TRANSGENIC NON-HUMAN ANIMALS CAPABLE OF PRODUCING HETEROLOGOUS ANTIBODIES

Abstract: The invention relates to transgenic non-human animals capable of producing heterologous antibodies, i.e., antibodies encoded by immunoglobulin heavy and light chain genes not normally found in the genome of that species of non-human animal. In one aspect of the invention, transgenes encoding unrearranged heterologous human immunoglobulin heavy and light chains are introduced into a non-human animal thereby forming a transgenic animal capable of producing antibodies encoded by human immunoglobulin genes. Such heterologous human antibodies are produced in B-cells which are thereafter immortalized, e.g., by fusing with an immortalizing cell line such as a myeloma or by manipulating such B-cells by other techniques to perpetuate a cell line capable of producing a monoclonal heterologous antibody. The invention also relates to heavy and light chain immunoglobulin transgenes for making such transgenic non-human animals as well as methods and vectors for disrupting endogenous immunoglobulin loci in the transgenic animal. The invention also includes methods to generate a synthetic immunoglobulin variable region gene segment repertoire used in transgene construction and methods to induce heterologous antibody production using animals containing heterologous rearranged or unrearranged heavy and light chain immunoglobulin transgenes.

Description: TRANSGENIC NON-HUMAN ANIMALS CAPABLE OF PRODUCING HETEROLOGOUS ANTIBODIES TECHNICAL FIELD The invention relates to transgenic non-human animals capable of producing heterologous antibodies, transgenes used to produce such transgenic animals, immortalized B-cells capable of producing heterologous antibodies, methods and vectors for disrupting endogenous immunoglobin loci, methods to generate a synthetic immunoglobulin variable region gene segment repertoire, and methods to induce heterologous antibody production. BACKGROUND OF THE INVENTION One of the major impediments facing the development of in vivo applications for monoclonal antibodies in humans is the intrinsic i munogenicity of non-human immunoglobulins. Patients respond to therapeutic doses of rodent monoclonal antibodies by making antibodies against the rodent immunoglobulin sequences. These human anti-mouse antibodies (HAMA) neutralize the therapeutic antibodies and can cause acute toxicity. The HAMA response is less dramatic in immunodeficient patients. Therefore, intrinsic immunogenicity has not prevented the use of rodent monoclonal antibodies for the treatment of graft rejection, which involves the temporary attenuation of the patient's immune response. Rodent antibodies may also be useful for treating certain lymphomas that involve immunodeficiencies. However, even immunodeficient patients can mount a HAMA response which leads to a reduction in safety and efficacy. The present technology for generating monoclonal antibodies involves pre-exposing, or priming, an animal (usually a rat or mouse) with antigen. This pre-exposure leads to the formation of splenic B-cells that secrete immunoglobulin molecules with high affinity for the antigen. Spleen cells from a primed animal are then fused with myeloma cellε to form immortal, antibody secreting, hybridoma cells. Individual hybridoma clones are screened to identify those cells producing immunoglobulins directed against a particular antigen. The genetic engineering of individual antibody genes has been proposed. Two genetic engineering approaches have been reported: chimeric antibodies and complementarity- determining-region (CDR) grafting. The simplest approach, chimeric antibodies, takes advantage of the fact that the variable and constant portions of an antibody molecule are encoded on separate exons. By simply fusing the variable region exons of a rearranged mouse antibody gene with a human constant region exons, a hybrid antibody gene can be obtained (Morrison, S.L., et al. (1984), Proc. Natl. Acad. Sci. USA, 81, 6851-6855) . The major problem with this approach iε that while the highly immunogenic mouse Fc region is eliminated, the remaining mouse Fab sequences are still immunogenic (Brugge ann, et al. (1989), J_ EXP. Med. , 170, 2153-2157) . The CDR grafting approach uses computer modeling to generate a completely artificial antibody in which the only mouse sequences are those involved in antigen binding (Riechmann, L. , et al. (1988) , Nature, 332. 323-327) . Each of these approaches requires the prior characterization of a rodent monoclonal antibody directed against the antigen of interest, and both require the generation of a stable transfected cell line that produces high levels of the engineered antibody. Another approach. to the production of human antibodies is a proposal involving the construction of bacterial expression libraries containing immunoglobulin cDNA sequences (Orlandi, et al. (1989), Proc. Natl. Acad. Sci. USA, 86, 3833-3837, and Huse, et al. (1989), Science, 246, 1275-1281). This technique reportedly has only been used to generate antibody fragments derived from mouse cDNA sequences. A number of experiments have reported the use of transfected cell lines to determine the specific DNA sequences required for Ig gene rearrangement (reviewed by Lewiε and Gellert (1989) , Cell. 59, 585-588) . Such reports have identified putative sequences and concluded that the accessibility of these sequences to the recombinase enzymes used for rearrangement is modulated by transcription (Yancopoulos and Alt (1985) , Cell, 40, 271-281) . The sequences for V(D)J joining are reportedly a highly conserved, near-palindromic heptamer and a less well conserved AT-rich nanomer separated by a spacer of either 12 or 23 bp (Tonegawa (1983) , Nature, 302, 575-581; Hesse, et al. (1989) , Genes in Dev.. 3_, 1053-1061) . Efficient recombination reportedly occurs only between sites containing recombination signal sequences with different length spacer regions. The production of transgenic mice containing various forms of immunoglobulin genes has also been reported. Rearranged mouse immunoglobulin heavy or light chain genes have been used to produce transgenic mice. Such transgenes reportedly are capable of excluding the rearrangement of endogenous Ig genes. See e.g. Weaver et al. (1985), Cell, 42, 117-127; Iglesias, et al. (1987), Nature, 330, 482-484; Storb et al. (1985), Banbury Reports, 20, 197-207; Neuberger et al. (1989), Nature. 338, 350-352; Hagman et al. (1989), J. Exp. Med. , 169, 1911-1929; and Storb (1989) in Immunoglobulin Genes, Academic Press, T. Honjo, F.W. Alt and T.H. Rabbittε edε. pp. 303-326. In addition, functionally rearranged human Ig genes including the μ or I conεtant region have been expressed in transgenic mice. Yamamura, et al. (1986) , Proc. Natl. Acad. Sci. USA, 83, 2152-2156; Nussenzweig, et al. (1987), Science, 236, 816-819. In the case of the μ rearranged heavy chain gene, allelic exclusion of endogenous immunoglobulin gene loci was reported. Allelic exclusion, however, does not always occur in all transgenic B-cells. See e.g. Rath, et al. (1989) , J___ Immunol. , 143. 2074-2080 (rearranged μ gene construct); Manz, et al. (1988), J. Exp. Med. r 168, 1363-1381 (μ transgenes lacking transmembrane exons did not prevent rearrangement of the endogenous genes) ; Ritchie, et al. (1984) , Nature, 312, 517-520 and Storb, et al. (1986), Immunol. Rev. , 89, 85-102 (transgenic mice expressing rearranged n tranεgene capable of forming stable heavy/light chain complex only rearrange endogenous K genes in B-cells that fail to correctly rearrange endogenous heavy chain gene); and Manz, et al. (1988), J. Ex . Med.. 168, 1363-1381 (transgenic mice containing K gene encoding light chain incapable of combining with heavy chains, show only a low level of allelic exclusion) . See alεo Nussenzweig, et al. (1988) , Nature, 336, 446-450) ; Durdik, et al. (1989) , Proc. Natl. Acad. Sci. USA, 86. 2346-2350; and Shimizu, et al. (1989), Proc. Natl. Acad. Sci. USA, 86, 8020-8023. Somatic mutation has also been reported in a 15 kb mouse K gene construct in hyperimmunized transgenic mice (O'Brien, et al. (1987), Nature, 326, 405-409; Storb (1989) in Immunoglobulin Genes, Academic Press, T. Honjo, F.W. Alt, and T.H. Rabbitts, eds. pp. 303-326) and in the variable portion of a μ heavy chain transgene (Durdik, et al. (1989) , Proc. Natl. Acad. Sci. USA, 86, 2346-2350) . Ig gene rearrangement, though studied in tissue culture cells, has not been extensively examined in transgenic mice. Only a handful of reportε have been publiεhed describing rearrangement test constructε introduced into mice [Buchini, et al. (1987) , Nature, 326, 409-411 (unrearranged chicken λ transgene) ; Goodhart, et al. (1987) , Proc. Natl. Acad. Sci. USA, 84 , 4229-4233) (unrearranged rabbit K gene) ; and Bruggemann, et al. (1989), Proc. Natl. Acad. Sci. USA, 86 , 6709-6713 (hybrid mouse-human heavy chain) ] . The resultε of such experiments, however, have been variable, in some cases, producing incomplete or minimal rearrangement of the transgene. Based on the foregoing, it is clear that a need exiεtε for heterologous monoclonal antibodies, e.g. antibodies of human origin, derived from a species other than human. Thus, it is an object of the invention herein to provide a source of monoclonal antibodies that may be used therapeutically in the particular species for which they are designed. In accordance with the foregoing object tranεgenic nonhuman animals are provided which are capable of producing a heterologous antibody, such as a human antibody. Further, it is an object to provide B-cellε from such transgenic animals which are capable of expressing heterologous antibodies wherein such B-cells are immortalized to provide a source of a monoclonal antibody specific for a particular antigen. In accordance with this foregoing object, it is a further ob j ect of the invention to provide hybridoma cellε that are capable of producing such heterologous monoclonal antibodies. Still further, it is an object herein to provide heterologous unrearranged and rearranged immunoglobulin heavy and light chain transgenes useful for producing the aforementioned non-human transgenic animals. Still further, it iε an object herein to provide methods to disrupt endogenous immunoglobulin loci in the transgenic animals. Still further, it is an object herein to provide methods to induce heterologous antibody production in the aforementioned transgenic non-human animal. A further object of the invention is to provide methods to generate an immunoglobulin variable region gene segment repertoire that is used to construct one or more transgenes of the invention. The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein iε to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention. SUMMARY OF THE INVENTION In accordance with the foregoing objects, in one aspect of the invention, transgenic non-human animals are provided that contain rearranged, unrearranged or a combination of rearranged and unrearranged heterologous immunoglobulin heavy and light chain transgenes in the germline of the transgenic animal. For each of the foregoing animalε, functionally rearranged heterologous heavy and light chain immunoglobulin transgeneε are found in the B-cellε of the transgenic animal. Heterologous heavy and/or light unrearranged immunoglobulin transgenes are introduced into a host non-human animal to produce a transgenic non-human animal containing a heavy and a light heterologous immunoglobulin gene or an intermediate animal containing one or the other transgene. When incorporated into the germline of such intermediate animals, crosses between one containing a heavy chain tranεgene and one containing a light chain transgene produces a transgenic non-human animal containing both heavy and light heterologous immunoglobulin transgenes. The transgenes of the invention include a heavy chain transgene comprising DNA encoding at least one variable gene segment, one diversity gene segment, one joining gene segment and one constant region gene segment. The immunoglobulin light chain transgene compriseε DNA encoding at least one variable gene segment, one joining gene segment and one constant region gene segment. The gene segmentε encoding the light and heavy chain gene segments are heterologous to the transgenic non-human animal in that they are derived from, or correspond to, DNA encoding immunoglobulin heavy and light chain gene segments from a specieε not consisting of the transgenic non-human animal. In one aspect of the invention, the transgene is constructed such that the individual gene segments are unrearranged, i.e., not rearranged so aε to encode a functional immunoglobulin light or heavy chain. Such unrearranged transgenes permit recombination of the gene segments (functional rearrangement) and somatic mutation of the resultant rearranged immunoglobulin heavy and/or light chains within the transgenic non-human animal when exposed to antigen. In one aspect of the invention, heterologous heavy and light immunoglobulin transgenes comprise relatively large fragments of unrearranged heterologous DNA. Such fragments typically comprise a substantial portion of the C, J (and in the case of heavy chain, D) segments from a heterologous immunoglobulin locus. In addition, such fragments also comprise a substantial portion of the variable gene segmentε. In an alternate embodiment, HP LaserJet Serieε IIHPLASEII.PRSegments. In such transgene constructε, the various regulatory sequences, e.g. promoters, enhancers, class switch regions, recombination signals and the like, comprise corresponding sequences derived from the heterologous DNA. Alternatively, such regulatory sequences may be incorporated into the transgene from the same or a related specieε of the non-human animal used in the invention. For example, human immunoglobulin gene segments may be combined in a transgene with a rodent immunoglobulin enhancer sequence for use in a transgenic mouse. In a method of the invention, a tranεgenic non-human animal containing germline unrearranged light and heavy immunoglobulin transgenes - that undergo VDJ joining during D-cell differentiation - is contacted with an antigen to induce production of a heterologous antibody in a εecondary repertoire B-cell. Such induction causes εo atic mutation in the rearranged heavy and/or light chain transgenes contained in primary repertoire B-cells to produce a heterologous antibody having high affinity and specificity for the antigen. Such antibody producing B-cells may be immortalized by transforming with a virus, or with an oncogene containing DNA construct, or alternatively, immortalized by fusing with a myeloma cell line to form antibody secreting hybridomas. In each instance, cloneε having sufficient affinity and specificity for a particular antigen are selected to provide a source of monoclonal antibody having low immunogenicity in the specieε from which the immunoglobulin gene segmentε of the tranεgenes are derived. Also included in the invention are vectors and methods to disrupt the endogenous immunoglobulin loci in the non-human animal to be used in the invention. Such vectors and methods utilize a transgene, preferably positive-negative εelection vector, which is constructed such that it targets the functional disruption of a class of gene segments encoding a heavy and/or light immunoglobulin chain endogenous to the non-human animal used in the invention. Such endogenous gene segments include diversity, joining and constant region gene segments. In this aspect of the invention, the positive-negative selection vector is contacted with at least one embryonic stem cell of a non-human animal after which cells are selected wherein the positive-negative selection vector has integrated into the genome of the non-human animal by way of homologous recombination. After transplantation, the resultant transgenic non-human animal is substantially incapable of mounting an immunoglobulin— ediated immune response as a result of homologous integration of the vector. Such immune deficient non-human animals may thereafter be used for study of immune deficiencies or used as the recipient of heterologous immunoglobulin heavy and light chain transgenes. The invention also includes methods for generating a synthetic variable region gene segment repertoire to be used in the transgenes of the invention. The method comprises generating a population of immunoglobulin V segment DNAs wherein each of the V segment DNAs encodes an immunoglobulin V segment and contains at each end a cleavage recognition site of a restriction endonucleaεe. The population of immunoglobulin V segment DNAs is thereafter concatenated to form the synthetic immunoglobulin V segment repertoire. Another aspect of the invention includes transgenic nonhuman animals that contain functionally rearranged heterologous heavy and light chain immunoglobulin transgeneε in the germline of the tranεgenic animal. Such animalε contain primary repertoire B-cells that express such rearranged heavy and light transgeneε. Such B-cells are capable of undergoing somatic mutation when contacted with an antigen to form a heterologous antibody having high affinity and specificity for the antigen. The invention also includes transgenic animalε containing germ line cells having a heavy and light transgene wherein one of the said transgenes contains rearranged gene segments with the other containing unrearranged gene segments. In the preferred embodiments, the rearranged transgene is a light chain immunoglobulin transgene and the unrearranged transgene is a heavy chain immunoglobulin transgene. The invention also includes methods for producing heterologous antibodies in a transgenic animal containing primary repertoire B-cells having rearranged heavy and light heterologous immunoglobulin transgeneε. Such tranεgenic animals may be obtained from any of the aforementioned transgenic animals. Thus, the transgenic animal containing unrearranged heavy and light transgenes, the transgenic animal containing rearranged heavy and light transgenes or the animal containing one rearran g ed and one unrearran g ed transcrene in the germline of the animal, each contain primary repertoire B-cellε having