucleotides off of both ends of single-stranded DNA, and exonuclease lambda which removes nucleotides from the 5' end of duplex DNA which have 5'-phosphate groups attached to them. The term "recombinase" encompasses enzymes that induce, mediate or facilitate recombination, and other nucleic acid modifying enzymes that cause, mediate or facilitate the rearrangement of a nucleic acid sequence, or the excision or insertion of a first nucleic acid sequence from or into a second nucleic acid sequence. The "target site" of a recombinase is the nucleic acid sequence or region that is recognized (e.g., specifically binds to) and/or acted upon (excised, cut or induced to recombine) by the recombinase. As used herein, the expression "enzyme-directed site-specific recombination" is intended to include the following three events: 1. deletion of a pre-selected DNA segment flanked by recombinase target sites; 2. inversion of the nucleotide sequence of a pre-selected DNA segment flanked by recombinase target sites; and 3. reciprocal exchange of DNA segments proximate to recombinase target sites located on different DNA molecules. Brief Description of the Drawings Figure 1 is a schematic depicting one method of constructing a targeting vector of the present invention. The plasmid PCR method is described in Examples 9 and 10. Figure 2A is a schematic depicting the pDG2 vector. The vector contains an ampicillin resistance gene and a neomycin (Neo 1 ) gene. On each side of the Neo r gene are two sites for ligation-independent cloning along with restriction sites. The sequence of pDG2 is shown in Figure 2B and SEQ ID NO:l. Figure 3A is schematic depicting the pDG4 vector. The vector contains an ampicillin resistance gene, a neomycin (Neo 1 ) gene and a green fluorescent protein (GFP) gene. On each side of the Neo r gene are two sites for ligation-independent cloning along with restriction enzyme recognition sites. The sequence of pDG4 is shown in Figure 3B and SEQ ID NO:2. Figure 4 (SEQ ID NO:3 through SEQ ID NO: 10) shows the nucleic acid sequence before and after T4 DNA polymerase treatment of annealing sites 1-4 contained on the ends of PCR- amplified genomic DNA. Figure 5 (SEQ ID NO: 11 through SEQ ID NO: 18) shows the nucleic acid sequence before and after T4 DNA polymerase treatment of annealing site 1-4 contained within the pDG2 vector. Figure 6 shows the arrangement of 5' and 3' flanking DNA relative to annealing sites 1, 2, 3 and 4 within the pDG2 vector during an annealing reaction. Figure 7 shows the arrangement of 5' and 3' flanking DNA relative to annealing sites 1, 2, 3 and 4 and the GFP screening marker within the pDG4 vector during an annealing reaction. Figure 8 shows the sequences of the oligonucleotide primers (SEQ ID NO: 19 through SEQ ID NO:44) used in Examples 4 to 10. The lower case sequences are to cloning sites (e.g., ligation-independent cloning sequences). Figure 9 shows length, weight, and weight/length ratios for the progeny of mating #1799 between two heterozygous T243 knockout mice. Figure 10 shows length, weight, and weight/length ratios for the progeny of mating #1808 between two heterozygous T243 knockout mice. Figure 11 shows the nucleic acid sequence (SEQ ID NO:47) encoding a murine TRP (SEQ ID NO:52)(specifically, the expression product of T243); and the nucleic acid sequence (SEQ ID NO:57) encoding a human TRP (SEQ ED NO:58). Figure 12 shows the amino acid sequence of a murine TRP (SEQ ID NO:52) and the amino acid sequence of a human TRP (SEQ ID NO:58). Figure 13 shows the nucleic acid sequences of oligonucleotide primers (SEQ ID NO:45; SEQ ID NO:46) used in PCR amplification of sequences homologous to target gene T243. Further shown are the same primers with cloning sites (SEQ ID NO:48; SEQ ID NO:49); and nucleic acid sequences of primers (SEQ ID NO:55; SEQ ID NO:56) used to identify the aliquot of a library contained in target gene T243. Figure 14 shows the nucleic acid sequences of sequences homologous (SEQ ID NO:50; SEQ ID NO:51) to target gene T243 generated by PCR amplification. Figure 15 shows the nucleic acid sequence of the deleted gene fragment (SEQ ID NO:59) of target gene T243 using a construct comprising homologous sequences (SEQ ID NO: 50; SEQ ID NO:51). Further shown are the nucleic acid sequence of an expanded T243 gene (SEQ ID NO:53) and the amino acid sequence of the corresponding expression product (SEQ ID NO:54). Detailed Description of the Invention The invention is based, in part, on the evaluation of the expression and role of genes and gene expression products, primarily those associated with trinucleotide repeat proteins. Among others, this permits the definition of disease pathways and the identification of targets in the pathway that are useful both diagnostically and therapeutically. For example, genes which are mutated or down-regulated under disease conditions may be involved in causing or exacerbating the disease condition. Treatments directed at up-regulating the activity of such genes or treatments which involve alternate pathways, may ameliorate the disease condition. As used herein, "gene" refers to (a) a gene containing at least one of the DNA sequences disclosed herein; (b) any DNA sequence that encodes the amino acid sequence encoded by the DNA sequences disclosed herein and or; (c) any DNA sequence that hybridizes to the complement of the coding sequences disclosed herein. Preferably, the term includes coding as well as noncoding regions, and preferably includes all sequences necessary for normal gene expression including promoters, enhancers and other regulatory sequences. The invention also includes nucleic acid molecules, preferably DNA molecules, that hybridize to, and are therefore the complements of, the DNA sequences (a) through (c), in the preceding paragraph. Such in vitro hybridization conditions may be highly stringent or less highly stringent. Highly stringent conditions, for example, include hybridization to filter-bound DNA in 0.5M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C, and washing in O.lx SSC/0.1% SDS at 68° C (see Ausubel F. M., et al, eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3; Sambrook, Fritsch, and Maniatis, Molecular Cloning; A Laboratory Manual, Second Edition, Volume 2, Cold Springs Harbor Laboratory, Cold Springs, N.Y., pages 8.46- 8.47 (1995), both of which are herein incoφorated by reference) while less highly stringent conditions, such as moderately stringent conditions, e.g., washing in 0.2 x SSC/0.1% SDS at 42° C (Ausubel, et al, 1989, supra; Sambrook, et al, 1989, supra). In instances wherein the nucleic acid molecules are deoxyoligonucleotides ("oligos"), highly stringent conditions may refer, e.g., to washing in 6x SSC/0.05% sodium pyrophosphate at 37°C (for 14-base oligos), 48°C (for 17-base oligos), 55°C (for 20-base oligos), and 60°C (for 23-base oligos). These nucleic acid molecules may act in vivo as target gene antisense molecules, useful, for example, in target gene regulation and/or as antisense primers in amplification reactions of target gene nucleic acid sequences. Further, such sequences may be used as part of ribozyme and/or triple helix sequences, also useful for target gene regulation. Still further, such molecules may be used as components of diagnostic methods whereby the presence of a disease- causing allele, may be detected. The invention also encompasses (a) DNA vectors that contain any of the foregoing coding sequences and/or their complements (i.e., antisense); (b) DNA expression vectors that contain any of the foregoing coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences; and (c) genetically engineered host cells that contain any of the foregoing coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell. As used herein, regulatory elements include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression. The invention includes fragments of any of the DNA sequences disclosed herein. In addition to the gene sequences described above, homologues of such sequences, as may, for example be present in other species, may be identified and may be readily isolated, without undue experimentation, by molecular biological techniques well known in the art. Further, there may exist genes at other genetic loci within the genome that encode proteins which have extensive homology to one or more domains of such gene products. These genes may also be identified via similar techniques. For example, the isolated differentially expressed gene sequence, or portion thereof, may be labeled and used to screen a cDNA library constructed from mRNA obtained from the organism of interest. Hybridization conditions will be of a lower stringency when the cDNA library was derived from an organism different from the type of organism from which the labeled sequence was derived. Alternatively, the labeled fragment may be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions. Such low stringency conditions will be well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook, et al, 1989, Ausubel, et al, 1989. In cases where the gene identified is the normal, or wild type, gene, this gene may be used to isolate mutant alleles of the gene. Such an isolation is preferable in processes and disorders which are known or suspected to have a genetic basis. Mutant alleles may be isolated from individuals either known or suspected to have a genotype which contributes to disease symptoms. Mutant alleles and mutant allele products may then be utilized in therapeutic and diagnostic assay systems. A cDNA of the mutant gene may be isolated, for example, by using PCR, a technique which is well known to those of skill in the art. In this case, the first cDNA strand may be synthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolated from tissue and known or suspected to be expressed in an individual putatively carrying the mutant allele, and by extending the new strand with reverse transcriptase. The second strand of the cDNA is then synthesized using an oligonucleotide that hybridizes specifically to the 5' end of the normal gene. Using these two primers, the product is then amplified via PCR, cloned into a suitable vector, and subjected to DNA sequence analysis through methods well known to those of skill in the art. By comparing the DNA sequence of the mutant gene to that of the normal gene, the mutation(s) responsible for the loss or alteration of function of the mutant gene product can be ascertained. Alternatively, a genomic or cDNA library can be constructed and screened using DNA or RNA, respectively, from a tissue known to or suspected of expressing the gene of interest in an individual suspected of or known to carry the mutant allele. The normal gene or any suitable fragment thereof may then be labeled and used as a probe to identify the corresponding mutant allele in the library. The clone containing this gene may then be purified through methods routinely practiced in the art, and subjected to sequence analysis. Any technique known in the art may be used to introduce a target gene transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten, et al, Proc. Natl Acad. Sci., USA, 82:6148-6152 (1985)); gene targeting in embryonic stem cells (Thompson, et al, Cell, 56:313-321 (1989)); electroporation of embryos (Lo, Mol Cell. Biol, 3:1803-1814 (1983)); and sperm-mediated gene transfer (Lavitrano, et al, Cell, 57:717-723 (1989)); etc. For a review of such techniques, see Gordon, Transgenic Animals, Intl. Rev. Cytol, 115:171-229 (1989), which is incoφorated by reference herein in its entirety. In a preferred embodiment, homologous recombination is used to generate the knockout mice of the present invention. Preferably, the construct is generated in two steps by (1) amplifying (for example, using long-range PCR) sequences homologous to the target sequence, and (2) inserting another polynucleotide (for example a selectable marker) into the PCR product so that it is flanked by the homologous sequences. Typically, the vector is a plasmid from a plasmid genomic library. The completed construct is also typically a circular plasmid. Thus, as shown in Figure 1, using long-range PCR with "outwardly pointing" oligonucleotides results in a vector into which a selectable marker can easily be inserted, preferably by ligation-independent cloning. The construct can then be introduced into ES cells, where it can disrupt the function of the homologous target sequence. Homologous recombination may also be used to knockout genes in stem cells, and other cell types, which are not totipotent embryonic stem cells. By way of example, stem cells may be myeloid, lymphoid, or neural progenitor and precursor cells. Such knockout cells may be particularly useful in the study of target gene function in individual developmental pathways. Stem cells may be derived from any vertebrate species, such as mouse, rat, dog, cat, pig, rabbit, human, non-human primates and the like. In cells which are not totipotent it may be desirable to knock out both copies of the target using methods which are known in the art. For example, cells comprising homologous recombination at a target locus which have been selected for expression of a positive selection marker (e.g., Neor) and screened for non-random integration, can be further selected for multiple copies of the selectable marker gene by exposure to elevated levels of the selective agent (e.g., G418). The cells are then analyzed for homozygosity at the target locus. Alternatively, a second construct can be generated with a different positive selection marker inserted between the two homologous sequences. The two constructs can be introduced into the cell either sequentially or simultaneously, followed by appropriate selection for each of the positive marker genes. The final cell is screened for homologous recombination of both alleles of the target. In another aspect, two separate fragments of a clone of interest are amplified and inserted into a vector containing a positive selection marker using ligation-independent cloning techniques. In this embodiment, the clone of interest is generally from a phage library and is identified and isolated using PCR techniques. The ligation-independent cloning can be performed in two steps or in a single step. According to a preferred method, constructs are used having multiple sites where 5 '-3' single-stranded regions can be created. These constructs, preferably plasmids, include a vector capable of directional, four-way ligation-independent cloning. The constructs typically include a sequence encoding a positive selection marker such as a gene encoding neomycin resistance; a restriction enzyme site on either side of the positive selection marker and a sequence flanking the restriction enzyme sites which does not contain one of the four base pairs. This configuration allows single-stranded ends to be created in the sequence by digesting the construct with the appropriate restriction enzyme and treating the fragments with a compound having exonuclease activity, for example T4 DNA polymerase. In one preferred embodiment, a construct suitable for introducing targeted mutations into ES cells is prepared directly from a plasmid genomic library. Using long-range PCR with specific primers, a sequence of interest is identified and isolated from the plasmid library in a single step. Following isolation of this sequence, a second polynucleotide that will disrupt the target sequence can be readily inserted between two regions encoding the sequence of interest. Using this direct method a targeted construct can be created in as little as 72 hours. In another embodiment, a targeted construct is prepared after identification of a clone of interest in a phage genomic library as described in detail below. The methods described herein obviate the need for hybridization isolation, restriction mapping and multiple cloning steps. Moreover, the function of any gene can be determined using these methods. For example, a short sequence (e.g., EST) can be used to design oligonucleotide probes. These probes can be used in the direct amplification procedure to create constructs or can be used to screen genomic or cDNA libraries for longer full-length genes. Thus, it is contemplated that any gene can be quickly and efficiently prepared for use in ES cells. In a preferred embodiment, constructs are prepared directly from a plasmid genomic library. The library can be produced by any method known in the art. Preferably, DNA from mouse ES cells is isolated and treated with a restriction endonuclease which cleaves the DNA into fragments. The DNA fragments are then inserted into a vector, for example a