rearranged, heterologous heavy and light immunoglobulin transgenes. In the method of the invention, a desired first heterologous antibody is produced which is capable of binding a first antigen. The rearranged immunoglobulin heavy and light transgenes in the primary repertoire B-cells of such animals are known to produce primary repertoire antibodies having sufficient affinity for a second known antigen. In this method, the transgenic non-human animal is contacted, sequentially or simultaneously, with the first and εecond antigen to induce production of the first heterologouε antibody by somatic mutation of the rearranged transgenes. The secondary repertoire B-cells so produced are then manipulated as previously described to immortalize the production of the desired monoclonal antibody capable of binding the first antigen. The present invention also includes plas ids, useful in cloning large DNA fragments (e.g., immunoglobulin genomic fragments) , that have an origin of replication (ORI) , a copy control region (e.g., ROP, or the copy control region of pACYC177, or others known to those skilled in the art), and a cloning site. The plasmids also include a transcription terminator (e.g., trpR or others known to those skilled in the art) downstream of endogenous plasmid-derived promoters such as that of the ampicillin resistance gene (amp R ) . The transcription termination is located upstream of the cloning site so that transcripts originating at the promoter are terminated upstream of the cloning site. In a preferred embodiment, the cloning site iε flanked by rare restriction sites, which are sites consisting of seven, eight, or more nucleotides, instead of the six or fewer nucleotides that make up more common restriction sites; e.g., Not I, Sfi I, and Pac I. Rare restriction sites also include sites that contain nucleotide sequences occurring rarely in natural DNA sequences; i.e., less frequently than about once in every 8,000-10,000 nucleotides. Fig. 1 depicts the complementarity determining regions CDRl, CDR2 and CDR3 and framework regions FRl, FR2, FR3 and FR4 in unrearranged genomic DNA and mRNA expressed from a rearranged immunoglobulin heavy chain gene, Fig. 2 depicts the human λ chain locus, Fig. 3 depicts the human K chain locus, Fig. 4 depicts the human heavy chain locus, Figs. 5 and 6 depict the strategy for generating a synthetic V segment repertoire. Fig. 7 depicts the strategy for functional diεruption of endogenous immunoglobulin loci. Fig. 8 depicts the T-cell mediated secondary response leading to maturation of the B-cell. Fig. 9 depicts somatic mutation and clonal expansion of B-cells in response to two different antigenε. Fig. 10 depicts a transgene construct containing a rearranged IgM gene ligated to a 25 kb fragment that contains human ^3 and 7I constant regions followed by a 700 bp fragment containing the rat chain 3' enhancer sequence. Fig. 11 is a restriction map of the human K chain locus depicting the fragments to be used to form a light chain transgene by way of in vivo homologous recombination. Fig. 12 depicts the construction of pGPl. Fig. 13 depicts the construction of the polylinker contained in pGPl. Fig. 14 depicts the fragments used to construct a human heavy chain transgene of the invention. Fig. 15 depicts the construction of pHIGl and pCONl. Fig. 16 depicts the human C7I fragments which are inserted into pRE3 (rat enhancer 3') to form pREG2. Fig. 17 depicts the construction of pHIG3 ' and PCON. Fig. 18 depicts the fragment containing human D region segments used in construction of the transgenes of the invention. Fig. 19 depicts the construction of pHIG2 (D segment containing plasmid) . Fig. 20 depicts the fragments covering the human Jc and human Cc gene segments used in constructing a transgene of the invention. Fig. 21 depicts the structure of pEμ. Fig. 22 depicts the construction of pKapH. Figs. 23A through 23D depict the construction of a positive-negative selection vector for functionally disrupting the endogenous heavy chain immunoglobulin locuε of mouse. Figs. 24A through 24C depict the construction of a poεitive-negative selection vector for functionally disrupting the endogenous immunoglobulin light chain loci in mouse. Figs. 25 a through e depict the structure of a kappa light chain targeting vector. Figs. 26 a through f depict the structure of a mouse heavy chain targeting vector. Fig. 27 depicts the map of vector pGPe. Fig. 28 depicts the structure of vector pJM2. Fig. 29 depicts the structure of vector pCORl. Fig. 30 depicts the transgene constructs for pIGMl, pHCl and pHC2. Fig. 31 depicts the structure of P β2. Fig. 32 depicts the structure of pVGEl. Fig. 33 depicts the assay resultε of human Ig expresεion in a pHCl transgenic mouεe. Fig. 34 depicts the structure of pJCKl. Fig. 35 depicts the construction of a synthetic heavy chain variable region. Table 1 depicts the sequence of vector pGPe. Table 2 depicts the sequence of gene V R 49.8. DETAILED DESCRIPTION The design of a transgenic non-human animal that responds to foreign antigen εtimulation with a heterologous antibody repertoire, requires that the heterologous immunoglobulin transgenes contained within the transgenic animal function correctly throughout the pathway of B-cell development. Accordingly, the transgenes in one aspect of the invention are constructed so as to produce one or all of the following: (1) high level and cell-type εpecific expression, (2) functional gene rearrangement, (3) activation of and response to allelic exclusion. (4) expression of a sufficient primary repertoire, (5) signal transduction, (6) class switching, (7) somatic hypermutation, and (8) domination of the transgene antibody locus during the immune response. As will be apparent from the following diεcloεure, not all of the foregoing criteria need be met. For example, in those embodiments wherein the endogenous immunoglobulin loci of the transgenic animal are functionally disrupted, the transgene need not activate allelic excluεion. Further, in thoεe embodiments wherein the transgene compriεeε a functionally rearranged heavy and/or light chain immunoglobulin gene, the second criteria of functional gene rearrangement is unnecessary, at least for that transgene which iε already rearranged. For background on molecular immunology, see, Fundamental Immunology, 2nd edition (1989) , Paul William E., ed. Raven Press, N.Y. The Structure and Generation of Antibodies Immunoglobulinε, alεo known as antibodies, are a group of glycoproteins present in the serum and tisεue fluids of all mammals. They are produced in large amountε by plaεma cells (also referred to herein as "secondary repertoire B-cells") which develop from precursor B lymphocytes (referred to herein as "primary repertoire B-cells") . Such primary repertoire B-cells carry membrane-bound immunoglobulin which is similar to that produced by the fully differentiated secondary repertoire B-cell. Contact between primary repertoire B-cells and foreign antigen is required for the induction of antibody formation. The basic structure of all immunoglobulins iε baεed upon a unit consisting of two identical light polypeptide chains and two identical heavy polypeptide chains linked together by disulfide bonds. Each light chain comprises two regions known as the variable light chain region and the constant light chain region. Similarly, the immunoglobulin heavy chain comprises two regions deεignated the variable heavy chain region and the constant heavy chain region. The constant region for the heavy or light chain is encoded by genomic sequences referred to as heavy or light constant region gene segments. The use of a particular heavy chain gene εegment defines the class of immunoglobulin. For am p le in humans the μ constant region gene segments define the IgM class of antibody whereas the use of a 7, 72, 73 or 7 4 constant region gene segment defines the IgG class of antibodies as well as the IgG subclasses IgGl through IgG4. The variable regions of the heavy and light immunoglobulin chains together contain the antigen binding domain of the antibody. Becauεe of the need for diverεity in this region of the antibody to permit binding to a wide range of antigens, the DNA encoding the initial or primary repertoire variable region comprises a number of different DNA segments derived from familieε of εpecific variable region gene εeg ents. In the case of the light chain variable region, such families comprise variable (V) gene εegments and joining (J) gene segments. Thus, the initial variable region of the light chain is encoded by one V gene segment and one J gene segment each selected from the family of V and J gene segments contained in the genomic DNA of the organism. In the case of the heavy chain variable region, the DNA encoding the initial or primary repertoire variable region of the heavy chain comprises one heavy chain V gene segment, one heavy chain diversity (D) gene εegment and one J gene segment, each selected from the appropriate V, D and J families of immunoglobulin gene segments in genomic DNA. The Primary Repertoire The process for generating DNA encoding the heavy and light chain immunoglobulin genes occurs primarily in developing B-cells. Prior to the joining of various immunoglobulin gene segments, the V, D, J and constant (C) gene segments are found, for the most part, in clusters of V, D, J and C gene segmentε in the precursors of primary repertoire B-cellε. Generally, all of the gene segmentε for a heavy or light chain are located in relatively close proximity on a εingle chromosome. Such genomic DNA prior to recombination of the variouε immunoglobulin gene segments iε referred to herein aε "unrearranged" genomic DNA. During B-cell differentiation, one of each of the appropriate family memberε of the V, D, J (or only V and J in the case of light chain genes) gene εegments are recombined to form functionally rearranged heavy and light immunoglobulin genes. Such functional rearrangement is of the variable region segments to form DNA encoding a functional variable region. This gene segment rearrangement proceεε appears to be sequential. First, heavy chain D-to-J joints are made, followed by heavy chain V-to-DJ joints and light chain V-to-J joints. The DNA encoding this initial form of a functional variable region in a light and/or heavy chain iε referred to aε "functionally rearranged DNA" or "rearranged DNA". In the case of the heavy chain, such DNA is referred to aε "rearranged heavy chain DNA" and in the case of the light chain, such DNA is referred to aε "rearranged light chain DNA". Similar language iε used to describe the functional rearrangement of the transgenes of the invention. The recombination of variable region gene segments to form functional heavy and light chain variable regions is mediated by recombination signal sequences (RSS's) that flank recombinationally competent V, D and J segments. RSS'ε necessary and sufficient to direct recombination, comprise a dyad-symmetric heptamer, an AT-rich nonamer and an intervening spacer region of either 12 or 23 base pairs. These signals are conserved among the different loci and species that carry out D-J (or V-J) recombination and are functionally interchangeable. See Oettinger, et al. (1990), Science. 248 , 1517-1523 and references cited therein. The heptamer comprises the sequence CACAGTG or its analogue followed by a spacer of unconserved εequence and then a nonamer having the εequence ACAAAAACC or its analogue. These sequences are found on the J, or downstream side, of each V and D gene segment. Immediately preceding the germline D and J segments are again two recombination signal sequences, first the nonamer and then the heptamer again separated by an unconserved sequence. The heptameric and nonameric sequences following a ^ τ . , V„ or D segment are complementary to those preceding the J τ , D cr J κ segments with which they recombine. The spacers between the heptameric and nonameric sequences are either 12 base pairs long or between 22 and 24 base pairs long. In addition to the rearran g ement of V D and J segments, further diversity is generated in the primary repertoire of immunoglobulin heavy and light chain by way of variable recombination between the V and J segmentε in the light chain and between the D and J segments of the heavy chain. Such variable recombination is generated by variation in the exact place at which such segments are joined. Such variation in the light chain typically occurs within the last codon of the V gene εegment and the firεt codon of the J segment. Similar imprecision in joining occurs on the heavy chain chromosome between the D and J H segments and may extend over as many as 10 nucleotides. Furthermore, several nucleotides may be inεerted between the D and J H and between the V R and D gene segmentε which are not encoded by genomic DNA. The addition of these nucleotides is known as N-region diversity. After VJ and/or VDJ rearrangement, transcription of the rearranged variable region and one or more constant region gene segments located downstream from the rearranged variable region produces a primary RNA transcript which upon appropriate RNA splicing resultε in an mRNA which encodeε a full length heavy or light immunoglobulin chain. Such heavy and light chains include a leader signal sequence to effect secretion through and/or insertion of the immunoglobulin into the transmembrane region of the B-cell. The DNA encoding this signal sequence is contained within the first exon of the V segment used to form the variable region of the heavy or light immunoglobulin chain. Appropriate regulatory sequences are also present in the mRNA to control translation of the mRNA to produce the encoded heavy and light immunoglobulin polypeptides which upon proper aεεociation with each other form an antibody molecule. The net effect of such rearrangements in the variable region gene segmentε and the variable recombination which may occur during such joining, iε the production of a primary antibody repertoire. Generally, each B-cell which haε differentiated to this εtage, produces a εingle primary repertoire antibody. During thiε differentiation process, cellular events occur which suppress the functional rearrangement of gene segments other than those contained within the functionally rearranged Ig gene. The procesε by which diploid B-cells maintain such mono-specificity is termed allelic exclusion. The Secondary Repertoire B-cell clones expreεεing immunoglobulinε from within the set of sequences comprising the primary repertoire are immediately available to respond to foreign antigens. Because of the limited diversity generated by simple VJ and VDJ joining, the antibodies produced by the so-called primary response are of relatively low affinity. Two different types of B-cells make up thiε initial reεponεe: precursors of primary antibody-forming cells and precursors of secondary repertoire B-cells (Linton, et al. (1989), Cell, 59, 1049-1059). The first type of B-cell matures into IgM-secreting plaεma cellε in reεponse to certain antigenε. The other B-cells respond to initial exposure to antigen by entering a T-cell dependent maturation pathway. It iε during this T-cell dependent maturation of B-cells that a second level of diversity iε generated by a process termed somatic mutation (εometimes also referred to as hypermutation) . These primary repertoire B-cells use the immunoglobulin molecules on their surfaces to bind and internalize the foreign antigen. If the foreign antigen is a protein or is physically linked to another protein antigen, that protein antigen is then processed and presented on the cell surface by a major histocompatibility complex (MHC) molecule to a helper T-cell which in turn induces maturation of the B-cell. Lanzavecchia (1985), Nature, 314 , 537. This overall maturation of the B-cell iε known as the secondary response. During the T-cell dependent maturation of antigen stimulated B-cell clones, the structure of the antibody molecule on the cell surface changes in two ways: the constant region switches to a non-IgM subtype and the sequence of the variable region is modified by multiple single amino acid substitutions to produce a higher affinity antibody molecule. Tt is this process of somatic mutation, followed by the selection of higher affinity clones, that generates highly specific and tightly binding immunoglobulinε characterized by the Ig mediated immune response. As previously indicated, each variable region of a heavy or light Ig chain contains an antigen binding domain. It has been determined by amino acid and nucleic acid sequencing that somatic mutation during the secondary response occurs throughout the V region including the three complementary determining regions (CDRl, CDR2 and CDR3) alεo referred tc as hypervariable regionε 1, 2 and 3. The CDRl and CDR2 are located within the variable gene εegment whereas the CDR3 iε largely the reεult of recombination between V and J gene segmentε or V, D and J gene segments. Those portions of the variable region which do not consist of CDRl, 2 or 3 are commonly referred to as framework regions designated FRl, FR2, FR3 and FR . See Fig. 1. During hypermutation, the rearranged DNA is mutated to give rise to new clones with altered Ig molecules. Those clones with higher affinities for the foreign antigen are selectively expanded by helper T-cells, giving rise to affinity maturation of the expressed antibody. Clonal selection typically results in expression of clones containing new mutation within the CDRl, 2 and/or 3 regions. However, mutations outside these regions also occur which influence the specificity and affinity of the antigen binding domain. Transgenic Non-Human Animals Capable of Producing Heterologous Antibody Transgenic non-human animals in one aspect of the invention are produced by introducing at leaεt one of the immunoglobulin tranεgenes of the invention (discussed hereinafter) into a zygote or early embryo of a non-human animal. The non-human animals which are used in the invention generally comprise any mammal which is capable of rearranging immunoglobulin gene segments to produce a primary antibody response and, which, in addition, are capable of mounting a secondary response by way of somatic mutation of such rearranged Ig genes. A particularly preferred non-human animal is the mouse or other members of the rodent family. Mice are particularly useful since their immune system has been extensively studied, including the genomic organization of the mouse heavy and light immunoglobulin loci. See e.g. Immunoglobulin Genes, Academic Press, T. Honjo, F.W. Alt and T.H. Rabbitts, eds. (1989). However, the invention is not limited to the use of mice. Rather, any non-human mammal which is capable of mounting a primary and secondary antibody response may be used. Such animals include non-human primates, such aε chimpanzee, bovine, ovine and porcine species, other members of the rodent family, e.g. rat, as well as rabbit and guinea pig. Particular preferred animals are mouse, rat, rabbit and guinea pig, most preferably mouse. As used herein, the term "antibody" refers to a glycoprotein comprising at least two identical light polypeptide chains and two identical heavy polypeptide chainε linked together by diεulfide bondε. Each of the heavy and light polypeptide chainε contains a variable region (generally the amino terminal portion of the polypeptide chain) which contains a binding domain which interacts with antigen. Each of the heavy and light polypeptide chains also comprises a constant region of the polypeptide chains (generally the carboxyl terminal portion) some of which sequences mediate the binding of the immunoglobulin to host tissues including various cells of the immune system, some phagocytic cells and the first component (Clq) of the classical complement system. As used herein, a "heterologous antibody" iε defined in relation to the transgenic non-human organism producing such an antibody. It is defined as an antibody having an amino acid sequence or an encoding DNA sequence corresponding to that found in an organism not consiεting of the tranεgenic non-human animal. Thus, prior to rearrangement of a transgene containing various heavy or light chain gene segments, such gene segmentε may be readily identified, e.g. by hybridization or DNA sequencing, as being from a species of organism other than the transgenic animal. For example, in one embodiment of the invention, various gene segments from the human genome are used in heavy and light chain transgenes in an unrearranged form. In this embodiment, such transgenes are introduced into mice. The unrearranged gene segments of the light and/or heavy chain transgene have DNA sequences unique to the human species which are distinguishable from the endogenous immunoglobulin gene segmentε in the mouse genome. They may be readily detected in unrearranged form in the germ line and εomatic cells not consiεting of B-cells and in rearranged form in B-cellε. In an alternate embodiment of the invention, the transgeneε comprise rearranged heavy and/or light immunoglobulin transgeneε. Specific εegmentε of such transgenes corresponding to functionally rearranged VDJ or VJ segments, contain immunoglobulin DNA sequences which are also clearly distinguishable from the endogenous immunoglobulin gene segmentε in the mouse. Such differences in DNA sequence are also reflected in the amino acid sequence encoded by such human immunoglobulin transgenes as compared to those encoded by mouse B-cellε. Thus, human immunoglobulin amino acid sequences may be detected in the tranεgenic non-human animals of the invention with antibodieε specific for immunoglobulin epitopes encoded by human immunoglobulin gene segmentε. Tranεgenic B-cells containing unrearranged transgenes from human or other species functionally recombine the appropriate gene segments to form functionally rearranged light and heavy chain variable regions. It is to be underεtood that the DNA of such rearranged transgenes for the most part will not correspond exactly to the DNA sequence of the gene segmentε from which such rearranged transgeneε were obtained. Thiε is due primarily to the variations introduced during variable recombination and because of mutations introduced by hypermutation during the εecondary reεponse. Notwithstanding such modifications in DNA (as well as in amino acid) sequence, it will be readily apparent that the antibody encoded by such rearranged transgeneε has a DNA and/or amino acid εequence which is heterologous to that normally encountered in the non- human animal used to practice the invention. The term "substantial identity", when referring to polypeptides, indicates that the polypeptide or protein in question exhibits at least about 30% identity with an entire naturally occurring protein or a portion thereof, usually at least about 70% identity, and preferably at least about 95% identity. As used herein, the terms "isolated", "substantially pure" and "substantially homogenous" are used interchangeably herein and describe a polypeptide protein which haε been separated from components which naturally accompany it. Typically, a monomeric protein is substantially pure when at least about 60 to 75% of a sample exhibits a εingle polypeptide backbone. Minor variants or chemical modifications typically share the same polypeptide sequence. A εubεtantially pure protein will typically compriεe over about 85 to 90% of a protein sample, more usually about 95%, and preferably will be over about 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such aε polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band on a polyacrylamide gel upon staining. For certain purpoεeε high resolution will be needed and HPLC or a similar means for purification utilized. A polypeptide is subεtantially free of naturally-associated components when it is separated from the native contaminants which- accompany it in its natural state. Thus, a polypeptide which is synthesized in a cellular syεtem different from the cell from which it naturally originates will be substantially free from its naturally-associated components. Unrearranged Transgenes As used herein, an "unrearranged immunoglobulin heavy chain transgene" comprises DNA encoding at least one variable gene segment, one diversity gene segment, one joining gene segment and one constant region gene segment. Each of the gene segments of said heavy chain transgene are derived from, or has a sequence corresponding to, DNA encoding immunoglobulin heavy chain gene segments from a species not consisting of the non-human animal into which said transgene iε introduced. Similarly, as used herein, an "unrearranged immunoglobulin light chain transgene" comprises DNA encoding at least one variable gene segment, one joining gene segment and at least one constant region gene segment wherein each gene segment of said light chain transgene is derived from, or has a sequence corresponding to, DNA encoding immunoglobulin light chain gene segmentε from a species not consiεting of the non-human animal into which said light chain transgene is introduced. Such heavy and light chain transgeneε in thiε aspect of the invention contain the above-identified gene segmentε in an unrearranged form. Thus, interposed between the V, D and J segments in the heavy chain transgene and between the V and J segmentε on the light chain tranεgene are appropriate recombination signal sequences (RSS's). In addition, such transgeneε alεo include appropriate RNA splicing εignalε to join a constant region gene segment with the VJ or VDJ rearranged variable region. To the extent that the heavy chain tranεgene containε more than one C region gene segment, e.g. Cμ and C 7 I from the human genome, as explained below "switch regions" are incorporated upεtream from each of the constant region gene segments and downstream from the variable region gene segmentε to permit recombination between such constant regions to allow for immunoglobulin class switching, e.g. from IgM to IgG. Such heavy and light immunoglobulin transgenes also contain transcription control sequences including promoter regions situated upstream from the variable region gene segments which contain OCTA and TATA motifs. In addition to promoters, other regulatory sequences which function primarily in B-lineage cells are used. Thus, for example, a light chain enhancer sequence εituated preferably between the J and constant region gene segments on the light chain tranεgene is used to enhance transgene expreεεion, thereby facilitating allelic exclusion. In the case of the heavy chain transgene, regulatory enhancers and also employed. Although the foregoing promoter and enhancer regulatory control sequences have been generically described, such regulatory sequences may be heterologous to the nonhuman animal being derived from the genomic DNA from which the heterologous transgene immunoglobulin gene segments are obtained. Alternately, such regulatory gene segmentε are derived from the corresponding regulatory sequences in the genome of the non-human animal, or closely related species, which contains the heavy and light transgene. Such regulatory sequences are used to maximize the transcription and translation of the transgene so as to induce allelic exclusion and to provide relatively high levelε of tranεgene expresεion. In the preferred embodimentε, each of the immunoglobulin gene segments contained on the heavy and light Ig transgenes are derived from, or have sequences corresponding to, genomic DNA, cDNA or portions thereof from a specieε or individual which is heterologous to the non-human animal into which the transgene is to be introduced. As a consequence, when such gene segments are functionally rearranged and hypermutated in the transgenic non-human animal, the heterologous antibody encoded by such heavy and light transgenes will have an amino acid sequence and overall secondary and terteriary structure which provides specific utility against a desired antigen when used therapeutically in the organism from which the Ig gene segmentε are derived. In addition, such antibodies demonstrate substantially reduced immunogenicity as compared to antibodies which are "foreign" to the organism treated. For example, in the preferred embodiments, gene segments are derived from human beings. The transgenic non-human animals harboring such heavy and light transgeneε are capable of mounting an Ig-mediated immune response to a specific antigen administered to such an animal. B-cells are produced within such an animal which are capable of producing heterologous human antibody. After immortalization, and the selection for an appropriate monoclonal antibody (Mab) , e.g. a hybridoma, a εource of therapeutic human monoclonal antibody iε provided. Such human Mabε have significantly reduced immunogenicity when therapeutically administered to humans. Examples of antigens which may be used to generate heterologous antibodies in the transσenic animals of the invention containing human immunoglobulin transgeneε include bacterial, viral and tumor antigen as well aε particular human B- and T-cell antigenε associated with graft rejection or autoimmunity. Although the preferred embodiments diεclose the construction of heavy and light transgeneε containing human gene segments, the invention is not so limited. In this regard, it is to be understood that the teachings described herein may be readily adapted to utilize immunoglobulin gene segments from a species other than human beings. For example, in addition to the therapeutic treatment of humans with the antibodies of the invention, therapeutic antibodieε encoded by appropriate gene segments may be utilized to generate monoclonal antibodies for use in the veterinary sciences. For example, the treatment of livestock and domestic animals with species-related monoclonal antibodies is also contemplated by the invention. Such antibodieε may be εimilarly generated by uεing tranεgeneε containing immunoglobulin gene εegmentε from εpecies such as bovine, ovine, porcine, equine, canine, feline and the like. Class Switching The use of μ or . constant regions is largely determined by alternate splicing, permitting IgM and IgD to be coexpreεsed in a single cell. The other heavy chain iεotypeε ( 7 , , and e) are only expresεed natively after a gene rearrangement event deletes the Cμ and Cδ exons. Thiε gene rearrangement process, termed class switching, occurs by recombination between so called switch segments located immediately upstream of each heavy chain gene (except δ ) . The individual switch segments are between 2 and 10 kb in length, and consist primarily of short repeated sequenceε. The exact point of recombination differε for individual claεs switching events. The ability of a transgene conεtruction to εwitch isotypes during B-cell maturation has not been directly tested in transgenic mice; however, transgenes should carry out this function. Durdik et al. (1989) Proc. Natl- Acad. Sci. USA, 86, 2346-2350) microinjected a rearranged mouse μ heavy chain gene construct and found that in four independent mouse lines, a high proportion of the transgenic B-cellε expressed the transgene-encoded variable region associated with IgG rather than IgM. Thus, isotype switching appears to have taken place between the transgene and the endogenous 7 constant region on another chromosome. As used herein, the term switch sequence thus refers to those DNA sequenceε reεponεible for εwitch recombination. A "εwitch donor" sequence, typically a μ εwitch region, will be 5' (i.e., upstream) of the construct region to be deleted during the switch recombination. The "switch acceptor" region will be between the construct region to be deleted and the replacement constant region (e.g., 7, e, etc.) . As there is no εpecific site where recombination alwayε occurε, the final gene sequence will typically not be predictable from the construct. The switch (S) region of the μ gene, S , is located about 1 to 2 kb 5 ' to the coding sequence and iε co poεed of numerouε tandem repeatε of sequenceε of the form (GAGCT) (GGGGT) , where n iε uεually 2 to 5 but can range aε high as 17. (See T. Nikaido, et al. (1981) : Nature, 292:845- 848.) Similar internally repetitive switch sequences spanning several kilobases have been found 5 ' of the other C H genes. The Sα region has been sequenced and found to consist of tandemly repeated 80-bp homology units, whereas S , S , and S all contain repeated 49-bp homology units very εimilar to each other. (See, P. Szurek, et al. (1985) : J. Immunol, 135:620-626 and T. Nikaido, et al. (1982) : J. Biol. Chem. , 257 :7322-7329. ) All the εequenced S regionε include numerouε occurrences of the pentamers GAGCT and GGGGT that are the basic repeated elementε of the S gene (T. Nikaido, et al. (1982) : J. Biol. Chem. , 257:7322-7329) ; in the other S regionε theεe pentamers are not precisely tandemly repeated aε in S , but instead are embedded in larger repeat unitε. The S region has an additional higher-order structure: two direct repeat sequences flank each of two clusters of 49-bp tandem repeats. (See M. R. Mowatt, et al (1986) : J. Immunol. , JL3_6:2674-2683) . Switch regions of human H chain genes have been found very similar to their mouse homologs. Generally, unlike the enzymatic machinery of V-J recombination, the εwitch machinery can apparently accommodate different alignments of the repeated homologous regionε of germline S precurεors and then join the sequences at different poεitions within the alignment. (See, T. H. Rabbitε, et al . (1981) : Nucleic Acidε Reε. , 9_:4509-4524 and J. Ravetch, et al . (1980) : Proc. Natl. Acad. Sci. USA, 77:6734-6738. ) Induction of class switching appears to be associated with sterile transcripts that initiate upstream of the εwitch segments (Lutzker et al., 1988 Mol. Cell. Biol. , 8 . , 1849; Stavnezer et al. 1988 Proc. Natl. Acad. Sci. USA, 85, 7704; Esεer and Radbruch 1989 EMBO J. , 8 . , 483; Berton et al. 1989 Proc. Natl. Acad. Sci. USA, 86, 2829; Roth an et al. 1990 Int. Immunol. 