bacteriophage or phagemid (e.g., Lamda ZAP™, Stratagene, La Jolla, CA) systems. When the library is created in the ZAP™ system, the DNA fragments are preferably between about 5 and about 20 kilobases. Preferably, the organism(s) from which the libraries are made will have no discernible disease or phenotypic effects. Preferably, the library is a mouse library. This DNA may be obtained from any cell source or body fluid. Non-limiting examples of cell sources available in clinical practice include ES cells, liver, kidney, blood cells, buccal cells, cerviovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy. Body fluids include urine, blood cerebrospinal fluid (CSF), and tissue exudates at the site of infection or inflammation. DNA extracted from the cells or body fluid using any method known in the art. Preferably, the DNA is extracted by adding 5 ml of lysis buffer (10 mM Tris-HCl pH 7.5), 10 mM EDTA (pH 8.0), 10 mM NaCl, 0.5% SDS and 1 mg/ml Proteinase K) to a confluent 100 mm plate of embryonic stem cells. The cells are then incubated at about 60°C for several hours or until fully lysed. Genomic DNA is purified from the lysed cells by several rounds of gentle phenol: chloroform extraction followed by an ethanol precipitation. For convenience, the genomic library can be arrayed into pools. In a preferred embodiment, a sequence of interest is identified from the plasmid library using oligonucleotide primers and long-range PCR. Typically, the primers are outwardly- pointing primers which are designed based on sequence information obtained from a partial gene sequence, e.g., a cDNA or an EST sequence. As depicted for example in Figure 1, the product will be a linear fragment that excludes the region which is located between each primer. PCR conditions found to be suitable are described below in the Examples. It will be understood that optimal PCR conditions can be readily determined by those skilled in the art. (See, e.g., PCR 2: A Practical Approach (1995) eds. M.J. McPherson, B.D. Hames and G.R. Taylor, IRL Press, Oxford; Yu, et al, Methods Mol Bio., 58:335-9 (1996); Munroe, et al, Proc. Nat'l Acad. Sci, USA, 92:2209-13 (1995)). PCR screening of libraries eliminates many of the problems and time-delay associated with conventional hybridization screening in which the library must be plated, filters made, radioactive probes prepared and hybridization conditions established. PCR screening requires only oligonucleotide primers to sequences (genes) of interest. PCR products can be purified by a variety of methods, including but not limited to, microfiltration, dialysis, gel electrophoresis and the like. It may be desirable to remove the polymerase used in PCR so that no new DNA synthesis can occur. Suitable thermostable DNA polymerases are commercially available, for example, Vent™ DNA Polymerase (New England Biolabs), Deep Vent™ DNA Polymerase (new England Biolabs), HotTub™ DNA Polymerase (Amersham), Thermo Sequenase™ (Amersham), rBst™ DNA Polymerase (Epicenter), Pfu™ DNA Polymerase (Stratagene), Amplitaq Gold™ (Perkin Elmer), and Expand™ (Boehringer- Mannheim). To form the completed construct, a sequence which will disrupt the target sequence is inserted into the PCR-amplified product. For example, as described herein, the direct method involves joining the long-range PCR product (i.e., the vector) and one fragment (i.e., a gene encoding a selectable marker). As discussed above, the vector contains two different sequence regions homologous to the target DNA sequence. Preferably, the vector also contains a sequence encoding a selectable marker, such as ampicillin. The vector and fragment are designed so that, when treated to form single stranded ends, they will anneal such that the fragment is positioned between the two different regions of substantial homology to the target gene. Although any method of cloning is suitable, it is preferred that ligation-independent cloning strategies be used to assemble the construct comprising two different homologous regions flanking a selectable marker. Ligation-independent cloning (LIC) is a strategy for the directional cloning of polynucleotides without the use of kinases or ligases. (See, e.g., Aslanidis et al., Nucleic Acids Res., 18:6069-74 (1990); Rashtchian, Current Opin. Biotech., 6:30-36 (1995)). Single-stranded tails (also referred to as cloning sites or annealing sequences) are created in LIC vectors, usually by treating the vector (at a digested restriction enzyme site) with T4 DNA polymerase in the presence of only one dNTP. The 3' to 5' exonuclease activity of T4 DNA polymerase removes nucleotides until it encounters a residue corresponding to the single dNTP present in the reaction mix. At this point, the 5' to 3' polymerase activity of the enzyme counteracts the exonuclease activity to prevent further excision. The vector is designed such that the single-stranded tails created are non-complementary. For example, in the pDG2 vector, none of the single-stranded tails of the four annealing sites are complementary to each other. PCR products are created by building appropriate 5' extensions into oligonucleotide primers. The PCR product is purified to remove dNTPs (and original plasmid if it was used as template) and then treated with T4 DNA polymerase in the presence of the appropriate dNTP to generate the specific vector-compatible overhangs. Cloning occurs by annealing of the compatible tails. Single-stranded tails are created at the ends of the clone fragments, for example using chemical or enzymatic means. Complementary tails are created on the vector; however, to prevent annealing of the vector without insert, the vector tails are not complementary to each other. The length of the tails is at least about 5 nucleotides, preferably at least about 12 nucleotides, even more preferably at least about 20 nucleotides. In one embodiment, placing the overlapping vector and fragment(s) in the same reaction is sufficient to anneal them. Alternatively, the complementary sequences are combined, heated and allowed to slowly cool. Preferably the heating step is between about 60°C and about 100°C, more preferably between about 60°C and 80°C, and even more preferably between 60°C and 70°C. The heated reactions are then allowed to cool. Generally, cooling occurs rather slowly, for instance the reactions are generally at about room temperature after about an hour. The cooling must be sufficiently slow as to allow annealing. The annealed fragment/vector can be used immediately, or stored frozen at -20°C until use. Further, annealing can be performed by adjusting the salt and temperature to achieve suitable conditions. Hybridization reactions can be performed in solutions ranging from about 10 mM NaCl to about 600 mM NaCl, at temperatures ranging from about 37°C to about 65°C. It will be understood that the stringency of the hybridization reaction is determined by both the salt concentration and the temperature. For instance, a hybridization performed in 10 mM salt at 37°C may be of similar stringency to one performed in 500 mM salt at 65°C. For the present invention, any hybridization conditions may be used that form hybrids between homologous complementary sequences. As shown in Figure 1, in one embodiment, a construct is made after using any of these annealing procedure where the vector portion contains the two different regions of substantial homology to the target gene (amplified from the plasmid library using long-range PCR) and the fragment is a gene encoding a selectable marker. After annealing, the construct is transformed into competent E. coli cells, for example DH5-α cells by methods known in the art, to amplify the construct. The isolated construct is then ready for introduction into ΕS cells. In another embodiment, a clone of interest is identified in a pooled genomic library using PCR. In one embodiment, the PCR conditions are such that a gene encoding a selectable marker can be inserted directly into the positively identified clone. The marker is positioned between two different sequences having substantial homology to the target DNA. Genomic phage libraries can be prepared by any method known in the art and as described in the Examples. Preferably, a mouse embryonic stem cell library is prepared in lambda phage by cleaving genomic DNA into fragments of approximately 20 kilobases in length. The fragments are then inserted into any suitable lambda cloning vector, for example lambda Fix II or lambda Dash II (Stratagene, La Jolla, Ca) In order to quickly and efficiently screen a large number of clones from a library, pools may be created of plated libraries. In a preferred embodiment, a genomic lambda phage library is plated at a density of approximately 1,000 clones (plaques) per plate. Sufficient plates are created to represent the entire genome of the organism several times over. For example, approximately 1 million clones (1000 plates) will yield approximately 8 genome equivalents. The plaques are then collected, for example by overlaying the plate with a buffer solution, incubating the plates and recollecting the buffer. The amount of buffer used will vary according to the plate size, generally one 100 mm diameter plate will be overlayed with approximately 4 ml of buffer and approximately 2 ml will be collected. It will be understood that the individual plate lysates can be pooled at any time during this procedure and that they can be pooled in any combinations. For ease in later identification of single clones, however, it is preferable to keep each plate lysate separately and then make a pool. For example, each 2 ml lysate can be placed in a 96 well deep well plate. Pools can then be formed by taking an amount, preferably about 100 μl, from each well and combining them in the well of a new plate. Preferably, 100 μl of 12 individual plate lysates are combined in one well, forming a 1.2 ml pool representative of 12,000 clones of the library. Each pool is then PCR-amplified using a set of PCR primers known to amplify the target gene. The target gene can be a known full-length gene or, more preferably, a partial cDNA sequence obtained from publicly available nucleic acid sequence databases such as GenBank or EMBL. These databases include partial cDNA sequences known as expressed sequence tags (ESTs). The oligonucleotide PCR primers can be isolated from any organism by any method known in the art or, preferably, synthesized by chemical means. Once a positive clone of the target gene has been identified in a genomic library, two fragments encoding separate portions of the target gene must be generated. In other words, the flanking regions of the small known region of the target (e.g., EST) are generated. Although the size of each flanking region is not critical and can range from as few as 100 base pairs to as many as 100 kb, preferably each flanking fragment is greater than about 1 kb in length, more preferably between about 1 and about 10 kb, and even more preferably between about 1 and about 5 kb. One of skill in the art will recognize that although larger fragments may increase the number of homologous recombination events in ES cells, larger fragments will also be more difficult to clone. In one embodiment, one of the oligonucleotide PCR primers used to amplify a flanking fragment is specific for the library cloning vector, for example lambda phage. Therefore, if the library is a lambda phage library, primers specific for the lambda phage arms can be used in conjunction with primers specific for the positive clone to generate long flanking fragments. Multiple PCR reactions can be set up to test different combinations of primers. Preferably, the primers used will generate flanking sequences between about 2 and about 6 kb in length. Preferably, the oligonucleotide primers are designed with 5' sequences complementary to the vector into which the fragments will be cloned. In addition, the primers are also designed so that the flanking fragments will be in the proper 3 '-5' orientation with respect to the vector and each other when the construct is assembled. Where the target gene is T243, in one embodiment, one of the primers comprises SEQ ID NO:48 and in another embodiment, the other primer comprises SEQ ID NO:49. Thus, using PCR-based methods, for example, positive clones can be identified by visualization of a band on an electrophoretic gel. In one aspect, the cloning involves a vector and two fragments. The vector contains a positive selection marker, preferably Neo r , and cloning sites on each side of the positive selection marker for two different regions of the target gene. Optionally, the vector also contains a sequence coding for a screening marker (reporter gene), preferably, positioned opposite the positive selection marker. The screening marker will be positioned outside the flanking regions of homologous sequences. Figure 3A shows one embodiment of the vector with the screening marker, GFP, positioned on one side of the vector. However, the screening marker can be positioned anywhere between Not I and Site 4 on the side opposite the positive selection marker, Neo r . One example of a suitable vector is the plasmid vector shown in Figure 2 having the sequence of SEQ ID NO:l. The specific nucleic acid ligation-independent cloning sites (also referred to herein as annealing sites) labeled "sites 1, 2, 3 or 4" in Figure 1 are also shown herein. Generally, the cloning sites are lacking at least one type of base, i.e., thymine (T), guanine (G), cytosine (C) or adenine (A). Accordingly, reacting the vector with an enzyme that acts as both a polymerase and exonuclease in presence of only the one missing nucleotide will create an overhang. For example, T4 DNA polymerase acts as both a 3'-5' exonuclease and a polymerase. Thus, when there are insufficient nucleotides available for the polymerase activity, T4 will act as an exonuclease. Specific overhangs can therefore be created by reacting the pDG2 vector with T4 DNA polymerase in the presence of dTTP only. Other enzymes useful in the practice of this invention will be known to those in the art, for instance uracil DNA glycosylase (UDG) (See, e.g., WO 93/18175). The vector exemplified herein has an overhand of 24 nucleotides. It will be known by those skilled in the art that as few as 5 nucleotides are required for successful ligation independent cloning. In another embodiment, a construct is assembled in a two-step cloning protocol. In the first step, each cloning region of homology is separately cloned into two of the annealing sites of the vector. For example, an "upstream" region of homology is cloned into annealing sites 1 and 2 while a separate cloning, a "downstream" region of homology is cloned into annealing sites 3 and 4. Once clones containing each single region of homology are identified, a targeting construct containing both regions of homology can be created by digesting each clone with restriction enzymes where one enzyme digests outside of annealing site 1 (e.g., Not I in Figure 2A) and another enzyme digests between the positive selection marker and annealing site 3 (e.g., Sal I in Figure 2A). The fragments containing the flanking homology regions from each construct will be purified (e.g., by gel electrophoresis) and combined using standard ligation techniques known in the art, to produce the resulting targeting construct. In yet another embodiment, a construct according to one aspect of the present invention can be formed in a single-step, four-way ligation procedure. The vector and fragments are treated as described above. Briefly, the vector is treated to form two pieces, each piece having a single-stranded tail of specific sequence on each end. Likewise, the PCR-amplified flanking fragments are also treated to form single-stranded tails complementary to those of the vector pieces. The treated vector pieces and fragments are combined and allowed to anneal as described above. Because of the specificity of the single-stranded tails, the final construct will contain the fragments separated by the positive selection marker in the proper orientation. The final plasmid constructs are amplified in bacteria, purified and can then be introduced into ES cells, or stored frozen at -20°C until use. Where so desired, the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see e.g., Li, et al, Cell, 69:91526 (1992)). The selected cells are then injected into a blastocyst (or other stage of development suitable for the puφoses of creating a viable animal, such as, for example, a morula) of an animal (e.g., a mouse) to form chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed., IRL, Oxford, pp. 113-152 (1987)). Alternatively, selected ES cells can be allowed to aggregate with dissociated mouse embryo cells to form the aggregation chimera. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Chimeric progeny harbouring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA. In one embodiment, chimeric progeny mice are used to generate a mouse with a heterozygous disruption in the target gene. Heterozygous knockout mice can then be mated. It is well know in the art that typically VΛ of the offspring of such matings will have a homozygous disruption in the target gene. The heterozygous and homozygous knockout mice can then be compared to normal, wild type mice to determine whether disruption of the target gene causes phenotypic change, especially pathological change. In one embodiment, where the target DNA sequence is T243, the homozygous knockout mouse is reduced in weight relative to an average normal, wild type adult mouse. Weight is typically reduced by at least about 15%; more typically by about 30-90%; even more typically by about 40-80%; and most typically by about 60-70%. In another embodiment, the length of homozygous knockout mouse is decreased relative to an average normal, wild type adult mouse. Length is generally decreased by at least about 10%; often by about 15-50%; more often by about 20-40%; and most often by about 25-35%. The ratio of weight to length may also be decreased, relative to a normal, wild type adult mouse. Commonly, the ratio of weight to length is decreased at least about 20%, more commonly about 25-75%; even more commonly, about 30-65%; and most commonly about 40- 55%. Mice having a phenotype including both decreased length and reduced weight, are also observed. Such mice may also demonstrate a decreased ratio of weight to length. In another embodiment of the invention, the knockout mouse has a phenotype including cartilage and or bone disease. Typically, in this embodiment, there is abnormal cartilage and a generalized reduction of bone formation. As used herein, "disease" refers to any alteration in the state of the body or of some of its organs, interrupting or disturbing the performance of the vital functions, and causing or threatening pain or weakness. Disease may also be considered as including any deviation from or interruption of the normal structure or function of any part, organ or system (or combination thereof) of the body that is manifested by a characteristic single or set of symptoms and/or signs and whose etiology, pathology and/or prognosis may be known or unknown. Commonly observed pathological conditions include shortening of both the axial and appendicular skeleton. Proximal and distal bones of the limbs are proportionally shortened. Joint cartilage lacks alcian blue staining. Further aspects of this embodiment include thin growth plates of the distal femur and thin to absent epiphyseal cartilage. The disease may also present microfractures suggestive of growth plate fragility. Within the physes chondrocyte columns in the proliferating and hypertrophic zones are short in this embodiment. Cartilaginous spicules within the metaphysis are short and widely spaced; and occasional spicules are haphazardly oriented. Osteoblasts are abundant and frequently pile up along cartilaginous spicules. Epiphyseal cartilage is thin and often replaced by fibrous connective tissue. There is also decreased alcian blue staining of the epiphyseal surface. Cartilage at the epiphyseal/physeal junction is slightly flared with an irregular, prominent edge that overhangs the physis. Also included in this embodiment are irregular sternebrae; and growth plates are either lacking or are discontinuous. Large, irregular islands of cartilage extend into the shaft of the stemebra and occasionally have secondary ossification centers. Edges of the cartilage may also be flared. Another aspect includes variably ossified vertebral bodies which may be small and predominantly cartilaginous. Growth plates of these predominantly cartilaginous vertebrae are irregular and thin and the lateral processes are tapered. In one aspect of the invention, the disease is characterized as chondrodysplasia. In yet another embodiment of the invention, the phenotype of the knockout mouse includes kidney disease. Typically, the kidneys are small and lack normal architecture. The cortex is thin and some glomeruli may be subcapsular. Subcapsular glomeruli are small with shrunken, hypercellular glomerular tufts. The corticomedullary area may lack radiating arcuate vessels and distinct tubule formation. Tubular epithelial cells within the corticomedullary junction are haphazardly arranged into sheets, piles and clusters. Some tubular epithelial cells are small and darkly basophilic indicating regeneration. Dysplastic changes are typically present in both kidneys and are most prominent in the corticomedullary junction and to a lesser extent in the cortex. According to one aspect of this invention, the kidney disease is characterized as renal dysplasia. Other conditions of the pathological state may also be observed. An additional feature that may be incoφorated into the presently described vectors includes the use of recombinase target sites. Bacteriophage PI Cre recombinase and flp recombinase from yeast plasmids are two non-limiting examples of site-specific DNA recombinase enzymes which cleave DNA at specific target sites (lox P sites for cre recombinase and frt sites for flp recombinase) and catalyze a ligation of this DNA to a second cleaved site. A large number of suitable alternative site-specific recombinases have been described, and their genes can be used in accordance with the method of the present invention. Such recombinases include the Int recombinase of bacteriophage λ (with or without Xis) (Weisberg, R. et. al., in Lambda II, (Hendrix, R., et al, Eds.), Cold Spring Harbor Press, Cold Spring Harbor, NY, pp. 211-50 (1983), herein incoφorated by reference); Tpnl and the β-lactamase transposons (Mercier, et al, J. Bacteriol, 172:3745-57 (1990)); the Tn3 resolvase (Flanagan & Fennewald J. Molec. Biol, 206:295-304 (1989); Stark, et al, Cell, 58:779-90 (1989)); the yeast recombinases (Matsuzaki, et al, J. Bacteriol, 172:610-18 (1990)); the B. subtilis SpoIVC recombinase (Sato, et al, J. Bacteriol 172:1092-98 (1990)); the Flp recombinase (Schwartz & Sadowski, J. Molec.BioL, 205:647-658 (1989); Parsons, et al, J. Biol Chem., 265:4527-33 (1990); Golic & Lindquist, Cell, 59:499-509 (1989); Amin, et al, J. Molec. Biol, 214:55-72 (1990)); the Hin recombinase (Glasgow, et al, J. Biol. Chem., 264:10072-82 (1989)); immunoglobulin recombinases (Malynn, et al, Cell, 54:453-460 (1988)); and the Cin recombinase (Haffter & Bickle, EMBO J., 7:3991-3996 (1988); Hubner, et al, J. Molec. Biol, 205:493-500 (1989)), all herein incoφorated by reference. Such systems are discussed by Echols (J. Biol. Chem. 265:14697-14700 (1990)); de Villartay (Nature, 335:170-74 (1988)); Craig, (Ann. Rev. Genet., 22:77-105 (1988)); Poyart-Salmeron, et al, (EMBO J. 8:2425-33 (1989)); Hunger-Bertling, et al. (Mol Cell Biochem., 92:107-16 (1990)); and Cregg & Madden (Mol. Gen. Genet., 219:320-23 (1989)), all herein incoφorated by reference. Cre has been purified to homogeneity, and its reaction with the loxP site has been extensively characterized (Abremski & Hess J. Mol Biol. 259:1509-14 (1984), herein incoφorated by reference). Cre protein has a molecular weight of 35,000 and can be obtained commercially from New England Nuclear/Du Pont. The cre gene (which encodes the Cre protein) has been cloned and expressed (Abremski, et al. Cell 32:1301-11 (1983), herein incoφorated by reference). The Cre protein mediates recombination between two loxP sequences (Sternberg, et al. Cold Spring Harbor Symp. Quant. Biol. 45:297-309 (1981)), which may be present on the same or different DNA molecule. Because the internal spacer sequence of the loxP site is asymmetrical, two loxP sites can exhibit directionality relative to one another (Hoess & Abremski Proc. Natl Acad. Sci. U.S.A. 81:1026-29 (1984)). Thus, when two sites on the same DNA molecule are in a directly repeated orientation, Cre will excise the DNA between the sites (Abremski, et al. Cell 32:1301-11 (1983)). However, if the sites are inverted with respect to each other, the DNA between them is not excised after recombination but is simply inverted. Thus, a circular DNA molecule having two loxP sites in direct orientation will recombine to produce two smaller circles, whereas circular molecules having two loxP sites in an inverted orientation simply invert the DNA sequences flanked by the loxP sites. In addition, recombinase action can result in reciprocal exchange of regions distal to the target site when targets are present on separate DNA molecules. Recombinases have important application for characterizing gene function in knockout models. When the constructs described herein are used to disrupt target genes, a fusion transcript can be produced when insertion of the positive selection marker occurs downstream (3') of the translation initiation site of the target gene. The fusion transcript could result in some level of protein expression with unknown consequence. It has been suggested that insertion of a positive selection marker gene can affect the expression of nearby genes. These effects may make it difficult to determine gene function after a knockout event since one could not discern whether a given phenotype is associated with the inactivation of a gene, or the transcription of nearby genes. Both potential problems are solved by exploiting recombinase activity. When the positive selection marker is flanked by recombinase sites in the same orientation, the addition of the corresponding recombinase will result in the removal of the positive selection marker. In this way, effects caused by the positive selection marker or expression of fusion transcripts are avoided. Loss of function or null mutation models may be inadequate to characterize disease associated with TRP target genes. A number of published reports suggest that expansion of trinucleotide repeat regions in TRPs confer deleterious gains of function upon the resulting proteins. Such gains of function may involve novel or enhanced interaction with other proteins, increased resistance to proteolytic degradation, aberrant protein folding, and/or toxic accumulation of large, insoluble protein forms. It would therefore be of great value to mimic expansion of trinucleotide repeats in a TRP to determine whether expansion produces a phenotypic change that may be associated with a gain of function. Accordingly, one embodiment of the invention will involve the use of recombinases to bring about enzyme-assisted site-specific integration of a synthetic trinucleotide repeat at the site of disruption in a target gene. This embodiment will involve the reciprocal exchange ability of recombinase systems whereby a recombinase enzyme catalyzes the exchange of DNA distal to two target sites present on separate molecules. When the targeting construct used to generate a knockout stem cell includes a recombinase target site flanking the positive selection marker, recombination can occur between that site and a second site present on a synthetic nucleic acid in the presence of a recombinase enzyme. One of skill in the art will recognize that the synthetic nucleic acid can be readily synthesized to include both the recombinase target site and repeated trinucleotides of any desired sequence. For example, the synthetic nucleic acid sequence can include repeats of CTG, encoding leucine, or CAG, encoding glutamine. Preferably, the synthetic nucleic acid will have at least about 20 trinucleotide repeats; more preferably, about at least about 40 trinucleotide repeats; most preferably, at least about 100 trinucleotide repeats. The skilled artisan will also recognize the synthetic nucleic acid can be contacted with the disrupted gene by any standard laboratory methods for introducing DNA including, but not limited to, transfection, lipofection, or electroporation. In one embodiment, purified recombinase enzyme is provided to the cell by direct microinjection. In another embodiment, recombinase is expressed from a co-transfected construct or vector in which the recombinase gene is operably linked to a functional promoter. An additional aspect of this embodiment is the use of tissue-specific or inducible recombinase constructs which allow the choice of when and where recombination occurs. One method for practicing the inducible forms of recombinase-mediated recombination involves the use of vectors that use inducible or tissue-specific promoters or other gene regulatory elements to express the desired recombinase activity. The inducible expression elements are preferably operatively positioned to allow the inducible control or activation of expression of the desired recombinase activity. Examples of such inducible promoters or other gene regulatory elements include, but are not limited to, tetracycline, metallothionine, ecdysone, and other steroid- responsive promoters, rapamycin responsive promoters, and the like (No, et al. Proc. Natl Acad. Sci. USA, 93:3346-51 (1996); Furth, et al Proc. Natl. Acad. Sci. USA, 91:9302-6 (1994)). Additional control elements that can be used include promoters requiring specific transcription factors such as viral, promoters. Vectors incoφorating such promoters would only express recombinase activity in cells that express the necessary transcription factors. The TRP gene sequences may also be used to produce TRP gene products. TRP gene products may include proteins that represent functionally equivalent gene products. Such an equivalent gene product may contain deletions, additions or substitutions of amino acid residues within the amino acid sequence encoded by the gene sequences described herein, but which result in a silent change, thus producing a functionally equivalent TRP gene product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. "Functionally equivalent", as utilized herein, refers to a protein capable of exhibiting a substantially similar in vivo activity as the endogenous gene products encoded by the TRP gene sequences. Alternatively, when utilized as part of an assay, "functionally equivalent" may refer to peptides capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the corresponding portion of the endogenous gene product would. Other TRP protein products useful according to the methods of the invention are peptides derived from or based on TRP produced by recombinant or synthetic means (TRP-derived peptides). Mutant TRP proteins in which the trinucleotide regions are intentionally expanded, for example, by site-directed mutagensis, can also be produced. TRPs expanded by enzyme-assisted site-specific integration in stem cells can also be used. The TRP and expanded TRP gene products may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing the gene polypeptides and peptides of the invention by expressing nucleic acid encoding gene sequences are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing gene protein coding sequences and appropriate transcriptional/translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination (see, e.g., Sambrook, et al, 1989, supra, and Ausubel, et al, 1989, supra). Alternatively, RNA capable of encoding gene protein sequences may be chemically synthesized using, for example, automated synthesizers (see, e.g. Oligonucleotide Synthesis: A Practical Approach, Gait, M. J. ed., IRL Press, Oxford (1984)). A variety of host-expression vector systems may be utilized to express the gene coding sequences of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the gene protein of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing gene protein coding sequences; yeast (e.g. Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the gene protein coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the gene protein coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing gene protein coding sequences; or mammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter). In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the gene protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of antibodies or to screen peptide libraries, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther & Muller-Hill, EMBO J., 2:1791-94 (1983)), in which the gene protein coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res., 13:3101-09 (1985); Van Heeke & Schuster, J. Biol. Chem., 264:5503-9 (1989)); and the like. pGΕX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsoφtion to glutathione - agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene protein can be released from the GST moiety. In a preferred embodiment, full length cDNA sequences are appended with in-frame Bam HI sites at the amino terminus and Eco Rl sites at the carboxyl terminus using standard PCR methodologies (Innis, et al. (eds) PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego (1990)) and ligated into the pGEX-2TK vector (Pharmacia, Uppsala, Sweden). The resulting cDNA construct contains a kinase recognition site at the amino terminus for radioactive labeling and glutathione S-transferase sequences at the carboxyl terminus for affinity purification (Nilsson, et al, EMBO J., 4: 1075-80 (1985); Zabeau and Stanley, EMBO J., 1: 1217-24 (1982)). In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The gene coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of gene coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed (see, e.g., Smith, et al, J. Virol. 46: 584-93 (1983); Smith, U.S. Pat. No. 4,745,051). In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the gene coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing gene protein in infected hosts, (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:3655-59 (1984)). Specific initiation signals may also be required for efficient translation of inserted gene coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the gene coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bitter, et al, Methods in EnzymoL, 153:516-44 (1987)). In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the gene protein may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells which stably integrate the plasmid into their chromosomes and grow, to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the gene protein. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the gene protein. In a preferred embodiment, control of timing and/or quantity of expression of the recombinant protein can be controlled using an inducible expression construct. Inducible constructs and systems for inducible expression of recombinant proteins will be well known to those skilled in the art. Examples of such inducible promoters or other gene regulatory elements include, but are not limited to, tetracycline, metallothionine, ecdysone, and other steroid- responsive promoters, rapamycin responsive promoters, and the like (No, et al, Proc. Natl. Acad. Sci. USA, 93:3346-51 (1996); Furth, et al, Proc. Natl. Acad. Sci. USA, 91:9302-6 (1994)). Additional control elements that can be used include promoters requiring specific transcription factors such as viral, particularly HIV, promoters. In one in embodiment, a Tet inducible gene expression system is utilized. (Gossen & Bujard, Proc. Natl. Acad. Sci. USA, 89:5547-51 (1992); Gossen, et al, Science, 268:1766-69 (1995)). Tet Expression Systems are based on two regulatory elements derived from the tetracycline-resistance operon of the E. coli TnlO transposon — the tetracycline repressor protein (TetR) and the tetracycline operator sequence (tetO) to which TetR binds. Using such a system, expression of the recombinant protein is placed under the control of the tetO operator sequence and transfected or transformed into a host cell. In the presence of TetR, which is co-transfected into the host cell, expression of the recombinant protein is repressed due to binding of the TetR protein to the tetO regulatory element. High-level, regulated gene expression can then be induced in response to varying concentrations of tetracycline (Tc) or Tc derivatives such as doxycycline (Dox), which compete with tetO elements for binding to TetR. Constructs and materials for tet inducible gene expression are available commercially from CLONTECH Laboratories, Inc., Palo Alto, CA. When used as a component in an assay system, the gene protein may be labeled, either directly or indirectly, to facilitate detection of a complex formed between the gene protein and a test substance. Any of a variety of suitable labeling systems may be used including but not limited to radioisotopes such as 125 I; enzyme labeling systems that generate a detectable calorimetric signal or light when exposed to substrate; and fluorescent labels. Where recombinant DNA technology is used to produce the gene protein for such assay systems, it may be advantageous to engineer fusion proteins that can facilitate labeling, immobilization and/or detection. Indirect labeling involves the use of a protein, such as a labeled antibody, which specifically binds to either a gene product. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Described herein are methods for the production of antibodies capable of specifically recognizing one or more gene epitopes. Such antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Such antibodies may be used, for example, in the detection of a target TRP gene in a biological sample, or, alternatively, as a method for the inhibition of abnormal target gene activity. Thus, such antibodies may be utilized as part of disease treatment methods, and or may be used as part of diagnostic techniques whereby patients may be tested for abnormal levels of target TRP gene proteins, or for the presence of abnormal forms of the such proteins. For the production of antibodies to a gene, various host animals may be immunized by injection with a TRP protein, or a portion thereof. Such host animals may include but are not limited to rabbits, mice, and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as target gene product, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals such as those described above, may be immunized by injection with gene product supplemented with adjuvants as also described above. Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Kohler and Milstein, Nature, 256:495-7 (1975); and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosbor, et al, Immunology Today, 4:72 (1983); Cote, et al, Proc. Natl. Acad. Sci. USA, 80:2026-30 (1983)), and the EBV-hybridoma technique (Cole, et al, in Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., New York, pp. 77-96 (1985)). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production. In addition, techniques developed for the production of "chimeric antibodies" (Morrison, et al, Proc. Natl. Acad. Sci., 81:6851-6855 (1984); Takeda, et al, Nature, 314:452-54 (1985)) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-26 (1988); Huston, et al, Proc. Natl Acad. Sci. USA, 85:5879-83 (1988); and Ward, et al, Nature, 334:544-46 (1989)) can be adapted to produce gene-single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the F v region via an amino acid bridge, resulting in a single chain polypeptide. Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments. Alternatively, Fab expression libraries may be constructed (Huse, et al, Science, 246:1275-81 (1989)) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. Described herein are cell- and animal-based systems which can be utilized as models for diseases. Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate disease animal models. In addition, cells from humans may be used. These systems may be used in a variety of applications. For example, the cell- and animal-based model systems may be used to further characterize TRP genes. Such assays may be utilized as part of screening strategies designed to identify compounds which are capable of ameliorating disease symptoms. Thus, the animal- and cell-based models may be used to identify drugs, pharmaceuticals, therapies and interventions which may be effective in treating disease. Cells that contain and express target gene sequences which encode TRPs, and, further, exhibit cellular phenotypes associated with disease, may be utilized to identify compounds that exhibit anti-disease activity. Such cells may include non-recombinant monocyte cell lines, such as U937 (ATCC# CRL-1593), THP-1 (ATCC# TIB-202), and P388D1 (ATCC# TIB-63); endothelial cells such as HUVEC's and bovine aortic endothelial cells (BAEC's); as well as generic mammalian cell lines such as HeLa cells and COS cells, e.g., COS-7 (ATCC# CRL-1651). Further, such cells may include recombinant, transgenic cell lines. For example, the knockout mice of the invention may be used to generate cell lines, containing one or more cell types involved in a disease, that can be used as cell culture models for that disorder. While cells, tissues, and primary cultures derived from the disease transgenic animals of the invention may be utilized, the generation of continuous cell lines is preferred. For examples of techniques which may be used to derive a continuous cell line from the transgenic animals, see Small, et al, Mol. Cell Biol, 5:642-48 (1985). Target gene sequences may be introduced into, and overexpressed in, the genome of the cell of interest, or, if endogenous target gene sequences are present, they may be either overexpressed or, alternatively disrupted in order to underexpress or inactivate target gene expression. In order to overexpress a target gene sequence, the coding portion of the target gene sequence may be ligated to a regulatory sequence which is capable of driving gene expression in the cell type of interest. Such regulatory regions will be well known to those of skill in the art, and may be utilized in the absence of undue experimentation. For underexpression of an endogenous target gene sequence, such a sequence may be isolated and engineered such that when reintroduced into the genome of the cell type of interest, the endogenous target gene alleles will be inactivated. Preferably, the engineered target gene sequence is introduced via gene targeting such that the endogenous target sequence is disrupted upon integration of the engineered target gene sequence into the cell's genome. Cells transfected with target genes can be examined for phenotypes associated with a disease. Compounds identified via assays may be useful, for example, in elaborating the biological function of the target gene product, and for ameliorating a disease. In instances whereby a disease condition results from an overall lower level of target gene expression and/or target gene product in a cell or tissue, compounds that interact with the target gene product may include compounds which accentuate or amplify the activity of the bound target gene protein. Such compounds would bring about an effective increase in the level of target gene product activity, thus ameliorating symptoms. In vitro systems may be designed to identify compounds capable of binding a target TRP gene or an expanded TRP gene. Such compounds may include, but are not limited to, peptides made of D-and/or L-configuration amino acids (in, for example, the form of random peptide libraries; see e.g., Lam, et al, Nature, 354:82-4 (1991)), phosphopeptides (in, for example, the form of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, et al, Cell, 72:767-78 (1993)), antibodies, and small organic or inorganic molecules. Compounds identified may be useful, for example, in modulating the activity of target gene proteins, preferably mutant target gene proteins, may be useful in elaborating the biological function of the target gene protein, may be utilized in screens for identifying compounds that disrupt normal target gene interactions, or may in themselves disrupt such interactions. The principle of the assays used to identify compounds that bind to the target gene protein involves preparing a reaction mixture of the target gene protein or expanded target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring the target or expanded target gene protein or the test substance onto a solid phase and detecting target or expanded target gene protein/test substance complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the target gene protein may be anchored onto a solid surface, and the test compound, which is not anchored, may be labeled, either directly or indirectly. In practice, microtitre plates are conveniently utilized. The anchored component may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored. In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously nonimmobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for target gene product or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes. Compounds that are shown to bind to a particular target gene product through one of the methods described above can be further tested for their ability to elicit a biochemical response from the target gene protein. Cell-based systems may be used to identify compounds which may act to ameliorate a disease symptoms. For example, such cell systems may be exposed to a compound suspected of exhibiting an ability to ameliorate a disease symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of disease symptoms in the exposed cells. After exposure, the cells are examined to determine whether one or more of the disease cellular phenotypes has been altered to resemble a more normal or more wild type, non-disease phenotype. In addition, animal-based disease systems, such as those described herein, may be used to identify compounds capable of ameliorating disease symptoms. Such animal models may be used as test substrates for the identification of drugs, pharmaceuticals, therapies, and interventions which may be effective in treating a disease or other phenotypic characteristic of the animal. For example, animal models may be exposed to a compound or agent suspected of exhibiting an ability to ameliorate disease symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of disease symptoms in the exposed animals. The response of the animals to the exposure may be monitored by assessing the reversal of disorders associated with the disease. Exposure may involve treating mother animals during gestation of the model animals described herein, thereby exposing embryos or fetuses to the compound or agent which may prevent or ameliorate the disease or phenotype. Neonatal, juvenile, and adult animals can also be exposed. Similar disease symptoms can arise from a variety of etiologies. Chondrodysplasias, for example, comprise a broad group of bone malformations that can result from defective collagen formation, disruption of signaling molecules [insulin-like growth factor (IGF), parathyroid hormone related protein (PTHrP), Indian hedgehog (Ihh), bone moφhogenic proteins (BMPs)], or abnormal proteoglycans comprising the cartilage matrix (i.e. aggrecan). Primary bone diseases described in humans include osteogenesis imperfecta (defective type I collagen synthesis), mucopolysaccharidoses (lysosomal storage diseases that result in abnormal matrix), Blomstrand chondrodysplasia (defect of PTH/PTHrP hormone and/or receptor), multiple epiphyseal dysplasia (defective type IX collagen), and Schmid metaphyseal chondrodysplasia (defective type X collagen synthesis). Because of defective cartilage and/or cartilaginous matrix, there is reduced mineralization and bone formation. The term osteoporosis is used to denote a general reduction in bone mass and encompasses primary and secondary conditions. Primary osteoporotic conditions include idiopathic juvenile, idiopathic middle adulthood, postmenopausal, and senile osteoporosis. Secondary conditions that can result in osteoporosis include endocrine disorders (hypeφarathyroidism, hyperthyroidism, hypothyroidism, hypogonadism, acromegaly, Cushing's disease, type 1 Diabetes, and Addison's disease), gastrointestinal disorders (malabsoφtion, vitamin C, D deficiency, malnutrition, and hepatic insufficiency), chronic obstructive pulmonary disease, Gaucher's disease, anemia, and homocystinuria. In addition to chondrocytes, osteoblasts play a critical role in bone formation. Osteoblasts have receptors for hormones (PTH, Vitamin D, estrogen), cytokines, and growth factors, and secrete collagenous and noncollagenous proteins. The noncollaginous proteins include cell adhesion proteins (osteopontin, fibronectin, thrombospondin), calcium binding proteins (osteonectin, bone sialoprotein), proteins involved in mineralization (osteocalcin), enzymes (collagenase and alkaline phosphatase), growth factors (IGF-1, TGF-B, PDGF) and cytokines (prostaglandins, IL-1, IL-6). Furthermore, the aggregating proteoglycans of ground substance (aggrecan, versican, neurocan, and brevican) are important components of the extracellular matrix. The recently described ligand for aggrecan and versican, fibulin-1 (Aspberg, et al, J Biol Chem, 274:20444-9 (1999)), is strongly expressed in developing cartilage and bone. Another group of symptoms, renal dysplasias and hypoplasias, account for 20% of chronic renal failure in children (Cotran, et al, Robbins Pathologic Basis of Disease, Saunders, Philadelphia (1994)). Congenital renal disease can be hereditary but is most often the result of an acquired developmental defect that arises during gestation. In affected individuals, urogenital differentiation is evident by 8.5 to 9 days of gestation in the mouse (corresponding to gestational days 22-24 in humans). During development, dysplasias have been hypothesized to result from abnormal cell differentiation, leading to sustained cellular proliferation and transepithelial fluid secretion that may result in cyst formation (Grantham, et al. (1993) Adv Intern Med 38:409-20), or an extracellular matrix defect that, in turn, affects epithelial differentiation (Calvet, et al, J Histochem Cytochem, 41:1223-31 (1993)). Growth factors that are common to bone and renal development include Insulin-like growth factor and BMPs. However, chronic renal failure can also affect bone formation because of calcium/phosphorus and acid/base imbalances. One of skill in the art will recognize that a given agent may be effective in ameliorating similar symptoms caused by disparate etiologies. Thus, a given agent may be useful in the treatment of a variety of diseases. Among the agents which may exhibit the ability to ameliorate disease symptoms are antisense, ribozyme, and triple helix molecules. Such molecules may be designed to reduce or inhibit mutant target gene activity. Techniques for the production and use of such molecules are well known to those of skill in the art. Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. With respect to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between the - 10 and +10 regions of the target gene nucleotide sequence of interest, are preferred. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage. The composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see U.S. Pat. No. 5,093,246, which is incoφorated by reference herein in its entirety. As such within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences encoding target gene proteins. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the molecule of interest for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features, such as secondary structure, that may render the oligonucleotide sequence unsuitable. The suitability of candidate sequences may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays. Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription should be single stranded and composed of deoxyribonucleotides. The base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex. Nucleotide sequences may be pyrimidine- based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide base complementarity to a purine- rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, nucleic acid molecules may be chosen that are purine-rich, for example, containing a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex. Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex. It is possible that the antisense, ribozyme, and or triple helix molecules described herein may reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by both normal and mutant target gene alleles. In order to ensure that substantially normal levels of target gene activity are maintained, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal activity may be introduced into cells that do not contain sequences susceptible to whatever antisense, ribozyme, or triple helix treatments are being utilized. Alternatively, it may be preferable to coadminister normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity. Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incoφorated into a wide variety of vectors which incoφorate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines. Various well-known modifications to the DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone. Antibodies that are both specific for target gene protein and interfere with its activity may be used to inhibit target gene function. Antibodies that are specific for expanded target gene protein and interfere with the unique interactions of that protein, especially functions attributable novel gains of function associated with trinucleotide expansion, may also be used to inhibit expanded target gene function. Of particular interest are antibodies directed to expanded trinucleotide regions of TRPs. Such antibodies may be generated using standard techniques against the proteins themselves or against peptides corresponding to portions of the proteins. Such antibodies include but are not limited to polyclonal, monoclonal, Fab fragments, single chain antibodies, chimeric antibodies, etc. In instances where the target gene protein is intracellular and whole antibodies are used, internalizing antibodies may be preferred. However, lipofectin liposomes may be used to deliver the antibody or a fragment of the Fab region which binds to the target gene epitope into cells. Where fragments of the antibody are used, the smallest inhibitory fragment which binds to the target or expanded target protein's binding domain is preferred. For example, peptides having an amino acid sequence corresponding to the domain of the variable region of the antibody that binds to the target gene protein may be used. Such peptides may be synthesized chemically or produced via recombinant DNA technology using methods well known in the art (see, e.g., Creighton J Proteins : Structures and Molecular Principles (1984) W.H. Freeman, New York 1983, supra; and Sambrook, et al, 1989, supra). Alternatively, single chain neutralizing antibodies which bind to intracellular target gene epitopes may also be administered. Such single chain antibodies may be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco, et al, Proc. Natl. Acad. Sci. USA, 90:7889-93 (1993). Antibodies that are specific for one or more extracellular domains of the TRP or expanded TRP and that interfere with its activity, are particularly useful in treating disease. Such antibodies are especially efficient because they can access the target domains directly from the bloodstream. Any of the administration techniques described below which are appropriate for peptide administration may be utilized to effectively administer inhibitory target gene antibodies to their site of action. RNA sequences encoding target gene protein may be directly administered to a patient exhibiting disease symptoms, at a concentration sufficient to produce a level of target gene protein such that disease symptoms are ameliorated. Patients may be treated by gene replacement therapy. One or more copies of a normal target gene, or a portion of the gene that directs the production of a normal target gene protein with target gene function, may be inserted into cells using vectors which include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes. Additionally, techniques such as those described above may be utilized for the introduction of normal target gene sequences into human cells. Cells, preferably, autologous cells, containing normal target gene expressing gene sequences may then be introduced or reintroduced into the patient at positions which allow for the amelioration of disease symptoms. The identified compounds that inhibit target or expanded target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to treat or ameliorate the disease. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disease. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 . Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, topical, subcutaneous, intraperitoneal, intraveneous, intrapleural, intraoccular, intraarterial, or rectal administration. It is also contemplated that pharmaceutical compositions may be administered with other products that potentiate the activity of the compound and optionally, may include other therapeutic ingredients. For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. Oral ingestion is possibly the easiest method of taking any medication. Such a route of administration, is generally simple and straightforward and is frequently the least inconvenient or unpleasant route of administration from the patient's point of view. However, this involves passing the material through the stomach, which is a hostile environment for many materials, including proteins and other biologically active compositions. As the acidic, hydrolytic and proteolytic environment of the stomach has evolved efficiently to digest proteinaceous materials into amino acids and oligopeptides for subsequent anabolism, it is hardly suφrising that very little or any of a wide variety of biologically active proteinaceous material, if simply taken orally, would survive its passage through the stomach to be taken up by the body in the small intestine. The result, is that many proteinaceous medicaments must be taken in through another method, such as parenterally, often by subcutaneous, intramuscular or intravenous injection. Pharmaceutical compositions may also include various buffers (e.g., Tris, acetate, phosphate), solubilizers (e.g., Tween, Polysorbate), carriers such as human serum albumin, preservatives (thimerosol, benzyl alcohol) and anti-oxidants such as ascorbic acid in order to stabilize pharmacetical activity. The stabilizing agent may be a detergent, such as tween-20, tween-80, NP-40 or Triton X-100. EBP may also be incoφorated into particulate preparations of polymeric compounds for controlled delivery to a patient over an extended period of time. A more extensive survey of components in pharmaceutical compositions is found in Remington's Pharmaceutical Sciences, 18th ed., A. R. Gennaro, ed., Mack Publishing, Easton, Pa. (1990). In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. A variety of methods may be employed to diagnose disease conditions associated with a TRP. Specifically, reagents may be used, for example, for the detection of the presence of target gene mutations, or the detection of either over or under expression of target gene mRNA. The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one specific gene nucleic acid or anti-gene antibody reagent described herein, which may be conveniently used, e.g., in clinical settings, to diagnose patients exhibiting disease symptoms or at risk for developing disease. Any cell type or tissue, preferably monocytes, endothelial cells, or smooth muscle cells, in which the gene is expressed may be utilized in the diagnostics described below. DNA or RNA from the cell type or tissue to be analyzed may easily be isolated using procedures which are well known to those in the art. Diagnostic procedures may also be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Nucleic acid reagents may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, PCR In Situ Hybridization: Protocols and Applications. Raven Press, N.Y. (1992)). Gene nucleotide sequences, either RNA or DNA, may, for example, be used in hybridization or amplification assays of biological samples to detect disease-related gene structures and expression. Such assays may include, but are not limited to, Southern or Northern analyses, restriction fragment length polymoφhism assays, single stranded conformational polymoφhism analyses, in situ hybridization assays, and polymerase chain reaction analyses. Such analyses may reveal both quantitative aspects of the expression pattern of the gene, and qualitative aspects of the gene expression and/or gene composition. That is, such aspects may include, for example, point mutations, insertions, deletions, chromosomal rearrangements, and/or activation or inactivation of gene expression. Preferred diagnostic methods for the detection of gene-specific nucleic acid molecules may involve for example, contacting and incubating nucleic acids, derived from the cell type or tissue being analyzed, with one or more labeled nucleic acid reagents under conditions favorable for the specific annealing of these reagents to their complementary sequences within the nucleic acid molecule of interest. Preferably, the lengths of these nucleic acid reagents are at least 9 to 30 nucleotides. After incubation, all non-annealed nucleic acids are removed from the nucleic acid:fingeφrint molecule hybrid. The presence of nucleic acids from the fingeφrint tissue which have hybridized, if any such molecules exist, is then detected. Using such a detection scheme, the nucleic acid from the tissue or cell type of interest may be immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a microtitre plate or polystyrene beads. In this case, after incubation, non-annealed, labeled nucleic acid reagents are easily removed. Detection of the remaining, annealed, labeled nucleic acid reagents is accomplished using standard techniques well-known to those in the art. Alternative diagnostic methods for the detection of gene-specific nucleic acid molecules may involve their amplification, e.g., by PCR (the experimental embodiment set forth in Mullis U.S. Pat. No. 4,683,202 (1987)), ligase chain reaction (Barany, Proc. Natl. Acad. Sci. USA, 88:189-93 (1991)), self sustained sequence replication (Guatelli, et al., Proc. Natl. Acad. Sci. USA, 87:1874-78 (1990)), transcriptional amplification system (Kwoh, et al, Proc. Natl. Acad. Sci. USA, 86:1173-77 (1989)), Q-Beta Replicase (Lizardi, P. M., et al, Bio/Technology, 6:1197 (1988)), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In one embodiment of such a detection scheme, a cDNA molecule is obtained from an RNA molecule of interest (e.g., by reverse transcription of the RNA molecule into cDNA). Cell types or tissues from which such RNA may be isolated include any tissue in which wild type fingeφrint gene is known to be expressed, including, but not limited, to monocytes, endothelium, and/or smooth muscle. A sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like. The nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method may be chosen from among the gene nucleic acid reagents described herein. The preferred lengths of such nucleic acid reagents are at least 15-30 nucleotides. For detection of the amplified product, the nucleic acid amplification may be performed using radioactively or non-radioactively labeled nucleotides. Alternatively, enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid staining method. Antibodies directed against wild type, mutant, or expanded gene peptides may also be used as disease diagnostics and prognostics. Such diagnostic methods, may be used to detect abnormalities in the level of gene protein expression, or abnormalities in the structure and/or tissue, cellular, or subcellular location of fingeφrint gene protein. Structural differences may include, for example, differences in the size, electronegativity, or antigenicity of the mutant fingeφrint gene protein relative to the normal fingeφrint gene protein. Protein from the tissue or cell type to be analyzed may easily be detected or isolated using techniques which are well known to those of skill in the art, including but not limited to western blot analysis. For a detailed explanation of methods for carrying out western blot analysis, see Sambrook, et al. (1989) supra, at Chapter 18. The protein detection and isolation methods employed herein may also be such as those described in Harlow and Lane, for example, (Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988)). Preferred diagnostic methods for the detection of wild type, mutant, or expanded gene peptide molecules may involve, for example, immunoassays wherein fingeφrint gene peptides are detected by their interaction with an anti-fingeφrint gene-specific peptide antibody. For example, antibodies, or fragments of antibodies useful in the present invention may be used to quantitatively or qualitatively detect the presence of wild type, mutant, or expanded gene peptides. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorimetric detection. Such techniques are especially preferred if the fingeφrint gene peptides are expressed on the cell surface. The antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of fingeφrint gene peptides. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention. The antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the fingeφrint gene peptides, but also their distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection. Immunoassays for wild type, mutant, or expanded fingeφrint gene peptides typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying fingeφrint gene peptides, and detecting the bound antibody by any of a number of techniques well known in the art. The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled gene-specific antibody. The solid phase support may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on solid support may then be detected by conventional means. By "solid phase support or carrier" is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the puφoses of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation. The binding activity of a given lot of anti-wild type, -mutant, or -expanded fingeφrint gene peptide antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation. One of the ways in which the gene peptide-specific antibody can be detectably labeled is by linking the same to an enzyme and using it in an enzyme immunoassay (EIA) (Voller, Ric Clin Lab, 8:289-98 (1978) ["The Enzyme Linked Immunosorbent Assay (ELISA)", Diagnostic Horizons 2:1-7, 1978, Microbiological Associates Quarterly Publication, Walkersville, Md.]; Voller, et al, J. Clin. Pathol, 31:507-20 (1978); Butler, Meth. Enzymol., 73:482-523 (1981); Maggio (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla. (1980); Ishikawa, et al, (eds.) Enzyme Immunoassay, Igaku-Shoin, Tokyo (1981)). The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards. Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect fingeφrint gene wild type, mutant, or expanded peptides through the use of a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. The antibody can also be detectably labeled using fluorescence emitting metals such as 1 S9 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediamine- tetraacetic acid (EDTA). The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for puφoses of labeling are luciferin, luciferase and aequorin. Throughout this application, various publications, patents, and published patent applications are referred to by an identifying citation. The disclosures of these publications, patents and published patent specifications referenced in this application are hereby incoφorated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains. The following examples are intended only to illustrate the present invention and should in no way be construed as limiting the subject invention. Examples Example 1: Direct Construct Construction from a Plasmid Library Genomic libraries using the lambda ZAP™ system were prepared as follows. Embryonic stem cells were grown in 100 mm tissue culture plates. High molecular weight genomic DNA was isolated from these ES cells by adding 5 ml of lysis buffer (10 mM Tris-HCL pH7.5, 10 mM EDTA pH 8.0, 10 mM NaCl, 0.5% SDS, and 1 mg/ml Proteinase K) to a confluent 100 mm plate of embryonic stem cells. The cells were then incubated at 60°C for several hours or until fully lysed. Genomic DNA was purified from the lysed cells by several rounds of gentle phenol: chloroform extractions followed by ethanol precipitation. The genomic DNA was partially digested with the restriction enzyme Sau 3A I to generate fragments of approximately 5-20 kb. The ends of these fragments were partially filled in by addition of dATP and dGTP in the present of Klenow DNA polymerase, creating incompatible ends on the genomic fragments. Size fragments of between 5 and 10 kb were then purified by agarose gel electrophoresis (lx TAE, 0.8% gel). The DNA was then isolated from the excised agarose pieces using a QIAquick gel extraction kit (Qiagen, Inc., Valencia, CA). The genomic fragments were ligated into the Lambda Zap™ II vector (Stratagene, Inc., La Jolla, CA) that had been cut with Xho I and partially filled in using dTTP, dCTP, and Klenow DNA polymerase. After ligation, the DNA was packaged using a lambda packaging mix (Gigapack III gold, Stratagene, Inc., La Jolla, CA) and the titer was determined. Circular phagemid DNA was derived from the lambda library by growing the lambda clones on the appropriate bacterial strain (XL-1 Blue MRF 1 , Stratagene, Inc.) in the presence of the M13 helper phage, ExAssist (Stratagene, Inc.). Specifically, approximately 100,000 lambda clones were incubated with a 10-100 fold excess of both bacteria and helper phage for 20 minutes at 37°C. One ml of LB media + 10 mM MgSO 4 was added to each excision reaction and it was incubated overnight at 37°C with shaking. Typically 24-96 of these reactions were set up at a time in a 96 well deep-well block. The following morning, the block was heated to 65°C for 15 minutes to kill both the bacteria and the lambda phage. Bacterial debris was removed by centrifugation at approximately 3000g for 15 minutes. The supernatant containing the circular phagemid DNA, was retained and used directly in plasmid PCR experiments (see Examples 9 and 10 for plasmid PCR experiments). The pools of phagemid DNA described above were screened for specific genes of interest using long-range PCR and "outward pointing" oligos, chosen as described above based on the known sequence (depicted in Figure 1). The PCR reactions contains 2 μl of a pool phagemid DNA sample, 3 μl of lOx PCR Buffer 3 (Boehringer Mannheim), 1.1 μl 10 mM dNTPs, 50 nM primers, 0.3 μl of EXPAND Long Template PCR Enzyme Mix (Boehringer-Mannheim) and 30 μl of H 2 0. Cycling conditions were 94°C for 2 minutes (1 cycle); 94°C for 10 seconds, 65°C for 30 seconds, 68°C for 15 seconds (15 cycles); 94°C for 10 seconds, 60°C for 30 seconds, 68°C for 15 seconds plus 20 seconds increase per each additional cycle (25 cycles); 68°C for 7 minutes (1 cycle) and holding at 4°C. The products of the PCR reactions were separated by electrophoresis through agarose gels containing IX TAE buffer and visualized with ethidium bromide and UV light. Any large fragments indicative of successful long-range PCR were excised from the gel and purified using QIAquick PCR purification kit (Qiagen). In order to eliminate the need to restriction map the PCR fragments, the following ligation-independent cloning strategy was employed. The long-range PCR fragment of interest was "purified" using a QIAquick PCR purification kit (Qiagen, Inc., Santa Clarita, California). Single-stranded ends of the PCR fragments were generated by mixing: 0.1-2 μg of the fragment; 2 μl of NEB (New England BioLabs) Buffer 4; 1 μl of 2 mM dTTP, 6 units of T4 DNA polymerase (NEB), H 2 O to total volume of 20 μl and incubating at 25 °C for 30 minutes. The polymerase was inactivated by heating at 75°C for 20 minutes. Single-stranded ends were also created on the Neo r selectable marker fragment by digesting the plasmid vector pDG2 at the unique restriction sites, with Sac I and Sac II (pDG2 depicted in Figure 2A) and treating each reaction with T4 DNA polymerase as above. The vector shown in Figure 1 was prepared with single-stranded ends complementary to those on the long-range PCR fragment. The vector and fragments were then assembled into constructs using either a two-step cloning strategy or a four-way, single-step protocol. Briefly, a reaction containing 10 ng of T4- treated Neo r cassette, 1 μl of T4-treated PCR fragment, 0.2 μl of 0.5 M EDTA, 0.3 μl of 0.5 M NaCl and H 2 O up to 4 μl was heated to 65 °C and allowed to cool to room temperature over approximately 45 minutes. The mixture was then transformed into subcloning efficiency DH5-α competent cells. Example 2: Generation of Constructs from Phage Libraries A mouse embryonic stem cell library was prepared in lambda phage as follows. Genomic libraries were constructed from genomic DNA by partial cleavage of DNA at Sau 3AI sites to yield genomic fragments of approximately 20 kb in length. The terminal sequences of these DNA fragments were partially filled in using Klenow enzyme in the presence of dGTP and dATP and the fragments were ligated using T4 DNA ligase into Xho I sites of an appropriate lambda cloning vector, e.g., lambda Fix II (Stratagene, Inc., La Jolla, California), which had been partially filled in using Klenow in the presence of dTTP and dCTP. Alternatively, the partially digested genomic DNA was size selected using a sucrose gradient and sequences of approximately 20 kb selected for. The enriched fraction was cloned into a Bam HI cut lambda vector, e.g., lambda Datsh II (Stratagene, Inc., La Jolla, California). The library was plated onto 1,152 plates, each plate containing approximately 1,000 clones. Thus, a total of 1.1 million clones (the equivalent of 8 genomes) was plated. The phage were eluted from each plate by adding 4 ml of lambda elution buffer (10 mM MgCl 2 , 10 mM Tris-pH 8.0) to each plate and incubating for 3 to 5 hours at room temperature. After incubation, 2 ml of buffer was collected from each plate and placed into one well of a 96 deep well plate (Costar, In.). Twelve 96-well plates were filled and referred to as the "sub-pool library." Using the sub-pool library, "pool libraries" were made by placing 100 μl of 12 different sub-pool wells into one well of a new 96 well plate. The 12 sub-pool plates were combined to form 1 plate of pool libraries. Using a pair of oligonucleotides that were known to PCR-amplify the gene of interest, supernatant from the 96 pools of the "large-pool library" were amplified. PCR was performed in the presence of 0.5 units of Amplitaq Gold™ (Perkin Elmer), 1 μM of each oligonucleotide, 200 μM dNTPs, 2 μl of a 1 to 5 dilution of the pool (or subpool) supernatant, 50 mM KC1, 100 mM Tris-HCl (pH 8.3), and either 1.5 mM or 1.25 mM MgCl 2 . Cycling conditions were 95°C for 8 minutes (1 cycle); 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 45 seconds (55 cycles); 72°C for 7 minutes (1 cycle) and holding at 4°C. Depending on the gene, between about 3 and 12 pool yielded positive signals as identified on agarose gels as described in Example 1. In cases where further purification was necessary (i.e. where a clear signal was not present after amplification), the 12 sub-pools making up the pool were subjected to amplification using the same primers and a single sub-pool (1000 clones) was identified. Generation of flanking fragments As described above, knock-out constructs contain two blocks of DNA sequence homologous to the target gene, flanking a positive selection marker. Long-range PCR was performed from the pools of lambda clones positively identified as described above in Example 2. Each fragment was generated using a pair of oligonucleotides with predetermined sequences lacking one type of base and complementary to predetermined sequences on the vector. The fragments obtained were between 1 and 5 kb. A third fragment, longer than 5 kb, is also generated using appropriate oligonucleotides. This third fragment was then used to obtain DNA sequences near the gene to be knocked out but outside of the vector. Example 3: Two-Step Cloning- General Procedure The pDG2 plasmid vector (Figure 2A) contains unique restriction sites Sac π and Sac I. Appropriate single-stranded annealing sites were generated by digesting the pDG2 vector with either restriction enzyme Sac II or Sac I and treating each reaction with T4 DNA polymerase and dTTP as described above. Four reactions were set up in microtitre plates for each vector, the reaction containing 1 μl of either (1) T4 DNA polymerase-treated fragments; (2) a 1:10 dilution of the T4-treated fragments reaction; (3) a 1: 100 dilution of the T4-treated fragments or (4) H 2 O (no insert control). The microtitre plates were sealed, placed in-between two temperature blocks heated to 65°C, and allowed to cool slowly at room temperature for 30 to 45 minutes. The microtitre plate was then placed on ice and 20-25 μl of subcloning efficiency competent cells added to each well. The plate was incubated on ice for 20-30 minutes. The microtitre plate was then placed between two temperature blocks heated to 42°C for 2 minutes, followed by 2 minutes on ice. 100 μl of LB was added to each well, the plate covered with parafilm and incubated 30-60 minutes at 37 °C. The entire contents of each well were plated on one LB-Amp plate and incubated at 37°C overnight. Between about 12-24 colonies were picked from plates which had at least 2-4 times more colonies than the no insert control. The colonies were grown in deep well plates overnight at 37°C and then the plasmid DNA extracted using a Qiagen mini-prep kit. The plasmid DNA was digested with Not I and Sal I enzymes. As shown in Figure 2A, a Not I Sal I digestion will generate a large fragment containing cloning sites 3 and 4 and a smaller fragment containing cloning sites 1 and 2 and the Neo r gene. After digestion, the reactions were run on a 0.8% agarose gel containing 0.2 μg/ml ethidium bromide. For no inserts, two bands were present, one of 1975 base pairs and one of 2793 base pairs. When an insert fragment was present, at least one of these bands would be larger because it would also contain a fragment (insert 1 or 2) either at the annealing site 1/2 or the site 3/4. The insert bands were excised and treated with a QIAquick gel extraction kit. A second ligation reaction was performed containing 1 μl of 10X ligase buffer (50 mM Tris-HCI pH 7.5, 10 mM MgCl 2 , 10 mM dithiothreitol, 1 mM ATP, 25 μg/ml bovine serum albumin), 1 μl T4 DNA ligase, 1-2 μl fragment (site 3/4 band), 5 μl of site 1/2 band and H 2 O up to 10 μl. Controls were also set up replacing either the site 3/4 fragment or the site 1/2 fragment with water. The reactions were incubated 1 to 2 hours at room temperature and transformed with 25 μl of competent cells. The following description applies to the Examples that follow. Sequences of many of the target genes are known and publicly available and were primarily obtained from the EST database. The oligonucleotide primers for PCR amplification of the target genes were prepared based on these sequences. "Flanking DNA" in the context of these examples refers to the genomic sequences flanking the region in the target gene that is to be deleted or mutated. "Flanking DNA" is also described above as the blocks of DNA sequence homologous to the target gene. Rl genomic library refers to a genomic library prepared from the Rl ES cell line. Such libraries can be prepared such as described in Example 1. To date, the methods of the invention have been practiced in about 200 known and novel target genes. Example 4: Two-way Cloning of Targeting Construct for Target 2, a Metalloprotease Gene Identification of flanking DNA for Target 2, a metalloprotease gene. Individual pools of an Rl genomic library were PCR-amplified under standard conditions using Oligos #174 (SEQ ID NO: 19) and #180 (SEQ ID NO:20) in order to identify individual wells containing genomic DNA of target #2 as indicated by the presence of a 500 bp band. A total of 12 pools, each containing approximately 12,000 clones were identified (pools A5, A7, C2, D2 E5, E10, F7, Gl, G7, H2, H4, H7). Pool C2 was then amplified using oligos 454 (SEQ ID NO:21) and 463 (SEQ ID NO:22) to generate a 2000 bp band, and pool H2 was amplified using oligos 464 (SEQ ID NO:23) and 42 (SEQ ID NO:24) to generate a 2700 bp band. These two bands containing flanking DNA for target 2. Construction of targeting construct. Each band containing flanking DNA for target 2 was gel-purified from an agarose gel and the ends were treated individually with T4 DNA polymerase in the presence of dTTP in order to produce single stranded overhangs. Each of these bands was then cloned individually into plasmid vector pDG2 (shown in Figure 2A). The C2 band was cloned into Sac II-digested pDG2 that had been treated with T4 DNA polymerase in the presence of dATP, by ligation-independent cloning. In a separate reaction, the H2 band was cloned into SAC I-digested pDG2 that had been treated with T4 DNA polymerase in the presence of dATP by ligation-independent cloning. In order to move the two flanking arms into a single targeting vector, each vector above was digested with Not I/Sal I and the 4 kb fragment containing the C2 band and the 5 kb fragment containing the H2 band were gel-purified. These two fragments were ligated together with T4 DNA ligase using standard conditions, and recombinants containing both flanking arms were identified. Out of 12 colonies examined, all 12 were correct, i.e. contained both arms correctly flanking the positive selection marker, Neo r . Example 5: Two-way Cloning of Targeting construct for Target 54, a Serine Protease Gene Identification of flanking DNA for target 54 Individual pools of an Rl genomic library were PCR-amplified under standard conditions using oligos #151 (SEQ ID NO:25) and #155 (SEQ ID NO:26) in order to identify individual wells containing genomic DNA of target #54 as indicated by the presence of a 179 bp band. A total of 12 pools, each containing approximately 12,000 clones were identified (pools A4, A10, B2, B9, C9, El, E6, F8, G4, H6, H7, and H9). Pool G4 was then amplified using oligos 454 (SEQ ID NO:27) and 465 (SEQ ID NO:28) to generate a 1400 bp band and pool H7 was amplified using oligos 466 (SEQ ID NO:29) and 42 (SEQ ID NO: 24) to generate a 3000 bp band. These