2_, 621) . For example, the observed induction of the 7I sterile transcript by IL-4 and inhibition by IFN-7 correlates with the observation that IL-4 promotes clasε switching to 7 I in B-cells in culture, while IFN-7 inhibits 7 1 expression. Ideally then, transgene constructε that are intended to undergo class switching should include all of the cis-acting sequences necessary to regulate these εterile transcripts. An alternative method for obtaining class switching in transgenic mice (σμ and eμ) involves the inclusion of the 400 bp direct repeat sequences that flank the human μ gene (Yasui et al. 1989 Eur. J. Immunol. , 19, 1399) . Homologous recombination between these two sequenceε deleteε the μ gene in IgD-only B-cells. Monoclonal Antibodieε Monoclonal antibodies can be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cellε from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol. , jS:511-519 (1976)) . Alternative methods of immortalization include transformation with Epstein Barr Virus, or.coger.es, or retroviruses, or other methods well known in the art. Colonies arising from εingle immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cellε may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Various techniques useful in these arts are discusεed, for example, in Harlow and Lane, Antibodies: A Laboratory Manual . Cold Spring Harbor, New York (1988) including: immunization of animalε to produce immunoglobulinε; production of monoclonal antibodieε; labeling immunoglobulins for use aε probeε; immunoaffinity purification; and immunoaεεayε. The Transgenic Primary Repertoire A. The Human Immunoglobulin Loci An important requirement for tranεgene function iε the generation of a primary antibody repertoire that iε diverεe enough to trigger a εecondary immune reεponεe for a wide range of antigens. The size of the human immunoglobulin loci encoding the various gene segmentε for heavy and light chains is quite large. For example, in the human genome the three separate loci for the λ light chain locus, the K light chain locus and the heavy chain locus together occupy over 5 Mb of DNA or almost 0.2% of the entire genome. Each locuε conεiεtε of multiple variable segments that recombine during B-cell development with a joining region segment (and, the heavy chain locus with diversity region segments) to form complete V region exons. Such rearranged light chain genes consiεt of three exons: a signal peptide exon, a variable region exon and a constant region exon. . The rearranged heavy chain gene iε somewhat more complex. It consists of a signal peptide exon, a variable region exon and a tandem array of multi-domain constant region regionε, each of which iε encoded by several exons. Each of the constant region geneε encode the conεtant portion of a different class of immunoglobulins. During B-cell development, V region proximal constant regions are deleted leading to the expresεion of new heavy chain claεseε. For each heavy chain class, alternative patterns of RNA. splicing give rise to both transmembrane and secreted immunoglobulins. Approximately 40% of human serum antibody molecules contain λ light chains. The structure of this locuε, which maps to chromosome 22, is the least well characterized (Fig. 2) . It consistε of an unknown number of V εegmentε upεtream of a tandem array of six constant region geneε, each of which is linked to a single J segment. In addition, two more constant region segmentε with aεsociated J segments have been isolated, although their linkage with the rest of the λ cluster has not been eεtablished, and it is not known if they are uεed. E. Seising, et al. , "Immunoglobulin Genes", Academic Press, T. Honjo, F.W. Alt and T.H. Rabbitts, eds. (1989). The K light chain locuε iε spread out over three clusters on chromosome 2 (Fig. 3) . The first two cluεters, covering 850 and 250 kb respectively contain only variable region gene segments. The third cluster, covering about 1 Mb, contains approximately 40 V gene segmentε upstream of a cluster of 5 J segments followed by a single conεtant region gene segment. A total of 84 V gene segments have been identified, and approximately half of these are thought to be pseudogeneε (Zachau (1989) in Immunoglobulin Geneε, Academic Press, T. Honjo, F.W. Alt, and T.H. Rabbitts, eds. pp. 91-110) . Approximately 25 kb downstream of the CK region there is a "k deleting element" (cde) . The cde sequence recombines with upstream sequences, causing the deletion of the K constant region in λ light chain expressing B-cells. Thiε leadε to isotopic exclusion in cells that succeεεfully rearrange both K. and λ geneε. The human heavy chain locus is the largest and most diverse. It consists of approximately 200 V gene segmentε spanning 2 Mb, approximately 30 D gene segments spanning about 40 kb, six J segments clustered within a 3 kb span, and nine constant region gene segments spread out over approximately 300 kb. The entire locus spans approximately 2.5 Mb of the distal portion of the long arm of chromosome 14 (Fig. 4) . The heavy chain V segments can be grouped into six familieε on the baεiε of sequence similarity. There are approximately 60 members of the V H 1 family, 30 V H 2 segments, 80 V H 3 segments, 30 V H segments, three V H 5 segments, and one V H 6 segment. Berman, J.E., et al. (1988), EMBO J. , 1_, 727-738. In the human heavy chain locus, the members of individual V families are intermingled, unlike the mouse locuε where related V εegmentε are clustered. The single member of the VH6 family is the most proximal of the V segments, mapping to within 90 kb of the constant region gene segments. Sato, T. , et al. (1988) , Biochem. Biophys. Res. Comm. , 154 , 265-271. All of the functional D and J segments appear to lie in thiε 90 kb region (Siebenlist, et al. (1981), Nature, 294 , 631-635; Matsuda, et al. (1988) , EMBO J. , 2, 1047-1051; Buluwela, et al. (1988) , EMBO J. , 7_, 2003-2010; Ichihara, et al. (1988), EMBO J. , 7, 4141-4150; Berman, et al. (1988) , EMBO J.. 1_, 727-738) . B. Gene Fragment Tranεgenes 1. Heavy Chain Transgene In a preferred embodiment, immunoglobulin heavy and light chain transgenes compriεe unrearranged genomic DNA from humans. In the case of the heavy chain, a preferred transgene compriseε a Notl fragment having a length between 670 to 830 kb. The length of this fragment is ambiguouε becauεe the 3' restriction site has not been accurately mapped. It is known, however, to reside between the αl and ψ gene segments (see Fig. 4) . This fragment contains members of all six of the known V H families, the D and J gene segments, as well as the μ, 5, 73, 7I and αl constant regions. Berman, et al. (1988), EMBO J. , 1_, 727-738. A transgenic mouse line containing this transgene correctly expresses all of the heavy chain claεεeε required for B-cell development as well aε a large enough repertoire of variable regionε to trigger a εecondary reεponεe for most antigens. 2. Light Chain Transgene A genomic fragment containing all of the necessary gene segments and regulatory sequences from a human light chain locus ma be similarl' 17 constructed. Such a construct iε described in the Examples. C. Transgeneε Generated Intracellularly by In Vivo Recombination It iε not necessary to isolate the all or part of the heavy chain locus on a single DNA fragment. Thus, for example, the 670-830 kb Notl fragment from the human immunoglobulin heavy chain locus may be formed in vivo in the non-human animal during transgeneεis. Such in vivo transgene construction is produced by introducing two or more overlapping DNA fragments into an embryonic nucleus of the non-human animal. The overlapping portions of the DNA frag entε have DNA εequenceε which are substantially homologous. Upon exposure to the recombinases contained within the embryonic nucleus, the overlapping DNA fragmentε homologously recombined in proper orientation to form the 670-830 kb Notl heavy chain fragment. It iε to be understood, however, that in vivo transgene construction can be used to form any number of immunoglobulin transgeneε which becauεe of their εize are otherwise difficult, or imposεible, to make or manipulate by present technology. Thus, in vivo transgene construction is useful to generate immunoglobulin transgenes which are larger than DNA fragments which may be manipulated by YAC vectors (Murray and Szostak (1983), Nature, 305, 189-193) . Such in vivo transgene construction may be used to introduce into a non-human animal substantially the entire immunoglobulin loci from a species not consisting of the transgenic non-human animal. Thus, although several groups have successfully constructed libraries containing 50-200 kb of DNA fragments in YAC vectors (Burke, et al. (1987) , Science, 236, 806-812; Traver, et al. (1989), Proc. Natl. Acad. Sci. USA, 86 , 5898— 5902) and used polyamine condensation to produce YAC libraries ranging in εize from 200 to approximately 1000 kb (McCormick, et al. (1989) , Proc. Natl. Acad. Sci USA, 86, 9991-9995; , multiple overlapping fragments covering substantially more than the 670-830 kb Notl fragment of the human constant region immunoglobulin loci are expected to readily produce larger trans g enes b v the methods disclosed herei . In addition to forming genomic immunoglobulin transgeneε, in vivo homologous recombination may also be utilized to form "mini-locuε" transgeneε as described in the Exampleε. In the preferred embodiments utilizing in vivo transgene construction, each overlapping DNA fragment preferably has an overlapping substantially homologous DNA sequence between the end portion of one DNA fragment and the end portion of a εecond DNA fragment. Such overlapping portionε of the DNA fragmentε preferably compriεe about 500 bp to about 2000 bp, moεt preferably 1.0 kb to 2.0 kb. Homologous recombination of overlapping DNA fragmentε to form transgenes in vivo iε further described in commonly asεigned U.S. Patent Application entitled "Intracellular Generation of DNA by Homologouε Recombination of DNA Fragmentε" filed Auguεt 29, 1990, under U.S.S.N. 07/574,747. D. Minilocuε Transgeneε Aε uεed herein, the term "immunoglobulin minilocuε" refers to a DNA sequence (which may be within a longer sequence) , usually of lesε than about 150 kb, typically between about 25 and 100 kb, containing at leaεt one each of the following: a functional variable (V) gene segment, a functional joining (J) region segment, a functional constant (C) region gene segment, and—if it is a heavy chain minilocuε- -a functional diversity (D) region segment, such that εaid DNA sequence contains at least one substantial discontinuity (e.g., a deletion, usually of at least about 2 to 5 kb, preferably 10- 25 kb or more, relative to the homologous genomic DNA sequence) . A light chain minilocus transgene will be at leaεt 25 kb in length, typically 50 to 60 kb. A heavy chain transgene will typically be about 70 to 80 kb in length, preferably at least about 60 kb with two constant regionε operably linked to switch regions, versus at least about 30 kb with a single constant region and incomplete switch regions. Furthermore, the individual elements of the minilocus are preferably in the germline configuration and capable of lindorπn . net π&nez. τ-<s3 T-τ-a - ff a τrι«3 -(" -i fh*3 r.τ"(= — B f' l 1 rsΨ 3 -I- anc rpr. . animal so as to express functional antibody moleculeε with diverse antigen specificities encoded entirely by the elements of the minilocuε. In an alternate preferred embodiment, immunoglobulin heavy and light chain transgenes compriεe one or more of each of the V, D, J and C gene εegments. At least one of each appropriate type gene segment iε incorporated into the minilocus transgene. With regard to the C segments for the heavy chain transgene, it iε preferred that the transgene contain at least one μ gene segment and at least one other conεtant region gene segment, more preferably a 7 gene segment, and most preferably 7 3 or 7 1. This preference is to allow for clasε switching between IgM and IgG formε of the encoded immunoglobulin to provide for somatic mutation and the production of a secretable form of high affinity non-IgM immunoglobulin. Other constant region gene εegments may also be used such as those which encode for the production of IgD, IgA and IgE. The heavy chain J region segments in the human comprise six functional J segmentε and three pεeudo geneε cluεtered in a 3 kb εtretch of DNA. Given itε relatively compact εize and the ability to isolate these εegmentε together with the μ gene and the 5' portion of the <5 gene on a εingle 23 kb SFil/Spel fragment (Sado, et al. (1988) , Biochem. Bioεhyε. Reε. Comm. , 154 , 264271) , it is preferred that all of the J region gene segments be used in the mini-locus construct. Since this fragment spans the region between the μ and <5 genes, it is likely to contain all of the 3' ciε-linked regulatory elements required for μ expression. Furthermore, because thiε fragment includeε the entire J region, it containε the heavy chain enhancer and the μ εwitch region (Millε, et al . (1983) , Nature, 306, 809; Yancopouloε and Alt (1986), Ann. Rev. Immunol. , 4 . , 339-368) . It alεo containε the tranεcription start siteε which trigger VDJ joining to form primary repertoire B-cellε (Yancopoulos and Alt (1985) , Cell, 40, 271-281) . Alternatively, a 36 kb BssHII/Spell fragment, which includes part on the D region, may be used in place of the 23 kb Sfil/Spell fragment. The use of such a fragment increases the amount of 5' flanking sequence to facilitate efficient D-to-J joining. The human D region consistε of 4 or 5 homologouε 9 kb subregions, linked in tandem (Siebenlist, et al. (1981), Nature, 294. 631-635) . Each εubregion containε up to 10 individual D segmentε. Some of these segmentε have been mapped and are εhown in Fig. 4. Two different strategieε are uεed to generate a mini-locus D region. The first εtrategy involveε using only those D εegmentε located in a εhort contiguouε stretch of DNA that includes one or two of the repeated D subregions. A candidate is a εingle 15 kb fragment that containε 12 individual D segments. Thiε piece of DNA conεiεts of 2 contiguouε EcoRI fragments and has been completely sequenced (Ichihara, et al. (1988), EMBO J. , 2, 4141-4150) . Twelve D segments should be sufficient for a primary repertoire. However, given the dispersed nature of the D region, an alternative strategy is to ligate together several non-contiguous D-segment containing fragments, to produce a smaller piece of DNA with a greater number of segments. At least one, and preferably more than one V gene segment is used to conεtruct the heavy chain minilocuε transgene. A 10-15 kb piece of DNA containing one or two unrearranged V segments together with flanking sequenceε iε isolated. A clone containing such DNA is selected using a probe generated from unique 5 ' sequenceε determined from the transcribed V region of a characterized human hybridoma εuch aε that which produces anti-cytomegalovirus antibody (Newkirk et al. (1988) J. Clin. Invest. , 81, 1511-1518) . The 5' untranslated sequence of the heavy chain mRNA is used to construct a unique nucleotide probe (preferably about 40 nucleotides in length) for isolating the original germline V segment that generated this antibody. Using a V segment that is known to be incorporated in an antibody against a known antigen not only insures that this V segment iε functional, but aids in the analysis of transgene participation in secondary immune responses. This V segment is fused with the minilocus D region and constant region fragments, discuεεed previouεly, to produce a mini-locus heavy chain transσene. Alternatively, a large, contiguouε εtretch of DNA containing multiple V region segmentε iε isolated from a YAC library. Different sized pieces of DNA, containing different numbers of V region εegmentε, are teεted for their ability to provide a human antibody repertoire in the minilocuε transgene construct. It is also possible to build one large fragment from several non-contiguous V εegment containing fragments using YAC vectors (Murray and Szostak (1983) , Nature, 305 , 189-193) , F factor-based plasmids (O'Conner, et al. (1989) , Science, 244 , 1307-1312) or the aforementioned ;Ln vivo construction using recombination of overlapping fragments. Alternatively, a εynthetic V region repertoire (deεcribed hereinafter) may be uεed. A minilocus light chain transgene may be εi ilarly constructed from the human λ or . immunoglobulin locus. Construction of a c light chain mini-locus iε very similar to construction of the heavy chain mini-locus, except that it iε much simpler because of its smaller εize and lower complexity. The human K locus contains only one conεtant region segment; and thiε segment, together with 5' and 3' enhancerε, and all 5 of the functional J segmentε, can be isolated on a single 10 kb DNA fragment. This fragment is co-injected together with a minilocus V region constructed as described for the heavy chain minilocus. Thus, for example, an immunoglobulin heavy chain minilocus transgene construct, e.g., of about 75 kb, encoding V, D, J and constant region sequenceε can be formed from a plurality of DNA fragments, at least two, three or four of which each are either a V region sequence, a D region sequence, a J and conεtant region εequence, a D and J and conεtant region sequence or a constant region εequence, with each εequence being subεtantially homologous to human gene sequences. Preferably, the sequences are operably linked to transcription regulatory sequences and are capable of undergoing rearrangement. With two or more appropriately placed conεtant region sequences (e.g., μ and 7) and switch regions, εwitch recombination also occurs. An exemplary light chain tranεgene construct similarly formed from a plurality of DNA fragments, substantially homologous to human DNA and capable of undergoing rearrangement will include at least two, three or four DNA fragments, encoding V, D and constant regionε, each fragment comprising either a V region sequence, J and constant region sequence or a constant region sequence. E. Methods for Determining Functional V Gene Segments and for Generating Synthetic V Segment Repertoire Of the various families of gene segments, i.e., V, D, J and C region gene εegments, the number of V gene segmentε generally far εurpasses the number of corresponding gene segments for the D, J and C region gene εegmentε. By analogy to the rabbit εystem wherein a single V gene segmentε iε utilized by approximately 90% of the antibodieε produced (Knight and Becker (1990) , Cell, 60, 963-970) , it iε poεεible to produce heavy and light tranεgeneε containing a limited number of V region gene εegmentε, and as few aε one V region gene εegmentε. Therefore, it iε desirable to have a method tc determine which V region gene segmentε are utilized by a particular organiε , εuch aε the human being, when mounting an immunoglobulin-mediated immune response. According to thiε approach, a εingle V gene εegment when combining with the J or DJ gene segmentε is capable of providing sufficient diverεity at CDR3 for the generation of a primary repertoire which upon somatic mutation iε able to provide further diversity throughout the variable region, e.g. at CDRl and CDR2 for the production of high affinity antibodies. In this aεpect of the invention, methods and vectors are provided for determining which V gene segmentε are commonly utilized by an organiεm during an immune response. This method is based on determining which V εegmentε are found in cDNA synthesized from B-cell polyA+ RNA. Such methods and vectors may alεo be uεed to facilitate the conεtruction of a εynthetic V εegment repertoire. The outline of this strategy for identifying heavy chain V segments and for generating a εynthetic V εegment repertoire is depicted in Fig.s 5 and 6. It is similarly applicable for identifving light chain V segments with appropriate modification. The first step is the construction of a cloning vector. The preferred starting material is a DNA fragment (approximately 2 kb) containing an unrearranged V segment together with 5' and 3' flanking sequenceε. Thiε fragment is cloned into a plasmid such as pGPl or pGP2 described hereinafter containing a polylinker site flanked by the rare cutting reεtriction εiteε designated "w" and "z" in the Figs. 5 and 6 (the polylinkerε and reεtriction εiteε of pGPl and pGP2 are deεcribed in the Examples) . Oligonucleotide directed mutagenesiε is then used to introduce two new restriction siteε, "x" and "y" (generally each about 6 nucleotideε in length) . Reεtriction εite "x" iε placed approximately 20 nucleotideε from the 3 ' end of the intron between the signal and V segment exon. Reεtriction εite "y" is placed approximately 20 nucleotideε 3' of the V εegment junction, within the 23 bp εpacer between the heptamer and nonomer recombination εignal sequences. Cutting the resulting plasmid with enzymes "x" and "y" removes the second exon (V segment), leaving the 5' flanking sequences, the V region promoter, the signal peptide exon, the intron, a gap flanked by "x" and "y" ends, the outεide half of the recombination εignal sequence, and the 3' flanking sequenceε. Thiε plasmid is called pVHl. The second step is the synthesiε of four sets of oligonucleotide primers, PI through P4. PI and P2 are non-unique oligomers having approximately 50 nucleotideε each which are uεed to prime double stranded cDNA syntheεiε. PI εtartε (going 5' to 3') with about 20 nucleotideε of εequence homologous to the antisense strand of the recombination signal sequence in pVHl (including the recognition sequence of restriction enzyme "y") , and continues with approximately 30 nucleotides of antisense sequence hybridizing with about the last 30 nucleotideε of the VH framework region 3 (FR3). Randor, baseε are incorporated over about the laεt 30 nucleotides εo as to generate a set of primers that hybridize with all of the different VH families. The second oligonucleotide, P2, iε in the sense orientation, and is homologous to the approximately 50 nucleotides beginning with the restriction site "x" in pVHl - This includes the "x" restriction site, about the last 20 nucleotides of the intron, and about the first 30 nucleotides of FRl. Again, about the last 30 nucleotides are non-unique so as to accommodate different VH region segmentε. Oligonucleotides P3 and P4 are homologous to about the first 20 nucleotides of PI and P2 respectively. These oligoε are unique so as to avoid introducing new mutations into the V segments and are used to amplify double stranded cDNA by way of the poly eraεe chain reaction (PCR) . The 3 ' terminal portionε of pri erε PI and P2 which are capable of hybridizing to and priming the εyntheεiε of the variable segments of the heavy or light immunoglobulin locus may be readily determined by one εkilled in the art. For example, the nucleotide sequence for a number of human VH genes have been published, see e.g. Berman, J.E., et al. (1988) , EMBO J., 1_, 727-738 and Kabat, E.A., et al. (1987), Sequences of Protein of Immunological intereεts, U.S. Dept. Health & Human Serviceε, Washington, D.C. Similarly, when uεed to identify and/or generate V segments of the human light immunoglobulin locuε, the appropriate 3' εequence portions of primers PI and P2 may readily be determined from published sequences. See e.g Kabat, E.A. , et al., supra. In general, those nucleotide positions which are conserved amongst variouε V segments are also conserved in the 3 ' portion of the PI and P2 primers. For those nucleotide positionε wherein variation iε obεerved amongεt variable εegments, such nucleotide positionε in the corresponding PI and P2 primerε are εi ilarly varied to provide PI and P2 primers which comprise a pool of primers which are capable of hybridizing to different VH or VL segmentε. The next εtep iε to use these oligonucleotide primerε to generate a library of human heavy-chain V-region cDNA sequenceε in the vector pHVl. PI iε uεed to prime first strand cDNA synthesis from human B-cell polyA÷ RNA. The RNA iε base hydrolyzed, and second strand synthesis primed with P2. Full length, double stranded cDNA iε then purified on an acrylamide gel, electroeluted, and used as template for polymerase chain reaction (PCR) amplification using oligonucleotide primers PT and P4. Alternatively, cDNA is first synthesized by conventional methods and this cDNA iε used as a template for the PI primed reactor. The amplified product (approximately 0.3 kb) is then gel purified, cleaved with restriction enzymes "x" and "y", and cloned into pHVl. The resulting cDNA library represents a synthetic genomic library of variable region segments and offerε three advantages over a conventional genomic library of variable εegments. First, this library containε no pseudogeneε, while a conventional library would contain up to 50% pseudogene sequenceε. Second, the εynthetic library is more compact than a conventional library, containing one functional V segment per 2 kb of DNA, as opposed to one functional segment per 20 kb. Finally, this approach leaves the V segment promoter sequences accessible to manipulation. Such a cDNA library may be biased towards particular germline V segments becauεe of differential expreεεion. The two sources of bias are: (i) differential rates of V εegment recombination, and (ii) differential εelection of V εegment expressing B-cell clones. The first source of bias is dealt with in two ways. First, fetal tiεεue iε avoided aε a εource of B-cell RNA, aε the biaε is most pronounced in the fetal immunoglobulin repertoire. Second, the semi-random primers, PI and P2, are divided into pools, each of which selectively cross-hybridizes with different V segment families. Theεe primers are then uεed to generate 4 to 6 separate libraries, thus insuring that all of the V region families are represented. The second source of biaε, differential εelection of B-cell cloneε, iε also dealt with in two analogouε wayε. Firεt, a εource of RNA that includes the minimum fraction of antigen selected B-cells is used. Lymph nodeε and εpleen are avoided. Adult bone marrow is one εource of unεelected B-cellε. However, it may contain a high proportion of tranεcribed pseudogene sequenceε from pre-B-ceils. Another source of RNA is whole blood. Ninety percent of circulating B-cells are immature μ or μ, δ expresεing cellε, and are recent bone marrow immigrants. However, the level of antigen selected fc— -_..-.m._m-..».«-n.T...o— rv--_F-. the individual. Therefore, isolated polyA+ RNA is checked for selected B-cell sequences by northern blot hybridization with 7 specific probes. If it iε more practical to uεe εpleen RNA, and if thiε RNA contains a high fraction of IgG sequences, a second approach is used to minimize εelection bias. The first strand of cDNA synthesis is primed with about a 40 nucleotide constant-region exon 2 primer that is εpecific for IgM transcripts. Second strand εyntheses iε then primed with P2, and a third round of εyntheεiε primed with PI. The cDNA from this third round of synthesis provides the template for PCR amplification using P3 and P4. Once the variable region library has been generated, the V segments used therein may by identified by standard techniques, e.g. by way of sequencing and/or hybridization with family specific or segment specific oligonucleotides aε well aε differential amplification by PCR methodε. Such characterization of the V εegment library provideε information aε to the frequency and diεtribution of V εegment utilization in a particular organiεm and aε a consequence, the identification of V segmentε which may be uεed in the conεtruction of the variouε tranεgenes of the invention. Thus, one or more predominant V. gene εegments may be uεed in the above described mini-locus transgene conεtruct. Further, selected clones from such a library may be used to identify genomic fragments containing frequently used V εegmentε to facilitate identification of genomic fragments containing a particular desired V segment. In addition, a synthetic V segment repertoire may be constructed by concatenation of the library sequences. Large repeating transgene tandem arrayε, containing hundreds of copies of the injected sequence, are commonly generated in the production of transgenic mice. These tandem arrays are usually quite stable. However, to ensure the stability of the synthetic V region, blocks of random DNA between each 2 kb V region segment are preferably introduced. These blocks of random DNA are prepared by digesting and then religating genomic DNA, so as to prevent the insertion of dominant reσulatory elements= Genomic DNA is preferably digested with four frequent cutting restriction enzymes: Alul, Dpnl, Haelll, and Rsal. This digest produces blunt ended fragments with an average length of 64 nucleotides. Fragments in the size range of 50 to 100 nucleotides are eluted from an acrylamide gel, and religated. The relegated DNA is partially digested with Mbol and size fractionated. Fragments in the range of 0.5 to 2 Id- are cloned into the BamHI or Bglll site of the polylinker of the vector used to generate pVHl. The random sequence library is combined with the synthetic V segment library to create a synthetic V εegment repertoire. Inserts from the random sequence library are released with the enzymes "w" and "z", and purified away from vector sequenceε. Inserts from the εynthetic V εegment library are isolated by cutting with "w" and "z". Before purifying the V segment inserts, this DNA iε treated with calf-inteεtinal phosphatase, to prevent self ligation. The V segment inserts are then ligated together with the random insertε to generate an alternating tandem array compriεing a εynthetic V segment repertoire. This ligation mixture is ε.ize selected on a sucrose gradient, and the 50-100 kb fraction microinjected together with, for example, a D-J-constant mini-locus construct. By directly injecting the synthetic V εegment repertoire without an intervening cloning εtep, it iε poεεible to take advantage of the fact that tandem arrayε of injected fragments become inserted at a single site. In this case such tandem arrays are not completely redundant but lead to further diversity. Alternatively, the synthetic V εegment repertoire may be combined with a D-J-C minilocuε to form a heavy chain transgene. A synthetic light chain immunoglobulin segment repertoire may be similarly conεtructed using appropriate primers for the light chain locus. Functional Disruption of Endogenous Immunoglobulin Loci The expression of successfully rearranged immunoglobulin heavy and light transgenes. iε expected to have a dominant effect by suppressing the rearrangement of the endogenous immunoglobulin genes in the transgenic nonhuman animal. However, another way to generate a nonhuman that iε devoid of endogenouε antibodieε iε by mutating the endogenous immunoglobulin loci. Using embryonic stem cell technology and homologous recombination, the endogenous immunoglobulin repertoire can be readily eliminated. The following describes the functional description of the mouse immunoglobulin loci. The vectors and methods discloεed, however, can be readily adapted for use in other non-human animals. Briefly, this technology involveε the inactivation of a gene, by homologouε recombination, in a pluripotent cell line that is capable of differentiating into germ cell tiεεue. A DNA construct that contains an altered, copy of a mouse immunoglobulin gene iε introduced into the nuclei of embryonic stem cells. In a portion of the cells, the introduced DNA recombines with the endogenouε copy of the mouεe gene, replacing it with the altered copy. Cellε containing the newly engineered genetic lesion are injected into a host mouεe embryo, which iε reimplanted into a recipient female. Some of these embryos develop into chimeric mice that possesε germ cells entirely derived from the mutant cell line. Therefore, by breeding the chimeric mice it is posεible to obtain a new line of mice containing the introduced genetic lesion (reviewed by Capecchi (1989) , Science, 244, 1288-1292) . Because the mouse λ locuε contributeε to only 5% of the immunoglobulinε, inactivation of the heavy chain and/or κ-light chain loci iε εufficient. There are three wayε to disrupt each of these loci, deletion of the J region, deletion of the J-C intron enhancer, and disruption of conεtant region coding sequences by the introduction of a stop codon. The laεt option iε the most straightforward, in terms of DNA construct design. Elimination of the μ gene disrupts B-cell maturation thereby preventing clasε switching to any of the functional heavy chain segments. The strategy for knocking out these loci is outlined below. To disrupt the mouse μ and K genes, targeting vectors are used based on the design employed by Jaenisch and co— workers (Zijlstra, et al. (1989), Nature, 342, 435-438) for the successful disruption of the mouse /32-microglobulin gene. The neomycin resistance gene (neo) , from the plasmid pMCIneo is inserted into the coding region of the target gene. The pMCIneo insert uses a hybrid viral promoter/enhancer sequence to drive neo expression. This promoter is active in embryonic stem cellε. Therefore, neo can be uεed aε a selectable marker for integration of the knock-out construct. The HSV thymidine kinase (tk) gene is added to the end of the construct as a negative εelection marker againεt random insertion events (Zijlstra, et al., supra. ) . The targeting vectors for disrupting the heavy chain locus are illustrated in Fig. 7. The primary strategy for disrupting the heavy chain locus is the elimination of the J region. This region is fairly compact in the mouse, spanning only 1.3 kb. To construct a gene targeting vector, a 15 kb Kpnl fragment containing all of the secreted A constant region exons from mouse genomic library is isolated. The 1.3 kb J region is replaced with the 1.1 kb insert from pMCIneo. The HSV tk gene is then added to the 5' end of the Kpnl fragment. Correct integration of this construct, via homologous recombination, will result in the replacement of the mouse J H region with the neo gene (Fig. 7) . Recombinants are εcreened by PCR, using a primer based on the neo gene and a primer homologous to mouse sequences 5 ' of the Kpnl site in the D region. Alternatively, the heavy-chain locus is knocked out by disrupting the coding region of the μ gene. Thiε approach involves the same 15 kb Kpnl fragment uεed in the previouε approach. The 1.1 kb insert from pMCIneo is inserted at a unique BamHI εite in exon II, and the HSV tk gene added to the 3' Kpnl end. Double crossover events on either εide of the nee insert, that eliminate the tk gene, are then selected for. Theεe are detected from poolε of selected cloneε by PCR amplification. One of the PCR primers is derived from neo sequences and the other from mouse sequences outεide of the targeting vector. The functional disruption of the mouεe in__muno r 'lobulin loci iε '"■resented in the Exam p les. Transgenic Non-Human Animals Containing Rearranged Immunoglobulin Heavy and Light Transgenes A premise underlying the previously diεcuεεed transgenic animals containing unrearranged mini-locus Ig transgenes is that it is possible to generate a complete antibody repertoire without including all of the variable gene segments found in the natural immunoglobulin locuε. Theoretically, it iε poεsible to reduce the number of different sequenceε that contribute to the primary repertoire without reducing the εecondary repertoire. Aε long aε there iε enough diversity in the primary repertoire to trigger a T-cell dependent responεe for any given antigen, εomatic hypermutation should be capable of delivering a high affinity antibody against that antigen. Thiε concept iε taken a εtep further in thiε aspect of the invention wherein a full heterologous antibody repertoire is generated entirely by somatic mutation. The antigen combining site is created by the interface between the amino-terminal heavy chain domain and the amino-terminal light chain domain. The CDRl, 2 and 3 residues within each of theεe domainε that interact with the antigen are located on three different loopε that connect β εtrandε. Aε previouεly described, these regions have the greatest εequence diverεity between different antibody moleculeε recognizing different antigens. Thus, the antibody repertoire is determined by sequence diversity at CDRl, 2, and 3. The diversity at CDRl, 2, and 3 that gives rise to a complete antibody repertoire co eε from three sources: recombinational diversity, junctional diversity, and somatic mutation. Recombinational diversity at CDRl and 2 comeε from the choice of different V segmentε containing different CDRl and 2 εequences. Recombinational diversity at CDR 3 comes from the choice of different D and J segmentε. Junctional diverεity contributeε only tc CDR__ diverεity, while εomatic mutation, acting acroεε the entire V region, contributeε to diversity at all three complimentarity determining regions. Recombinational and junctional diversity to g ether constitute the diversit y of the r> ri_nar v re p ertoire (Fig. 1) . Thus VDJ joining generates a set of IgM expresεing primary B-cells. Any primary repertoire B-cell that expresses a cell surface IgM molecule with a certain minimal affinity for a foreign antigen, internalizes that antigen as IgM and cycle off the cell surface. The antigen is then processed and associated peptides are presented on the cell surface by class II MHC molecules. If enough foreign antigen is presented at the cell surface this, triggers a T-cell responεe that in turn triggers the T-cell dependent maturation of the B-cell. This iε the εc- called εecondary reεponεe (Fig. 8) . Part of thiε reεponεe involves the hypermutation of the variable portion of the immunoglobulin genes. Thus a B-cell clone undergoing a secondary response constantly gives rise to new cloneε with altered immunoglobulin molecules. Those clones with higher affinities for the foreign antigen are selectively expanded by helper T-cells, giving rise to affinity maturation of the expressed antibody. Because somatic hypermutation takes place across the entire V region, there is no theoretical limit to the process of affinity maturation. In thiε aεpect of the invention, CDRl and 2 diverεity iε not necessary for generating a complete antibody response. Rather, diversity at CDR3, created by VJ and VDJ joining provides sufficient minimal affinity to trigger the T-cell dependent maturation to give rise to high affinity antibodieε for a large number of different antigenε. Thuε, methodε and transgenic animalε are provided for generating a broad antibody repertoire without primary diverεity. Such diversity relies on somatic mutation for the generation of antibody diversity. During the process of affinity maturation, somatic mutation gives rise to a large number of clones with lower, rather than higher, affinities for the stimulating antigen. Most of theεe cloneε are not εelected for and die off. However, if one of these clones has affinity for a new antigen that is alεo present, thiε clone expandε and undergoeε affinity maturation for the new antigen (Fig. 9) . In this aspect of the invention, a transgenic non-human animal, such aε a mouεe, with rearranged human heavy and light chains combine to form an antibody that has a low affinity for a known antigen. If this animal is injected with the known antigen, its B-cells undergo a secondary response leading to the production of high affinity antibodieε for that antigen. However, if thiε mouεe iε firεt injected with a mixture of the known antigen and a new antigen, and then subsequently challenged with the new antigen alone, high affinity antibodies againεt the new antigen are produced by the branching proceεs described above. This approach haε two major advantageε: firεt the tranεgene conεtructε are eaεy to generate; and second, the rearranged transgeneε are capable of allelicly and isotypically excluding the rearrangement of the endogenouε mouse genes, thus making it unnecesεary to eliminate those genes by homologous recombination as previously described. The first step in thiε embodiment of the invention iε the isolation of rearranged heavy and light chain geneε from a human hybridoma that expreεεeε an IgM antibody directed against a known antigen. The ideal hybridoma recognizes a readily available antigen that is capable of generating a good mouse T-cell response. There are a number of such human hybridomaε in existence, including εeveral that react with promiεing antigenε such as tetanus toxoid, pεeudomonaε, or gram negative bacteria (reviewed by James and Bourla (1987) , J. Immunol. Methods. , 100. 