two bands contained flanking DNA for target 54. Construction of targeting construct. Each band was gel-purified from an agarose gel and the ends were treated individually with T4 DNA polymerase in the presence of dTTP in order to produce single stranded overhangs. Each of these bands was then cloned individually into pDG2. The G4 band was cloned into Sac II cut pDG2 that had been treated with T4 DNA polymerase in the presence of dATP, by ligation-independent cloning. In a separate reaction, the H7 band was cloned into Sac I cut pDG2 that had been treated with T4 DNA polymerase in the presence of dATP by ligation-independent cloning. In order to move the two flanking arms into a single targeting vector, each vector above was digested with Not I/Sal I and the 6 kb fragment containing the G4 band and the 8 kb fragment containing the H7 band were gel-purified. These two fragments were ligated together with T4 DNA ligase using standard conditions and recombinants containing both flanking arms were identified. Out of 24 colonies examined, 14 had the correct inserts. Example 6: Single-step (Four- Way) Cloning - General Procedure Because each single-stranded annealing site is unique, a four-way ligation strategy was also used to generate constructs in a single step. The annealing reactions were set up as described above except that each reaction contained a vector digested with both Sac I and Sac II, and both T4-treated fragments were added to these reactions. Example 7: Four-way Cloning of Targeting Construct for Target 43, a Gene for a G-protein Coupled Receptor Identification of flanking DNA for target 43 Individual pools of an Rl genomic library were PCR-amplified under standard conditions using oligos #1 (SEQ ID NO:30) and #2 (SEQ ID NO: 31) in order to identify individual wells containing genomic DNA of target #43 as indicated by the presence of a 414bp band. A total of 11 pools, each containing approximately 12,000 clones were identified (pools A32, A5, A9, B4, D4, D10, El, E9, F9, G7, and G8). Pool El was then amplified using oligos 41 (SEQ ID NO:32) and 38 (SEQ ID NO:33) to generate a 1500 bp band and pool D10 was amplified using oligos 40 (SEQ ID NO:34) and 37 (SEQ ID NO:35) to generate a 3500 bp band. These two bands contained flanking DNA for target 43. Construction of targeting construct: Each band was gel-purified from an agarose gel and the ends were treated individually with T4 DNA polymerase in the presence of dTTP in order to produce single stranded overhangs. These inserts were then mixed with -50 ng of pDG2 that had been digested with both Sac I and Sac II followed by treatment with T4 DNA polymerase in the presence of dATP. The DNA mixture was heated to 65 °C for 2 minutes followed by a 5 minute incubation on ice. The annealed DNA was then transformed into competent DH5-α cells and recombinant molecules were obtained by selection on ampicillin agarose plates. After incubation overnight at 37°C, individual colonies were picked and grown up for analysis. Recombinant molecules were identified by appropriate restriction enzyme digestion. Out of 52 colonies examined, 35 had the correct restriction pattern for the expected product. Example 8: Four- ay Cloning of Targeting Construct for Target 244, a Novel Gene Identification of flanking DNA for target 244 Individual pools of an Rl genomic library were PCR-amplified under standard conditions using oligos #540 (SEQ ID NO:36) and #546 (SEQ ID NO:37) in order to identify individual wells containing genomic DNA of target #244 as indicated by the presence of a 246bp band. A total of 16 pools, each containing approximately 12,000 clones were identified (pools Al, Bl, A3, A5, A6, B6, A8, C9, D10, El, F2, E5, E6, F10, G9, and H8). Pool G9 was then amplified using oligos 445 (SEQ ID NO:38) and 667 (SEQ ID NO:39) to generate a 1300 bp band and pool A6 was amplified using oligos 668 (SEQ ID NO:40) and 42 (SEQ ID NO:24) to generate a 1600 bp band. These two bands contained flanking DNA for target 244. Construction of targeting construct Each band was gel-purified from an agarose gel and the ends were treated individually with T4 DNA polymerase in the presence of dTTP in order to produce single stranded overhangs. These inserts were then mixed with ~50ng of pDG2 that had been digested with both Sac I and Sac II followed by treatment with T4 DNA polymerase in the presence of dATP. The DNA mixture was heated to 65°C for 2 minutes followed by a 5 minute incubation on ice. The annealed DNA was then transformed into competent DH5-α cells and recombinant molecules were obtained by selection on ampicillin agarose plates. After incubation overnight at 37°C, individual colonies were picked and grown up for analysis. Recombinant molecules were identified by appropriate restriction enzyme digestion. Out of 12 colonies examined, 2 had the correct restriction pattern for the expected product. Examples 9 and 10 below provide the plasmid PCR method (schematized in Figure 1) as an alternative and preferred method over the 2-way and 4-way strategies described in the Examples above. Example 9: Plasmid PCR Method of Cloning Targeting Construct for Target 227, a Novel Gene Amplification of genomic clone Individual pools of a plasmid PCR genomic library made from Rl ES cells, cloned into lambda Zap II and subsequently excised using Ml 3 helper phage ediated-excision, were amplified using oligos 907 (SEQ ID NO:41) and 908 (SEQ ID NO:42). These oligos amplified a product of approximately 9 kb from pool 6 of the library. This fragment, containing both flanking arms for target 227 as well as the plasmid pBluescript backbone, was isolated from an agarose gel. Construction of targeting construct The isolated DNA fragment was treated with T4 DNA polymerase in the presence of dTTP in order to generate appropriate single-stranded ends. This fragment was then annealed (ligation-independent) with a Neo r gene fragment obtained from pDG2 that had been digested with both Sac I and Sac II followed by treatment with T4 DNA polymerase in the presence of dATP. The digestion and polymerase treatment yielded a Neo r gene with ends that would specifically anneal to the target 227 fragment. Annealing reactions were set up essentially as described above and a target 227 construct was obtained (13 out of 14 clones were correct). Example 10: Plasmid PCR Method of Cloning Targeting Construct for Target 125, a Nuclear Hormone Receptor Gene Amplification of genomic clone Individual pools of a plasmid PCR library made from Rl ES cells, cloned into lambda Zap II and subsequently excised using M13 helper phage mediated excision were amplified using oligos 1157 (SEQ ID NO:43) and 1158 (SEQ ID NO:44). These oligos amplified a product of approximately lOkb from pool 10 of the library. This fragment, containing both flanking arms for target 125 as well as a pBluescript backbone, was isolated from an agarose gel. Construction of targeting construct The isolated DNA fragment was treated with T4 DNA polymerase in the presence of dTTP in order to generate appropriate single-stranded ends. This fragment was then annealed with a Neo r gene fragment obtained from pDG2 that had been digested with both Sac I and Sac II followed by treatment with T4 DNA polymerase in the presence of dATP. This yielded a Neo r gene with ends that would specifically anneal to the target 125 construct was obtained (12 out of 18 clones were correct). Example 11: Use of GFP as screening marker The addition of the GFP (Green Fluorescent Protein) gene outside the region of homology with the target gene allows one to enrich for homologous recombinants (recombination occurring between the targeting construct and the target gene in the ES cell) by screening ES cell colonies under a fluorescent light. Rapidly growing ES cells were trypsinized to make single cell suspensions. The respective targeting vector was linearized with a restriction endonuclease and 20 μg of DNA was added to 10 x 10° ES cells in ES medium {High Glucose DMEM (without L-Glutamine or Sodium Pyruvate) with LIF (Leukemia Inhibitory Factor-Gibco 13275-029 "ESGRO") 1,000 units/ml, and 12% Fetal Calf Serum}. Cells were placed into a 2 mm gap cuvette and electroporated on a BTX electroporator at 400 μF resistance and 200 volts. Immediately after electroporation, ES cells were plated at lx 10° cells per 100 mm gelatinized tissue culture plate. 48 hours later, media was changed to ES media + G418 (200 μg/ml). Media was changed on days 4, 6, and 8 with ES media + G418 (200 μg/ml). On days 10-12 the plates were then placed under an ultraviolet light and the ES cell colonies were scored on whether or not they were fluorescent. The basis of this experiment is that the fluorescent cells have randomly integrated the targeting vector and the GFP gene is intact. Cells that have undergone homologous recombination will have deleted the GFP gene and not fluoresce; these are the clones of interest. Example 12: Knockout of Target T243 and Analysis of Homozygous Knockout Mutant Mice Identification of Flanking DNA for Target T243 Individual pools of an Rl genomic library were PCR-amplified under standard conditions using oligos # 426 (SEQ ID NO:55) and # 432 (SEQ ED NO:56) to identify individual wells containing genomic DNA of target T243 as indicated by the presence of a 150 bp band. A total of 48 pools, each containing approximately 12,000 clones were identified (pools Al, A2, A9, B4, Bll, B12, C3, C8, Cll, C12, Dl, D3, E4, F3, G4, G5, G6, G12. H4, H5 and H12). Pool H10 was then amplified using oligos # 488 (SEQ ED NO:48) [primer with single-stranded tail sequences] and # 454 (see Figure 8) to generate a 2700 bp band. Pool A7 was then amplified using oligos # 489 (SEQ ED NO:49) [primer with single-stranded tail sequences] and # 42 (see Figure 8) to generate a 5200 bp band. These two bands contained flanking DNA for target T243, (SEQ ED NO:50) and (SEQ ID NO: 51). Construction of Targeting Construct Each band was gel-purified from an agarose gel and the ends were treated individually with T4 DNA polymerase in the presence of dTTP in order to produce single-stranded overhangs. These inserts were then mixed with -50 ng of pDG2 that had been digested with both Sac I and Sac II followed by treatment with T4 DNA polymerase in the presence of dATP. The DNA mixture was heated to 65°C for 2 minutes followed by a 5 minute incubation on ice. The annealed DNA was then transformed into competent DH5-α cells and recombinant molecules were obtained by selection on ampicillin agarose plates. After incubation overnight at 37°C, individual colonies were picked and grown up for analysis. Recombinant molecules were identified by appropriate restriction enzyme digestion. Introduction of Targeting Construct into ES cells and Homologous Recombination Rapidly growing ES cells were trypsinized to make single cell suspensions. The T243 targeting vector was linearized with a restriction endonuclease and 20 μg of DNA was added to 10x10° ES cell in ES medium {High Glucose DMEM (without L-glutamine or Sodium Pyruvate) with LEF (Leukemia Inhibitory Factor - Giboco 13275-029 "ESGRO") 1,00 units/ml, and 12% Fetal Calf Serum}. Cells were placed into a 2 mm gap cuvette and electroporated on a BTX electroporator at 400 μF resistance and 200 volts. Immediately after electroporation, ES cells were plated at lxlO 6 cells per 100 mm gelatinized tissue culture plate. 48 hours later, media was changed to ES media + G418 (200 mg/ml). Media was changed on days 4, 6, and 8 with ES media + G418 (200 mg/ml). On day 10-12, G418 -resistant colonies (average of 192 colonies) were picked into duplicate 96-well plates. After 2-5 days of culture in ES medium, one plate was frozen in 50% FBS, 40% DMEM, and 10% DMSO. The second plate was overgrown and refed for 8-10 days before lysis to prepare DNA for analysis (lysis buffer: 10 mM Tris pH 7.5, 10 mM EDTA pH 8.0, 10 mM NaCl, 0.5% Sarcosyl, and 1 mg/ml Poteinase K). The DNA was then precipitated with 2 volumes of ethanol and resuspended in the appropriate buffer. Upon confirmation of a homologous recombination event, a positive well from duplicate plates were thawed into 24-well tissue culture dishes that had previously been plated with mitomycin C-treated mouse embryonic fibroblasts (24 hours prior). The cells were grown to sufficient levels for diploid aggregation (CD-I host strain) and additional freezing of stock vials. For general procedures for the handling of ES cells and the production of chimeric mice from ES cells, refer to Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (E.J. Robertson, ed. IRL Press, Oxford (1987)). Reaggregation blastocysts were implanted into pseudo-pregnant female CD-I mouse. Highly chimeric mice were then bred to produce germline transmission of the mutated 243 gene. Generation of homozygous T243 knockout mice and analysis of mutant phenotype Heterozygous T243 knockout mice were bred and the homozygous knockout offspring compared to normal and heterozygous littermates for obvious phenotypic differences. Homozygotes were initially hyperactive as compared to normal littermates and had very dry skin. By about 15-17 days, homozygous knockout mice began to appear increasingly unstable and lethargic; by about 19-21 days, homozygotes showed signs of shivering and impending death. Homozygous knockout mice which were not found dead, were sacrificed at approximately 23-25 days for further analysis (see below). Figures 9 and 10 shows the results of daily measurements of length and weight, and the calculation of weight/length ratios for the progeny of two typical matings between two heterozygous 243 knockout mice. Homozygous pups were approximately the same size or slightly smaller than wild type or heterozygous littermates at birth. With age, however, both weight gain and lengthwise growth were markedly decreased in homozygous knockout pups. By 15-17 days, homozygotes began to lose weight, such weight loss continuing until death at approximately 3 weeks. Necropsy was performed on 6 homozygous mutants (4 female, 2 male) and 3 controls (2 female, 1 male). Significant differences attributable to the 243 mutation were observed in bone and kidney tissues. Bone Mutant mice had abnormal cartilage and a generalized reduction of bone formation. Specifically, shortening of both the axial and appendicular skeleton was observed. Proximal and distal bones of the limbs were proportionally shortened and joint cartilage lacked alcian blue staining. The distal femur had a thin growth plate and thin to absent epiphyseal cartilage. A single mutant mouse had a microfracture extending diagonally from the cortex through the metaphysis into the physis (suggestive of growth plate fragility). Within the physes of all mutant mice, chondrocyte columns in the proliferating and hypertrophic zones were short. Cartilaginous spicules within the metaphysis were short and widely spaced. Occasional spicules were haphazardly oriented. Osteoblasts were abundant and frequently piled up along cartilaginous spicules. Epiphyseal cartilage was thin and often replaced by fibrous connective tissue. The epiphyseal surface showed decreased staining with alcian blue. Cartilage at the epiphyseal/physeal junction was slightly flared with an irregular, prominent edge that overhung the physis. Mutant stemebrae were found to be irregular. Growth plates were either lacking or discontinuous. Large, irregular islands of cartilage extended into the shaft of the sternebra and occasionally had secondary ossification centers. Edges of the cartilage were flared. Based on alcian blue stains, vertebral bodies were variably ossified. Some were small and predominantly cartilaginous with irregular and thin growth plates showing tapered lateral processes. Kidney All of the mutant mice had dysplastic changes in both kidneys that were most prominent in the corticomedullary junction and to a lesser extent in the cortex. The kidneys were small and lacked normal architecture. The cortex was thin and some glomeruli were subcapsular. Subcapsular glomeruli were small with shrunken, hypercellular glomerular tufts indicating immaturity. The corticomedullary area lacked radiating arcuate vessels and distinct tubule formation. Tubular epithelial cells within the corticomedullary junction were haphazardly arranged into sheets, piles, and clusters. Some tubular epithelial cells were small and darkly basophilic, thus appearing to be regenerative. As is apparent to one of skill in the art, various modifications of the above embodiments can be made without departing from the spirit and scope of this invention. These modifications and variations are within the scope of this invention.