5-40) . The entire rearranged heavy chain gene is isolated on a single piece of DNA (approximately 20 kb) while the rearranged K light chain gene, including the 3' enhancer, iε isolated on a second DNA fragment (about 20 kb) . Each of these fragments are pieced together from clones iεolated from a phage λ library made from DNA iεolated from the hybridoma. Two constructs are generated, a heavy chain construct and a light chain construct. The heavy chain construct (Fig. 10) conεiεtε of the 20 kb hybridoma fragment, containing the rearranged IgM gene, ligated to a 25 kb fragment that containε the human 3 and 7 I constant regions followed by a 700 bp fragment containing the rat heavy chain 3* enhancer (Pettersson, et al. (1990), Nature, 344, 165-168) . The light chain conεtruct conεiεtε of the H L-αCJ < __ 3 ! enhancer. These two constructs are coinjected so that they are integrated at a single site in the mouεe genome. Transgenic mice are tested by Northern blot analyεiε for expression of the transgene mRNA. FACS analysiε is then carried-out on tail blood samples to detect cell surface expression of the transgene encoded protein. Mice are then immunized with the antigen recognized by the original hybridoma. ELISA and FACS analyεis are carried out on tail blood to detect class switching. Finally, the mice are tested for their ability to respond to a number of different antigenε by co-injecting a panel of antigens together with the original antigen. Tail blood are analyzed by ELISA to detect the production of high affinity human IgG antibodieε directed againεt individual antigenε. To use this transgenic mouεe to generate human antibodies directed against a given antigen, that antigen preferably is firεt coinjected together with the antigen associated with the hybridoma from which the genes were isolated. This hybridoma associated antigen is referred to aε the co-antigen (sometimeε as a εecond antigen) , and the new antigen simply as the antigen (or firεt antigen) . If poεεible, the second antigen is chemically cross-linked to the first antigen prior to injection. This causeε the first antigen to be internalized and presented by the primary transgene presenting B-cells, thuε ensuring the exiεtence of a pool of activated helper T-cellε that recognize the first antigen. A typical immunization schedule iε aε followε. Day l: Mice are injected ip with first antigen mixed with, or cross-linked to, second antigen in complete Freunds adjuvant. Day 14: firεt antigen (without second antigen) is injected ip in incomplete Freunds adjuvant. Day 35: repeat injection with firεt antigen in incomplete Freundε. Day 45: Test for antibody responεe by ELISA on tail blood εampleε. Day 56: repeat injection of good responders with antigen in incomplete Freunds. Day 59: Fuse spleenε of good responders. In an alternate aspect of thiε invention, the antigen recognized by the hybridoma from which the Ig genes were isolated, iε used as an immunogen. New transgenic hybridomaε are then isolated from the immunized animal that expresε somatically mutated verεions of the original antibody. These new antibodieε will have a higher affinity for the original antigen. Thiε antibody "sharpening" procedure can alεo be applied to antibody genes generated by CDR grafting (E.P. Pub. No. 239400, published Sept. 30, 1987) or isolated from bacterial (W.D. Huse et al. (1989) Science, 246 , 1275) or phage (T. Clackson et al. (1991) Nature, 352 , 624) expreεεion librarieε. Tranεgenic Non-Human Animalε Containing Rearranged and Unrearranged Immunoglobulin Heavy and/or Light Tranεgene The previous embodiments described the uεe of fully rearranged or fully unrearranged heavy and light immunoglobulin transgeneε to produce tranεgenic non-human animalε capable of producing a heterologouε antibody. In a further aεpect of the invention, tranεgenic animalε contain at leaεt one rearranged and at leaεt one unrearranged immunoglobulin tranεgene are produced by utilizing any of the aforementioned unrearranged and rearranged transgeneε in combination to provide heavy and light transgeneε in the tranεgenic animal. In thiε regard, the unrearranged transgene may comprise a heavy or light genomic or mini-locus transgene construct with the rearranged transgene comprising an appropriate rearranged tranεgene. For example, if a unrearranged mini-locus light chain transgene is used, the appropriate other tranεgene iε a fully rearranged heavy chain transgene. It iε preferred, however, that the rearranged tranεgene compriεe a rearranged immunoglobulin light chain transgene and that the unrearranged transgene comprise an immunoglobulin heavy chain genomic or mini-locus tranεgene, moεt preferably an unrearranged heavy chain tranεgene with associated A and y constant regions. The combination of rearranged and unrearranged transgene provideε an intermediate level of diversity within the primary repertoire B-cells. Thus, although primary diversity at CD1, CD2 and CD3 in the rearranged transgene is fixed in the primary repertoire B-cell, the primary diverεity at the CDRl, CDR2 and CDR3 produced by the rearrangement of the unrearranged tranεgene provideε a population of primary repertoire of B-cells having greater potential diversity than the B-cell clone obtained when rearranged heavy and light transgenes are used. Such primary diversity provides broadened secondary diverεity when εuch cells respond to foreign antigen by way of somatic mutation. Nucleic Acids The nucleic acidε, the term "substantial homology" indicates that two nucleic acidε, or deεignated εequenceε thereof, when optimally aligned and compared, are identical, with appropriate nucleotide inεertionε or deletions, in at least about 80% of the nucleotides, usually at leaεt about 90% to 95%, and more preferably at leaεt about 98 to 99.5% of the nucleotides. Alternatively, εubεtantial homology exists when the segmentε will hybridize under εelective hybridization conditions, to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or εubstantially pure form. A nucleic acid is "iεolated" or "rendered εubεtantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoreεis and others well known in the art. See, F. Ausubel, et al. , ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley- Interscience, New York (1987) . The nucleic acid compositionε of the preεent invention, while often in a native εequence (except for modified reεtriction sites and the like) , from either cDNA, genomic or mixtures may be mutated, thereof in accordance with standard techniques to provide gene sequenceε. For coding sequences, these mutations, may affect amino acid sequence aε deεired. In particular, DNA sequences substantially homologouε to or derived from native V, D, J, conεtant, εwitcheε and other such seTience≤ described herein are contemplated (where "derived" indicates that a sequence is identical or modified from another sequence) . A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affectε the tranεcription of the sequence. With respect to transcription regulatory sequenceε, operably linked meanε that the DNA εequenceε being linked are contiguouε and, where neceεεary to join two protein coding regionε, contiguouε and in reading frame. For εwitch sequences, operably linked indicates that the sequenceε are capable of effecting switch recombination. Specific Preferred Embodiments A preferred embodiment of the invention iε an animal containing a single copy of the transgene described in Example 14 (pHC2) bred with an animal containing a single copy of the transgene described in Example 16, and the offspring bred with the JH deleted animal described in Examples 9 and 12. Animalε are bred to homozygoεity for each of these three traitε. Such animalε have the following genotype: a εingle copy (per haploid set of chromosomeε) of a human heavy chain unrearranged mini-locus (described in Example 14) , a single copy (per haploid set of chromosomes) of a rearranged human K light chain construct (described in Example 16) , and a deletion at each endogenous mouse heavy chain locus that removes all of the functional JH segmentε (deεcribed in Exampleε 9 and 12) . Such animalε are bred with mice that are homozygous for the deletion of the JH segmentε (Exampleε 9 and 12) to produce offεpring that are homozygouε for the JH deletion and hemizygouε for the human heavy and light chain conεtructε. The reεultant animalε are injected with antigenε and used for production of human monoclonal antibodies against these antigenε. B cells isolated from such an animal are monospecific with regards to the human heavy and light chains because they contain only a single copy of each gene. Furthermore, they will be monos p eci ic with re g ards to human or mouse heavy chains because both endogenous mouse heavy chain gene copies are nonfunctional by virtue of the deletion spanning the JH region introduced aε deεcribed in Example 9 and 12. Furthermore, a εubstantial fraction of the B cells will be monospecific with regards to the human or mouse light chains because expression of the single copy of the rearranged human K light chain gene will allelically and iεotypically exclude the rearrangement of the endogenouε mouεe K and lambda chain geneε in a εignificant fraction of B-cellε. The transgenic mouse of the preferred embodiment will exhibit immunoglobulin production with a significant repertoire, ideally subεtantially εimilar to that of a native mouεe. Thus, for example, when the endogenouε Ig geneε have been inactivated, the total immunoglobulin levelε will range from about 0.1 to 10 mg/ml of εeru , preferably 0.5 to 5 mg/ml, ideally at least about 1.0 mg/ml. When a tranεgene capable of effecting a εwitch to IgG from IgM haε been introduced into the transgenic mouse, the adult mouse ratio of serum IgG to IgM is preferably about 10:1. Of course, the IgG to IgM ratio will te much lower in the immature mouse. In general, greater than about 10%, preferably 40 to 80% of the spleen and lymph node B cells express exclusively human IgG protein. The repertoire will ideally approximate that shown in a non-transgenic mouse, usually at least about 10% as high, preferably 25 to 50% or more. Generally, at least about a thousand different immunoglobulins (ideally IgG) , preferably 10 4 to 10 6 or more, will be produced, depending primarily on the number of different V, J and D regions introduced into the mouse genome. These immunoglobulins will typically recognize about one-half or more of highly antigenic proteins, including, but not limited to: pigeon cytochro e C, chicken lysozyme, pokeweed itogen, bovine serum albumin, keyhole limpit hemocyanin, influenza hemagglutinin, staphylococcuε protein A, εperm whale myoglobin, influenza neuraminidase, and lambda repressor protein. Some of the immunoglobulins will exhibit an affinity for preselected antigenε of at least about 10 "7 M _1 , preferably 10 "8 M _1 to 10 ~9 M _1 or greater. Although the foregoing describes a preferred embodiment of the tranεgenic animal of the invention, other embodiments are defined by the discloεure herein and more particularly by the tranεgeneε deεcribed in the Exampleε. Four categorieε of transgenic animal may be defined: I. Transgenic animals containing an unrearranged heavy and rearranged light immunoglobulin tranεgene. II. Transgenic animals containing an unrearranged heavy and unrearranged light immunoglobulin tranεgene III. Tranεgenic animal containing rearranged heavy and an unrearranged light immunoglobulin tranεgene, and IV. Transgenic animalε containing rearranged heavy and rearranged light immunoglobulin tranεgeneε. Of theεe categorieε of tranεgenic animal, the preferred order of preference iε aε followε I > II > III > IV. Within each of theεe categorieε of the tranεgenic animal, a number of poεεible combinationε are preferred. Such preferred embodimentε comprise the following: Category I (a) Example 1 and 2 or 19 and 20 animal bred with Example 7 or 16 animal. (b) Example 1 or 19 fragment coinjected with Example 7 or 16 fragment. (c) Example 5 (H, I or J) or 14, 17 or 21 animal bred with Example 7 or 16 animal. (d) Example 5(H) or 14 conεtruct coinjected with Example 7 or 16 construct. (e) All of the above bred with the animal of Example 9 or 11, 12 or 13. Particularly preferred embodiments are all of the above bred the with animal of Example 9 or 12 or 13. Category II (a) Example 1, 2, 19 or 20 animal bred with Example 6, 3, 4, 16, 22 or 23 animal. C . Fragment in Exam p le 1 or 19 coin j ected with fragment in Example 2 or 20. (c) Example 5 (H, I or J) or 14, 17 or 21 animal bred with Example 6(B, C or D) or 16 animal. (d) Conεtruct 5(H) or 14 coinjected with conεtruct 6(B) or 16. (e) Animal of Example 1, 2, 19 or 20 bred with animal of Example 6(B, C or D) or 16. (f) Animal of Example 3, 4, 22 or 23 bred with animal of Example 5(H, I or J) or 14, 17 or 21. (g) All of the above bred with animal of Example 9, 10, 11, 12 or 13. Category III (a) Example 3, 4, 22 or 23 animal bred with Example 8 or 15 animal. (b) Example 3 or 23 fragment coinjected with Example 8 or 15 fragment. (c) Example 6(B, C or D) or 16 animal bred with Example 8 or 15 animal. (d) Example 6(B) , or 15 conεtruct coinjected with Example 8 or 15 conεtruct. (e) All of the above bred with animal of Example 9 to 13. Category IV (a) Animal of Example 7 or 16, bred with animal of Example 8 or 15. (b) Conεtruct of Example 7 or 16 coinjected with conεtruct of Example 8 or 15. (c) All of the above bred with animal of Example 9 to 13. The following is presented by way of example and iε ot to be conεtrued aε a limitation to the εcope of the claims. METHODS AND MATERIALS Transgenic mice are derived according to Hogan, et al., "Manipulating the Mouse Embryo: A Laboratory Manual". Cold Spring Harbor Laboratory. Embryonic stem cells are manipulated according to published procedures (Teratocarcinomas and embryonic stem cells: a practical approach, E.J. Robertson, ed. , IRL Press, Washington, D.C., 1987; Zjilεtra, et al. (1989), Nature, 342, 435-438; and Schwartzberg, P., et al. (1989), Science, 246 , 799-803) . DNA cloning procedures are carried out according to J. Sambrook, et al. in Molecular Cloning: A Laboratory Manual, 2d ed. , 1989, Cold Spring Harbor Laboratory Preεε, Cold Spring Harbor, N.Y. Oligonucleotideε are syntheεized on an Applied Bio Systems oligonucleotide synthesizer according to εpecifications provided by the manufacturer. Hybridoma cells and antibodies are manipulated according to "Antibodies: A Laboratory Manual", Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988) . EXAMPLE 1 Genomic Heavy Chain Human Ig Transgene This Example describes the cloning and microinjection of a human genomic heavy chain immunoglobulin transgene which iε microinjected into a murine zygote. Nuclei are isolated from freεh human placental tiεsue as described by Marzluff, W.F., et al. (1985) , "Tranεcription and Translation: A Practical Approach", B.D. Hammes and S.J. Higgins, eds., pp. 89-129, IRL Presε, Oxford) . The isolated nuclei (or PBS washed human spermatocyteε) are embedded in a low melting point agarose matrix and lysed with EDTA and proteinaεe K to expose high molecular weight DNA, which is then digested in the agarose with the restriction enzyme Notl as described by M. Finney in Current Protocols in Molecular Biology (F. Ausubel, et al. , edε. John Wiley & Sonε, Supp. 4, 1988, Section 2.5.1) . The Notl digested DNA is then fractionated by pulsed field gel electrophoresis as described by Anand, R. , et al. (1989) , Nucl. Acids Reε. , 17, 3425-3433. Fractions enriched for the Notl fragment are assayed by Southern hybridization to detect one or more of the sequences encoded by this fragment. Such sequences include the heavy chain D εegments, J segmentε, μ and 71 constant regionε together with repreεentatives of all 6 VH families (although this fragment is identified as 670 kb fragment from HeLa cells by Berman, et al. (1988) , supra. , we have found it to be as 830 kb fragment from human placental an sperm DNA) . Those fractions containing this Notl fragment (εee Fig. 4) are pooled and cloned into the Notl εite of the vector pYACNN in Yeaεt cells. Plaεmid pYACNN is prepared by digestion of pYAC-4 Neo (Cook, H. , et al. (1988) , Nucleic Acids Res. , 16, 11817) with EcoRI and ligation in the presence of the oligonucleotide 5' - AAT TGC GGC CGC - 3'. YAC clones containing the heavy chain Notl fragment are isolated aε described by Brownstein, et al. (1989) , Science, 244. 1348-1351, and Green, E., et al. (1990), Proc. Natl. Acad. Sci. USA. 87, 1213-1217. The cloned Notl inεert is iεolated from high molecular weight yeaεt DNA by pulεe field gel electrophoresis as described by M. Finney, opcit. The DNA is condensed by the addition of 1 mM spermine and microinjected directly into the nucleus of single cell embryoε previouεly described. EXAMPLE 2 Discontinuous Genomic Heavy Chain Ig Transgene A 110 kb Spel fragment of human genomic DNA containing VH6, D segments, J segments, the μ constant region and part of the 7 conεtant region (see Fig. 4) is isolated by YAC cloning as described in Example 1. A 570 kb Notl fragment upstream of the 670-830 kb Notl fragment described above containing multiple copies of VI through V5 is isolated as described. (Berman, et al. (1988) , supra detected two 570 kb Notl fragments. Each of those contain multiple V segmentε.) The two fragments are coinjected into the nucleus of a mouse single cell embryo aε described in Example 1. Coinjection of two different DNA fragments will usually result in the integration of both fragmentε at the εame insertion site within the chromosome. Therefore, approximately 50% of the resulting transgenic animals that contain at least one copy of each of the two fragments will have the V segment fragment inserted upstream of the conεtant region containing fragment. Of these animals, 50% will carry out V to DJ joining by DNA inversion and 50% by deletion, depending on the orientation of the 570 kb Notl fragment relative to the position of the 110 kb Spel fragment. DNA is isolated from resultant tranεgenic animalε and thoεe animalε found to be containing both transgenes by Southern blot hybridization (specifically, thoεe animalε containing both multiple human V εegmentε and human conεtant region geneε) are teεted for their ability to expreεs human immunoglobulin moleculeε. EXAMPLE 3 Genomic K Light Chain Human Ig Tranεgene Formed bv In Vivo Homologouε Recombination A map of the human K light chain haε been described in Lorenz, W. , et al. (1987) , Nucl. Acids Res. , 15, 9667-9677 and is depicted in Fig. 11. A 450 kb Xhol to Notl fragment that includes all of Ck, the 3' enhancer, all J segments, and at leaεt five different V segments (a) is isolated and microinjected into the nucleus of single cell embryos as described in Example 1. EXAMPLE 4 Genomic K Light Chain Human Ig Tranεgene Formed by In Vivo Homologouε Recombination A 750 kb Mlul to Notl fragment that includeε all of the above pluε at leaεt 20 more V εegmentε (b) iε iεolated as described in Example 1 (see Fig. 11) and digested with BssHII to produce a fragment of about 400 kb (c) . The 450 kb Xhol to Notl fragment (a) plus the approximately 400 kb Mlul to BεsHII fragment (c) have sequence overlap defined by the BεsHII and Xhol restriction siteε shown in Fig. 11. Homologouε recombination of these two fragments upon microinjection of a mouse zygote results in a transgene containing at least an additional 15-20 V segments over that found in the 450 kb Xhol/Notl fragment (Example 3). EXAMPLE 5 Construction of Heavy Chain Mini-Locus A. Construction of pGPl and pGP2 pBR322 is digested with EcoRI and Styl and ligated with the following oligonucleotides to generate pGPl which containε a 147 baεe pair inεert containing the reεtriction sites shown in Fig. 13. The general overlapping of these oligos iε also shown in Fig. 13. The oligonucleotideε are: oligo-1 5' - CTT GAG CCC GCC TAA TGA GCG GGC TTT TTT TTG CAT ACT GCG GCC - 3 ' oligo-2 5' - GCA ATG GCC TGG ATC CAT GGC GCG CTA GCA TCG ATA TCT AGA GCT CGA GCA -3 ' θligo-3 5' - TGC AGA TCT GAA TTC CCG GGT ACC AAG CTT ACG CGT ACT AGT GCG GCC GCT -3 ' oligo-4 5' - AAT TAG CGG CCG CAC TAG TAC GCG TAA GCT TGG TAC CCG GGA ATT - 3 ' oligo-5 5' - CAG ATC TGC ATG CTC GAG CTC TAG ATA TCG ATG CTA GCG CGC CAT GGA TCC - 3* θligo-6 5' - AGG CCA TTG CGG CCG CAG TAT GCA AAA AAA AGC CCG CTC ATT AGG CGG GCT - 3 ' This plasmid contains a large polylinker flanked by rare cutting Notl εiteε for building large inεertε that can be isolated from vector sequences for microinjection. The plaεmid iε based on pBR322 which is relatively low copy compared to the pUC based plasmids (pGPl retains the pBR322 copy number control region near the origin of replication) . Low copy number reduces the potential toxicity of inεert sequences. In addition, pGPl contains a εtrong tranεcription terminator εequence derived from trpA (Christie, G.E., et al. (1981) , Proc. Natl. Acad. Sci. USA) inserted between the ampic l m resistance gene and the polylinker. This further reduces the toxicity associated with certain inserts by preventing readthrough transcription coming from the ampicillin promoters. Plasmid pGP2 iε derived from pGPl to introduce an additional restriction site (Sfil) in the polylinker. pGPl is digested with Mlul and Spel to cut the recognition sequences in the polylinker portion of the plasmid. The following adapter oligonucleotideε are ligated to the thus digested pGPl to form pGP2. 5 ' CGC GTG GCC GCA ATG GCC A 3 » 5' CTA GTG GCC ATT GCG GCC A 3 ' pGP2 is identical to pGPl except that it containε an additional Sfi I εite located between the Mlul and Spel εiteε. Thiε allows insertε to be completely exciεed with Sfil aε well as with Notl. B. Construction of pRE3 (rat enhancer 3') An enhancer sequence located downstream of the rat constant region is included in the heavy chain conεtructε. The heavy chain region 3' enhancer described by S. Pettersson, et al. (1990), Nature, 344 , 165-168) iε iεolated and cloned. The rat IGH 3' enhancer εequence iε PCR amplified by using the following oligonucleotides: 5 ' CAG GAT CCA GAT ATC AGT. ACC TGA AAC AGG GCT TGC 3 ' 5 • GAG CAT GCA CAG GAC CTG GAG CAC ACA CAG CCT TCC 3 ' The thus formed double stranded DNA encoding the 3 ' enhancer is cut with BamHI and Sphl and clone into BamHI/Sphl cut pGP2 to yield pRE3 (rat enhancer 3') . C. Cloning of Human J-u Region A substantial portion of this region is cloned by combining two or more fragmentε isolated from phage lambda inserts. See Fig. 14. A 6.3 kb BamHI/Hindlll fragment that includes all human J segments (Matsuda, et al. (1988), EMBO J. , 1_, 1047— 1051; Ravetech, et al. (1981), Cell, 27, 583-591) iε iεolated from human genomic DNA library using the oligonucleotide GGA CTG TGT CCC TGT GTG ATG CTT TTG ATG TCT GGG GCC AAG. An adjacent 10 kb Hindlll/Bamll fragment that containε enhancer, εwitch and conεtant region coding exonε (Yaεui, et al. (1989), Eur. J. Immunol. , 19 , 1399-1403) iε εimilarly isolated using the oligonucleotide: CAC CAA GTT GAC CTG CCT GGT CAC AGA CCT GAC CAC CTA TGA An adjacent 3' 1.5 kb BamHI fragment is εimilarly iεolated using clone pMUM insert as probe (pMUM is 4 kb EcoRI/Hindlll fragment isolated from human genomic DNA library with oligonucleotide: CCT GTG GAC CAC CGC CTC CAC CTT CAT CGT CCT CTT CCT CCT u membrane exon 1) and cloned into pUC19. pGPl is digested with BamHI and Bglll followed by treatment with calf inteεtinal alkaline phosphatase. Fragments (a) and (b) from Fig. 14 are cloned in the digested pGPl. A clone is then isolated which iε oriented εuch that 5' BamHI εite iε deεtroyed by BamHI/Bgl fuεion. It iε identified aε pMU (εee Fig. 15) . pMU iε digeεted with BamHI and fragment (c) from Fig. 14 iε inεerted. The orientation iε checked with Hindlll digest. The reεultant plaεmid pHIGl (Fig. 15) containε an 18 kb inεert encoding J and Cμ εegments. D. Cloning of Cμ Region pGPl iε digeεted with BamHI and Hindlll iε followed by treatment with calf inteεtinal alkaline phosphataεe (Fig. 14) . The so treated fragment (b) of Fig. 14 and fragment (c) of Fig. 14 are cloned into the BamHI/Hindlll cut pGPl. Proper orientation of fragment (c) iε checked by Hindlll digestion to form pCONl containing a 12 kb insert encoding the Cμ region. Whereas pHIGl contains J segments, switch and μ sequences in its 18 kb insert with an Sfil 3 ' εite and a Spel 5' εite in a polylinker flanked by Notl sites, will be uεed for rearranged VDJ εegmentε. pCONl iε identical except that it lackε the J region and containε only a 12 kb inεert. The use of pCONl in the construction of fragment containing rearranged VDJ εegmentε will be deεcribed hereinafter. E. Cloning of Ύ-1 Constant Region (pREG2) The cloning of the human 7-1 region iε depicted in Fig, 16. Yamamura, et al. (1986) , Proc. Natl. Acad. Sci. USA, 83 , 2152-2156 reported the expression of membrane bound human 7-1 from a transgene construct that had been partially deleted on integration. Their results indicate that the 3' BamHI εite delineateε a sequence that includes the transmembrane rearranged and switched copy of the gamma gene with a V-C intron of less than 5kb. Therefore, in the unrearranged, unswitched gene, the entire switch region is included in a sequence beginning less than 5 kb from the 5 ' end of the first 7 -1 conεtant exon. Therefore it iε included in the 5' 5.3 kb Hindlll fragment (Ellison, J.W. , et al. (1982), Nucleic Acidε Reε.. 10, 4071-4079). Takahaεhi, et al. (1982), Cell, 29, 671-679 also reports that thiε fragment containε the εwitch sequence, and this fragment together with the 7.7 kb Hindlll to BamHI fragment must include all of the sequences we need for the transgene construct. Phage clones containing the 7-1 region are identified and isolated using the following oligonucleotide which is specific for the third exon of 7 -1 (CH3) . 5' TGA GCC ACG AAG ACC CTG AGG TCA AGT TCA ACT GGT ACG TGG 3 ' A 7.7 kb Hindlll to Bglll fragment (fragment (a) in Fig. 16) iε cloned into Hindlll/Bglll cut pRE3 to form pREGl. The upstream 5.3 kb Hindlll fragment (fragment (b) in Fig. 16) is cloned into Hindlll digested pREGl to form pREG2.. Correct orientation iε confirmed by BamHI/Spel digeεtion. F. Combining C~t and Cμ The previouεly deεcribed plaεmid pHIGl containε human J εegmentε and the Cμ constant region exonε. To provide a transgene containing the Cμ constant region gene εegmentε, pHIGl waε digested with Sfil (Fig. 15) . The plaεmid pREG2 waε also digested with Sfil to produce a 13.5 kb insert containing human C7 exons and the rat 3' enhancer εequence. Theεe sequences were combined to produce the plaεmid pHIG3 ' (Fig. 17; containing the human J segments, the human Cμ constant region, the human C7I constant region and the rat 3 ' enhancer contained on a 31.5 kb insert. A second plasmid encoding human Cμ and human C 7 I without J segments is constructed by digesting pCONl with Sfil and combining that with the Sfil fragment containing the human C7 region and the rat 3' enhancer by digeεting pREG2 with Sfil. The resultant plasmid, pCON (Fig. 17) contains a 26 kb Notl/Spel insert containing human Cμ, human 7I and the rat 3' enhancer sequence. G. Cloning of D Segment The strategy for cloning the human D segments is depicted in Fig. 18. Phage clones from the human genomic library containing D segments are identified and isolated using probes specific for diversity region sequenceε (Y. Ichihara, et al. (1988), EMBO J. , 1_, 4141-4150) . The following oligonucleotideε are uεed: DXP1: 5' - TGG TAT TAC TAT GGT TCG GGG AGT TAT TAT AAC CAC AGT GTC - 3 ' DXP4: 5' - GCC TGA AAT GGA GCC TCA GGG CAC AGT GGG CAC GGA CAC TGT - 3 ' DN4: 5' - GCA GGG AGG ACA TGT TTA GGA TCT GAC- GCC GCA CCT GAC ACC - 3 ' A 5.2 kb Xhol fragment (fragment (b) in Fig. 18) containing DLR1, DXP1, DXP'l, and DAI is iεolated from a phage clone identified with oligo DXP1. A 3.2 kb Xbal fragment (fragment (c) in Fig. 18) containing DXP4, DA4 and DK4 iε iεolated from a phage clone identified with oligo DXP . Fragmentε (b) , (c) and (d) from Fig. 18 are combined and cloned into the Xbal/Xhol εite of pGPl to form pHIG2 which contains a 10.6 kb insert. This cloning is performed sequentially. First, the 5.2 kb fragment (b) in Fig. 18 and the 2.2 kb fragment (d) of Fig. 18 are treated with calf intestinal alkaline phosphatase and cloned into pGPl digested with Xhol and Xbal. The resultant clones are screened with the 5.2 and 2.2 kb insert. Half of those clones testing positive with the 5.2 and 2.2 kb inserts have the 5.2 kb insert in the proper orientation as determined by BamHI digestion. The 3.2 kb Xbal fragment from Fig. 18 is then cloned into thiε intermediate plaεmid containing fragmentε (b) and (d) to form pHIG2 (Fig. 9) . Thiε plasmid contains diverεity εegmentε cloned into the polylinker with a unique 5' Sfil εite and unique 3' Spel εite. The entire polylinker iε flanked by Notl sites. H. Construction of Heavy Chain Minilocus The following deεcribeε the construction of a human heavy chain mini-locus which contain one or more V εegmentε. An unrearranged V εegment corresponding to that identified as the V segment contained in the hybridoma of Newkirk, et al. (1988), J. Clin. Invest. , 81, 1511-1518, iε isolated using the following oligonucleotide: 5' - GAT CCT GGT TTA GTT AAA GAG GAT TTT ATT CAC CCC TGT GTC - 3 ' A restriction map of the unrearranged V segment is determined to identify unique restriction siteε which provide upon digestion a DNA fragment having a length approximately 2 kb containing the unrearranged V segment together with 5 ' and 3' flanking sequences. The 5' prime sequenceε will include promoter and other regulatory sequences whereas the 3' flanking sequence provides recombination sequences necesεary for V-DJ joining. This approximately 3.0 kb V segment insert is cloned into the polylinker of pGB2 to form pVHl. pVHl is digested with Sfil and the resultant fragment is cloned into the S il site of pHIG2 to form a riHlGS' _ Since pHIG2 contains D segments only, the resultant pHIG5' plasmid contains a single V segment together with D segments. The size of the insert contained in pHIG5 is 10.6 kb pluε the size of the V segment insert. The insert from pHIG5 is excised by digestion with Notl and Spel and isolated. pHIG3 ' which contains J, Cμ and C I segments is digested with Spel and Notl and the 3 ' kb fragment containing such sequences and the rat 3 ' enhancer sequence iε iεolated. Theεe two fragments are combined and ligated into Notl digested pGPl to produce pHIG which containε insert encoding a V segment, nine D εegmentε, εix functional J segments, Cμ, C 7 and the rat 3' enhancer. The size of thiε inεert iε approximately 43 kb pluε the size of the V segment insert. I. Construction of Heavy Chain Minilocus by Homologous Recombination As indicated in the previouε εection, the inεert of pHIG iε approximately 43 to 45 kb when a εingle V εegment iε employed. Thiε insert size is at or near the limit of that which may be readily cloned into plasmid vectors. In order to provide for the use of a greater number of V segments, the following describeε jLn vivo homologouε recombination of overlapping DNA fragmentε which upon homologous recombination within a zygote or ES cell form a transgene containing the rat 3' enhancer sequence, the human Cμ, the human C 7 I, human J segments, human D segments and a multiplicity of human V segments. A 6.3 kb BamHI/Hindlll fragment containing human J segmentε (see fragment (a) in Fig. 14) is cloned into Mlul/Spel digested pHIG5' using the following adapters: 5' GAT CCA AGC AGT 3' 5' CTA GAC TGC TTG 3 ' 5' CGC GTC GAA CTA 3' 5 ' AGC TTA GTT CGA 3 ' The resultant iε plasmid designated pHIG5 '0 (overlap) . The insert contained in this Plasmid contains human V D and J segments. When the single V segment from pVHl is used, the size of this insert is approximately 17 kb plus 2 kb. Thiε insert is isolated and combined with the insert from pHIG3 ' which contains the human J, Cμ, 71 and rat 3' enhancer sequences. Both inserts contain human J segments which provide for approximately 6.3 kb of overlap between the two DNA fragments. When coinjected into the mouse zygote, in vivo homologous recombination occurs generating a tranεgene equivalent to the insert contained in pHIG. This approach provides for the addition of a multiplicity of V εegmentε into the transgene formed In vivo. For example, instead of incorporating a single V εegment into pHIG5 ' , a multiplicity of V εegmentε contained on (1) iεolated genomic DNA, (2) ligated DNA derived from genomic DNA, or (3) DNA encoding a synthetic V εegment repertoire iε cloned into pHIG2 at the Sfil εite to generate pHIG5 ' V N . The J segments fragment (a) of Fig. 14 is then cloned into pHIG5 ' V N and the inεert iεolated. Thiε inεert now containε a multiplicity of V εegmentε and J segments which overlap with the J segments contained on the insert isolated from pHIG3 ' . When cointroduced into the nucleus of a mouεe zygote, homologouε recombination occurε to generate rn vivo the transgene encoding multiple V segments and multiple J segments, multiple D segments, the Cμ region, the C7I region (all from human) and the rat 3' enhancer εequence. J. Construction of Heavy Chain Mini-Locuε by Coinjection of Synthetic VH Region Fragment Together with Heavy Chain DJC Construct Synthetic H region fragments are generated and iεolated aε previously described. These fragmentε are coinjected with the purified Notl inεert of plaεmid pHIG (or a verεion of pHIG that doeε not contain any V εegmentε) . The coinjected DNA fragmentε are inεerted into a εingle εite in the chromoεome. Some of the reεulting tranεgenic animalε will contain transgene inserts that have synthetic V regions located adjacent and upstream of the sequences in the pHIG construct. These animals will have a larger human heavy chain primary repertoire than the animals described in Example 5 (H) . EXAMPLE 6 Conεtruction of Light Chain Minilocus A. Construction of pEμl The construction of pEμl is depicted in Fig. 21. The mouse heavy chain enhancer is isolated on the Xbal to EcoRI 678 bp fragment (J. Banerji, et al. (1983), Cell, 33 , 729-740) from phage clones uεing oligo: 5' GAA TGG GAG TGA GGC TCT CTC ATA CCC TAT TCA GAA CTG ACT 3 ' Thiε Eμ fragment is cloned into EcoRV/Xbal digested pGPl by blunt end filling in EcoRI site. The resultant plasmid is designated pEmul. B. Construction Of K Light chain Minilocus The K construct contains at leaεt one human V,. εegment, all five human J κ εegmentε, the human -C^ enhancer, human K conεtant region exon, and, ideally, the human 3 ' n enhancer (K. Meyer, et al. 1989), EMBO J. , 8 . , 1959-1964) . The K enhancer in mouse iε 9 kb downεtream from C κ . However, it is as yet