Claims: We claim: 1. A cell comprising a disruption in a target DNA sequence encoding a TRP. 2. The cell of claim 1, wherein the disruption is produced by the method comprising: (a) obtaining a first sequence homologous to a first region of the target DNA sequence; (b) obtaining a second sequence homologous to a second region of the target DNA sequence; (c) inserting the first and second sequences into a targeting construct; and (d) introducing the targeting construct into the cell to produce a homologous recombinant resulting in a disruption in the target DNA sequence. 3. The cell of claim 2, wherein the method further comprises: subsequent to step (b); (i) providing a vector having a gene encoding a positive selection marker; and (ii) using ligation-independent cloning to insert the first and second sequences into the vector to form the construct; wherein the positive selection marker is located between the first and second sequences in the construct. 4. The cell of claim 3, wherein the vector further comprises a gene coding for a screening marker. 5. The cell of claim 1, wherein said target DNA sequence comprises CTG trinucleotide repeats. 6. The cell of claim 5, wherein said CTG trinucleotide repeats encode leucine residues. 7. The cell of claim 1, wherein the target gene sequence is T243 or a naturally occurring allelic variation thereof. 8. The cell of claim 1, wherein the target DNA sequence comprises SEQ ID NO:47. 9. The cell of claim 1, wherein the target DNA sequence comprises SEQ ID NO:45 and SEQ ID NO:46. 10. The cell of claim 3, wherein the vector further comprises one or more recombinase target sites flanking the positive selection marker. 11. The cell of claim 2, wherein the first sequence is SEQ ID NO:50 and the second sequence is SEQ ID NO:51. 12. The cell of claim 2, wherein the first and second sequences are obtained by the method comprising: (a) obtaining two primers capable of hybridizing with said target, wherein the primers form the endpoints of amplification products; (b) providing a mouse genomic DNA library containing the target sequence; (c) annealing said primers to complementary sequences in said library; (d) amplifying said first and second sequences; and (e) isolating the products of the amplification reaction. 13. The cell of claim 12, wherein the first primer is SEQ ID NO:45. 14. The cell of claim 12, wherein the second primer is SEQ ID NO:46. 15. The cell of claim 12, wherein said amplification comprises PCR. 16. The cell of claim 15, wherein said amplification further comprises long-range PCR. 17. The cell of claim 12, wherein said mouse genomic library is a plasmid library. 18. The cell of claim 12, wherein said mouse genomic library is a bacteriophage library, said method further comprising obtaining two primers which are capable of hybridizing to bacteriophage vector sequences such that the amplification product terminates at one end with a target sequence primer and at the other end terminates with a vector primer. 19. The cell of claim 1, wherein said cell comprises a homozygous disruption in the target DNA sequence. 20. The cell of claim 1, wherein said cell is murine. 21. The cell of claim 1, wherein said cell is human. 22. The cell of claim 1, wherein said cell is a stem cell. 23. The stem cell of claim 22, wherein said stem cell is an embryonic stem cell. 24. A blastocyst containing the embryonic stem cell of claim 23. 25. The targeting construct used in the method of claim 2. 26. A non-human vertebrate comprising a heterozygous disruption in a gene encoding a TRP. 27. The vertebrate of claim 26, wherein said vertebrate is a mammal. 28. The vertebrate of claim 26, wherein said mammal is a mouse. 29. The mouse of claim 28, wherein said mouse is produced by the method comprising: (a) incoφorating a stem cell of claim 1 or 2 into a blastocyst; (b) implanting the resulting blastocyst into a pseudopregnant mouse wherein said pseudopregnant mouse gives birth to a chimeric mouse containing the disrupted gene encoding the TRP in its germ line; and (c) breeding said chimeric mouse to generate a mouse comprising a heterozygous disruption in the gene encoding the TRP. 30. The mouse of claim 28, said mouse produced by the method comprising: (a) incoφorating a stem cell of claim 3 into a blastocyst; (b) implanting the resulting blastocyst into a pseudopregnant mouse wherein said pseudopregnant mouse gives birth to a chimeric mouse containing the disrupted gene encoding the TRP in its germ line; and (c) breeding said chimeric mouse to generate a mouse comprising a heterozygous disruption in the gene encoding the TRP. 31. The mouse of claim 28, wherein said TRP is encoded by T243 or a naturally occurring allelic variation thereof. 32. A knockout mouse comprising a homozygous disruption in a gene encoding a TRP, wherein said disruption inhibits the production of the wild type TRP, said mouse produced by mating together two mice according to claim 28. 33. The knockout mouse of claim 32, wherein the disruption alters a TRP gene promoter, enhancer, or splice site such that the mouse does not express a functional TRP. 34. The knockout mouse of claim 32, wherein the disruption is an insertion, missense, frameshift or deletion mutation. 35. The knockout mouse of claim 32, wherein the phenotype of the adult mouse comprises reduced weight relative to a wild type adult mouse. 36. The knockout mouse of claim 35, wherein said phenotype further comprises weight reduced by at least about 15% relative to a wild type adult mouse. 37. The knockout mouse of claim 32, wherein the adult phenotype of the mouse decreased length relative to a wild type adult mouse. 38. The knockout mouse of claim 37, wherein said phenotype further comprises length decreased at least about 10% relative to a wild type adult mouse. 39. The knockout mouse of claim 32, wherein the adult phenotype of the mouse comprises a decreased ratio of weight to length relative to a wild type adult mouse. 40. The knockout mouse of claim 39, wherein said phenotype further comprises a ratio of weight to length decreased at least about 20% relative to a normal, wild type adult mouse. 41. The knockout mouse of claim 32, wherein the phenotype of the adult mouse relative to a wild type mouse adult comprises: (a) reduced weight; (b) decreased length; and (c) decreased ratio of weight to length. 42. The knockout mouse of claim 32, wherein the phenotype of the adult mouse comprises symptoms associated with cartilage disease. 43. The knockout mouse of claim 32, wherein the phenotype of the adult mouse comprises symptoms associated with bone disease. 44. The knockout mouse of claim 32, wherein the phenotype of the adult mouse comprises symptoms associated with kidney disease. 45. The knockout mouse according to claim 41, wherein the phenotype is not apparent at birth. 46. A cell or cell line derived from the mouse of claim 28 or 32 containing said disruption. 47. A method of identifying agents capable of affecting a phenotype of a knockout mouse comprising: (a) administering a putative agent to the knockout mouse of claim 32; (b) measuring the response of the knockout mouse to the putative agent; and (c) comparing the response with that of a wild type mouse; (d) thereby identifying the agent capable of affecting a phenotype of a knockout mouse. 48. An agent identified according to the method of claim 47. 49. A method of determining whether expansion of the trinucleotide repeat in a gene encoding a TRP produces a phenotypic change comprising: (a) providing the knockout cell of claim 10 and a synthetic nucleic acid comprising trinucleotide repeats flanked by recombinase target sites; (b) contacting said knockout stem cell with said synthetic nucleic acid in the presence of a recombinase which recognizes said recombinase target sites, such that recombination occurs between the synthetic nucleic acid, thereby producing a transgenic cell; and (c) comparing the phenotype of said transgenic cell with a wild type cell; thereby determining whether trinucleotide expansion produces a phenotypic change. 50. The method of claim 49, wherein said trinucleotide repeats comprise CTG. 51. The method of claim 49, wherein said method comprises the use of a Cre recombinase-lox target system. 52. The method of claim 49, wherein said method comprises the use of a FLP recombinase-FRT target system. 53. A knockout cell or cell line comprising a disruption in a target DNA sequence encoding a TRP. 54. The knockout cell or cell line of claim 53, wherein said cell is derived from the mouse of claim 32. 55. Tissue derived from the mouse of claim 28 or 32. 56. The knockout cell of claim 53 wherein the TRP is encoded by T243 or a naturally occurring allelic variation thereof. 57. A method of identifying agents capable of affecting a phenotype of a knockout cell line comprising: (a) contacting the knockout cell of claim 53 with a putative agent; (b) measuring the response of the cell to the putative agent; and (c) comparing the response with that of a wild type cell; (d) thereby identifying the agent capable of affecting a phenotype of a knockout cell. 58. A cell line comprising a nucleic acid sequence encoding a TRP operably linked to a promoter functional in said cell line. 59. The cell line of claim 58, wherein the TRP is encoded by T243 or a naturally occurring allelic variation thereof 60. The cell line according to claim 59, wherein the TRP consists essentially of the amino acid sequence SEQ ID NO:52 or a naturally occurring allelic variation thereof. 61. The Trinucleotide Repeat Protein encoded by T243 or a naturally occurring allelic variation thereof. 62. A murine TRP consisting essentially of the sequence of SEQ ID NO:52 or a naturally occurring allelic variation thereof. 63. A human TRP consisting essentially of the sequence of SEQ ED NO:58 or a naturally occurring allelic variation thereof. 64. A nucleic acid sequence encoding the murine TRP of claim 62, of the sequence SEQ ED NO:47 or a naturally occurring allelic variation thereof. 65. A nucleic acid sequence encoding the human TRP of claim 63, of the sequence SEQ ID NO:47 or a naturally occurring allelic variation thereof.

Inventors: ALLEN KEITH D, KLEIN ROBERT, MATTHEWS WILLIAM, MOORE MARK, ALLEN, KEITH, D., KLEIN, ROBERT, MATTHEWS, WILLIAM, MOORE, MARK
Applicants: ALLEN KEITH D, DELTAGEN INC, KLEIN ROBERT, MATTHEWS WILLIAM, MOORE MARK, ALLEN, KEITH, D., DELTAGEN, INC., KLEIN, ROBERT, MATTHEWS, WILLIAM, MOORE, MARK
Application year: 2000
Main classification: C07H21
IPC codes: A01K67, A61K45, A61P13, A61P19, C07K14, C12N5, C12N9, C12N15, C12Q1