unidentified in the human. In addition, the construct contains a copy of the mouse heavy chain J-Cμ enhancers. The minilocus is constructed from four component fragments: (a) A 16 kb Smal fragment that containε the human C κ exon and the 3' human enhancer by analogy with the mouεe locuε (fragment (a) in Fig. 20) ; (b) A 5' adjacent 5 kb Smal fragment, which contains all five J εegmentε (fragment (b) in Fig. 20) ; (c) The mouse heavy chain intronic enhancer isolated from pEμl (this sequence is included to induce expression of the light chain construct as early as poεεible in B-cell development. Becauεe the heavy chain geneε are tranεcribed earlier than the light chain geneε, thiε heavy chain enhancer is presumably active at an earlier stage than the intronic K βnhanrO l • anH (d) A fragment containing one or more V segments. The preparation of this conεtruct iε aε followε. Human placental DNA is digested with Smal and fractionated on agarose gel by electrophoresis. Similarly, human placental DNA is digested with BamHI and fractionated by electrophoresis. The 16 kb fraction is isolated from the Smal digested gel and the 11 kb region. is similarly isolated from the gel containing DNA digested with BamHI. The 16 kb Smal fraction is cloned into Lambda FIX II (Stratagene, La Jolla, California) which has been digested with Xhol, treated with klenow fragment DNA poly erase to fill in the Xhol reεtriction digeεt product. Ligation of the 16 kb Smal fraction deεtroys the Smal siteε and laεeε Xhol εiteε in tact. The 11 kb BamHI fraction iε cloned into λ EMBL3 (Strategene, La Jolla, California) which iε digeεted with BamHI prior to cloning. Clones from each library were probed with the Cc specific oligo: 5 ' GAA CTG TGG CTG CAC CAT CTG TCT TCA TCT TCC CGC CAT CTG 3 ' A 16 kb Xhol insert that was subcloned into the Xhol cut pEμl so that Cc is adjacent to the Smal site. The resultant plasmid was designated pKapl. See Fig. 22. The above C/c specific oligonucleotide iε uεed to probe the λ EMBL3/BamHI library to identify an 11 kb clone corresponding to fragment (d) of Fig. 20. A 5 kb Smal fragment (fragment (b) in Fig. 20) is subcloned and subsequently inserted into pKapl digested with Smal. Those plasmids containing the correct orientation of J segmentε, C/c and the Eμ enhancer are deεignated pKap2. One or more V/c εegmentε are thereafter εubcloned into the Mlul site of pKap2 to yield the plasmid pKapH which encodes the human V/c segments, the human Jc segmentε, the human Cc segments and the human Eμ enhancer. This inεert iε excised by digesting pKapH with Notl and purified by agarose gel electrophoresis. The thus purified insert is microinjected ι « tc the pronuc " ^ eus o"^ a mouse z rrτ ote aε ^r vio l^ 7 described. C. Construction of /c Light Chain Minilocuε by In Vivo Homologous Recombination The 11 kb BamHI fragment (fragment (d) in Fig. 20) is cloned into BamHI digested pGPl such that the 3 ' end is toward the Sfil εite. The reεultant plasmid is designated pKAPint. One or more V/c segments is inserted into the polylinker between the BamHI and Spel siteε in pKAPint to form pKapHV. The inεert of pKapHV iε exciεed by digestion with Notl and purified. The inεert from pKap2 iε excised by digeεtion with Notl and purified. Each of theεe fragmentε contain regionε of homology in that the fragment from pKapHV containε a 5 kb εequence of DNA that include the κ segments which iε εubstantially homologous to the 5 kb Smal fragment contained in the insert obtained fron pKap2. Aε εuch, theεe inεertε are capable of homologouεly recombining when microinjected into a mouεe zygote to form a transgene encoding V κ , J κ and C κ . D. Conεtruction of K Light Chain Mini-Locus by Coinjection of Synthetic Vc Region Fragment Together with Light Chain JC Construct Synthetic V/c, region fragments are generated and isolated as previouεly deεcribed. These DNA fragmentε are coinjected with the purified Notl insert of plaεmid pKap2 or plasmid pKapH. The coinjected DNA fragments are inserted into a single site in the chromosome. Some of the resulting transgenics will contain transgene insertε that have εynthetic V regionε located adjacent and upεtream of the εequenceε in the pKap2 or pKapH conεtruct. Theεe animalε will have a larger human K light chain primary repertoire than thoεe deεcribed in Example 6 (B) . EXAMPLE 7 Isolation of Genomic Clones Corresponding to Rearranged and Expreεsed Copies of Immunoglobulin /c Light Chain Genes This example describes the cloning of immunoglobulin K light chain genes from cultured cells that express an immunoglobulin of interest. Such cellε may contain multiple alleles of a given immunoglobulin gene. For example, a hybridoma might contain four copies of the /c light chain gene, two copies from the fusion partner cell line and two copieε from the original B-cell expreεsing the immunoglobulin of interest. Of these four copieε, only one encodeε the immunoglobulin of intereεt, deεpite the fact that εeveral of them may be rearranged. The procedure deεcribed in thiε example allowε for the εelective cloning of the expresεed copy of the K light chain. A. Double Stranded cDNA Cellε from human hybridoma, or lymphoma, or other cell line that synthesizes either cell surface or secreted or both forms of IgM with a /c light chain are used for the iεolation of polyA+ RNA. The RNA iε then uεed for the syntheεiε of oligo dT primed cDNA using the enzyme reverse tranεcriptaεe. The εingle εtranded cDNA iε then isolated and G residueε are added to the 3' end using the enzyme polynucleotide terminal tranεferase. The Gtailed εingle-stranded cDNA is then purified and used as template for second strand synthesis (catalyzed by the enzyme DNA polymerase) using the following oligonucleotide as a primer: 5' - GAG GTA CAC TGA CAT ACT GGC ATG CCC CCC CCC CCC - 3 ' The double stranded cDNA is isolated and used for determining the nucleotide sequence of the 5 ' end of the mRNAs encoding the heavy and light chains of the expresεed immunoglobulin molecule. Genomic cloneε of these expreεεed geneε are then iεolated. The procedure for cloning the expressed light chain gene iε outlined in part B below. B. Light Chain The double εtranded cDNA described in part A is denatured and used as a template for a third round of DNA synthesis using the following oligonucleotide primer: 5' - GTA CGC CAT ATC AGC TGG ATG AAG TCA TCA GAT GGC GGG AAG ATG AAG ACA GAT GGT GCA - 3 ' This primer contains sequences specific for the constant portion of the K light chain mesεage (TCA TCA GAT GGC GGG AAG ATG AAG ACA GAT GGT GCA) as well as unique sequences that can be used as a primer for the PCR amplification of the newly syntheεized DNA εtrand (GTA CGC CAT ATC AGC TGG ATG AAG) . The εequence iε amplified by PCR uεing the following two oligonucleotide primerε: 5 ' - GAG GTA CAC TGA CAT ACT GGC ATG -3 ' 5 1 - GTA CGC CAT ATC AGC TGG ATG AAG -3' The PCR amplified εequence iε then purified by gel electrophoreεis and used as template for dideoxy sequencing reactions using the following oligonucleotide aε a primer: 5 ' - GAG GTA CAC TGA CAT ACT GGC ATG -3 ' The firεt 42 nucleotideε of sequence will then be used to synthesize a unique probe for isolating the gene from which immunoglobulin message was transcribed. Thiε εynthetic 42 nucleotide εegment of DNA will be referred to below aε o-kappa. A Southern blot of DNA, isolated from the Ig expresεing cell line and digeεted individually and in pairwiεe co binationε with several different restriction endonucleaεes including Smal, is then probed with the 32-P labelled unique oligonucleotide o-kappa. A unique restriction endonuclease site is identified upstream of the rearranged V segment. DNA from the Ig expressing cell line is then cut with Smal and second enzyme (or BamHI or Kpnl if there is Smal εite inside V εegment) . Any resulting non-blunted ends are treated with the enzyme T4 DNA polymerase to give blunt ended DNA moleculeε. Then add restriction site encoding linkers (BamHI, EcoRI or Xhol depending on what site does not exist in fragment) and cut with the corresponding linker enzyme to give DNA fragmentε with BamHI, EcoRI or Xhol endε. The DNA is then size fractionated by agarose gel electrophoreεiε, and the fraction including the DNA fragment covering the expressed V segment iε cloned into lambda EMBL3 or Lambda FIX (Stratagene, La Jolla, California) . V εegment containing cloneε are isolated using the unique probe o-kappa. DNA is isolated from positive clones and subcloned into the polylinker of pKapl. The resulting clone is called pRKL. EXAMPLE 8 Isolation of Genomic Clones Corresponding to Rearranged Expressed Copies of Immunoglobuling Heavy Chain u Geneε Thiε example describes the cloning of immunoglobulin heavy chain μ genes from cultured cells of expresεed and immunoglobulin of intereεt. The procedure deεcribed in thiε example allows for the selective cloning of the expresεed copy of a μ heavy chain gene. Double-εtranded cDNA iε prepared and iεolated aε deεcribed in part A of Example 7. The double-εtranded cDNA iε denatured and used as a template for a third round of DNA synthesiε using the following oligonucleotide primer: 5' - GTA CGC CAT ATC AGC TGG ATG AAG ACA GGA GAC GAG GGG GAA AAG GGT TGG GGC GGA TGC - 3 • This primer contains sequences specific for the constant portion of the μ heavy chain message (ACA GGA GAC GAG GGG GAA AAG GGT TGG GGC GGA TGC) as well as unique sequenceε that can be used as a primer for the PCR amplification of the newly synthesized DNA strand (GTA CGC CAT ATC AGC TGG ATG AAG) . The sequence is amplified by PCR using the following two oligonucleotide primers: 5' - GAG GTA CAC TGA CAT ACT GGC ATG - 3' 5' - GTA CTC CAT ATC AGC TGG ATG AAG - 3 ' The PCR amplified sequence iε then purified by gel electrophoreεiε and used as template for dideoxy sequencing reactions using the following oligonucleotide as a primer: 5 ' - GAG GTA CAC TGA CAT ACT GGC ATG - 3 * The first 42 nucleotides of sequence are then used to synthesize a unique probe for isolating the gene from which immunoglobulin mesεage waε tranεcribed. Thiε εynthetic 42 nucleotide segment of DNA will be referred to below aε o-mu. A Southern blot of DNA, isolated from the Ig expressing cell line and digested individually and in pairwiεe combinations with several different restriction endonucleases including MluT (Mlul is a rare cutting enzyme that cleaves between the J segment and mu CHI) , is then probed with the 32-P labelled unique oligonucleotide o-mu. A unique restriction endonuclease site is identified upstream of the rearranged V segment. DNA from the IG expresεing cell line iε then cut with Mlul and second enzyme. Mlul or Spel adapter linkers are then ligated onto the ends and cut to convert the upstream site to Mlul or Spel. The DNA iε then size fractionated by agarose gel electrophoreεiε, and the fraction including the DNA fragment covering the expresεed V segment is cloned directly into the plasmid pGPl. V segment containing cloneε are isolated using the unique probe o-mu, and the inεert iε εubcloned into Mlul or Mlul/Spel cut plasmid pCON2. The resulting plaεmid iε called pRMGH. EXAMPLE 9 Deletion of the Mouεe Heavy Chain Gene by Homologous Recombination This example deεcribeε the deletion of the endogenous mouεe heavy chain gene by homologous recombination in embryonic stem (ES) cellε (Zjilstra, et al. (1989) , Nature, 342, 435-438; followed by the transplantation of those ES cells into a mouse blastocyst embryo such that the ES cells colonize the germline of the resultant chimeric mouεe (Teratocarcinomaε and embryonic stem cells: a practical approach, E.J. Robertεon, ed. , IRL preεε, Waεhington, D.C., 1987) . The construction of a DNA sequence that will homologously recombine into the mouεe chromoεome εo as to delete the heavy chain J εegmentε, thuε eliminating the poεsibility of εucceεεful gene rearrangement at the heavy chair. locuε. The deεign of thiε conεtruct iε outlined below. Plaεmid pGPl iε digeεted with the reεtriction endonucleases BamHI and Bglll and religated to form the plasr.i pGPldl. Thiε plasmid iε then used to build the εo-called gene knockout construct. To obtain sequences homologous to the deεired target rerrion of the mouse π-enome, mouse genomic cloneε are isolated from a phage library derived from non-lymphoid tisεue (εuch aε liver) using the J H specific oligonucleotide probe: 5' - GGT CTA TGA TAG TGT GAC TAC TTT GAC TAC TGG GGC CAA GGC - 3 ' A 3.5 kb Kpnl to EcoRI fragment that hybridizes with this probe is isolated from DNA derived from positive phage clones. This fragment is subcloned into Kpnl/EcoRI digested pGPldl to form the plasmid pMKOl. Neomycin resiεtance (Neo) and Herpeε Simplex Viruε thymidine k inase (TK) genes for drug selection of recombinants (M. Capecchi (1989), Science, 244, 1288-1292) are then iεolated as follows. The plasmid pGEM7(KJl) (M.A. Rudnicki, 3/15/89) is digested with Hindlll and the ends blunted with the klenow form of DNA pol I. The DNA is then cut with EcoRI and the pGKNeo fragment is isolated and cloned into Sphl/Nael cut pMKOl using the following oligonucleotide as an adapter: 5 ' - AATTCATG -3 • The resulting.plaεmid iε deεignated pMK02. Thiε plasmid contains the neomycin reεiεtance gene flanked by εequenceε that flank the mouεe J H εegmentε. Thiε plaεmid alone can be used for deletion of the heavy chain gene. Alternatively the Herpes TK gene can be added to the construct to improve the frequency of homologouε recombination eventε in Neo resistant clones (M. Capecchi (1989) , Science, 244, 1288-1292). This is done as follows. The EcoRI to Hindlll PGKTK fragment of pGEM7 (TK) (M.A. Rudnicki) is isolated and cloned into the Kpnl site of pMK02 using the following oligonucleotide as adapterε: 5 ' - AATTGTAC - 3 ' 5' - AGCTGTAC - 3' The resulting plasmid is designated pMK03. To further improve the overall efficiency of homologous recombination, a large segment of DN that iε homologouε to the target sequence is then added to the construct. A 13 kb EcoRI fragment, that hybridizes with the Cμ specific oligonucleotide deεcribed below: 5' - GCA TCC TGG AAG GTT CAG ATG AAT ACC TTG TAT GCA AAA TCC - 3 ' Thiε 12 kb fragment includeε the Cμ coding exonε, or a substantial portion of that fragment which includeε the 5 ' EcoRI end, ε iεolated from a mouεe genomic phage library and εubcloned into the EcoRI εite of pMK03. The reεultant plasmid is designated pMK04. The insert of pMK04 is isolated by digestion with Notl and electroporated into ES cellε. Homologous recombinant clones are isolated uεed to generate a J H deleted mouεe aε deεcribed by Zjilεtra, et al . (1989) , Nature, 342 , 435-438. EXAMPLE 10 Deletion of the Mouεe Light Chain Gene by Homologouε Recombination Thiε example describeε the deletion of the endogenouε mouse light chain gene by homologous recombination in embryonic stem cellε (see previous Example) . A DNA sequence that homologouεly recombineε into the mouse chromosome to delete the _c light chain conεtant region exon iε constructed. The design of this construct iε outlined below. A 2 kb BamHII to EcoRI thymidine kinaεe fragment from pGEM7(TK)Sal (M.A. Rudnicki, Whitehead Inεtitute) is isolated and subcloned into the BamHI/Sfil digeεted pGPl using the following oligonucleotide adapter: 5' - AATTTTG - 3' The resulting plasmid is deεignated pKKOl. To obtain sequences homologous to the desired target region of the mouse genome, mouεe genomic cloneε are iεolated from a phage library derived from non-lymphoid tissue (such aε liver) uεing the mouεe /c light chain εpecific oligo designated o-MKC given below: 5 ' - GGC TGA TGC TGC ACC AAC TGT ATC CAT CTT CCC ACC ATC CAG - 3 ' DNA is iεolated from positive clone and a 2.3 kb Bglll fragment (P.S. Neumaier and H.G. Zachau (1983) , Nucl . Acidε Res. , 11, 3631-3656) that hybridizeε with probe o-MK3 iε isolated. The sequence of probe o-MK3 is aε follows: 5' - CAT TCT GGG TAT GAA GAG CCC ACG TAT CAA AGG TTA CAT TAG . - 3 ' This 2.3 kb Bglll fragment is subcloned into BamHI digested pKKOl such that the 3' end of the fragment iε adjacent to the polylinker Sfil site. The resulting plasmid iε designated pKK02. The 4 kb Sphl to Hpal DNA fragment that hybridizeε with oligonucleotide o-MKC is isolated from positive phage clone and subcloned into EcoRV to Sphl digested plasmid pKK02. The resulting plasmid is designated pKK03. A 2 b Sail to EcoRI fragment of pGEM7 (KJ1) Sal (M.A. Rudnicki, 3/15/89) is isolated and cloned into the BsεHII εite of plaεmid pKK03 using linker adapters. This is carried out by firεt ligating a mixture of the following three oligonucleotideε to the 2 kb Sail to EcoRI fragment: 5' - CAGCGCGC - 3 « 5' - GATCGCGCGCTG - 3 5' - AATTGCGCGCTG - 3' The ligation mixture iε then digeεted with the enzyme BεεHII and ligated to BεsHII digested plasmid pKK03. The resulting plasmid is deεignated pKK04. The inεert of pKK04 iε iεolated by digeεting with Notl and electroporated into ES cellε. Homologouε recombinant clones are isolated and used to generate a C κ deleted mouse as described by Zjilstra, et al. (1989), Nature, 342, 435-438. EXAMPLE 11 Inactivation of the Mouse Kappa Light Chain Gene by Homologous Recombination This example describes the inactivation of the mouse endogenous kappa locus by homologouε recombination in embryonic stem (ES) cells followed by introduction of the mutated gene into the mouse germ line by injection of targeted ES cells bearing an inactivated kappa allele into early mouse embryoε (blastocysts) . The strategy is to delete J κ and C κ by homologous recombination with a vector containing DNA εequenceε homologous to the mouse kappa locus in which a 4.5 kb segment of the locus, snanning the J αene and C segments, is deleted and replaced by the selectable marker neo. Construction of the kappa targeting vector The plasmid pGEM7 (KJl) (M. A. Rudnicki, Whitehead Institute) contains the neomycin resistance gene (neo) , used for drug selection of tranεfected ES cellε, under the transcriptional control of the mouεe phosphoglycerate kinase (pgk) promoter (Xbal/I/Taql fragment; Adra, CN. et al. , (1987) Gene. 60. 65-74) in the cloning vector pGEM-72f(+) . The plasmid also includes a heterologous polyadenylation site for the neo gene, derived from the 3' region of the mouεe pgk gene (PvuII/Hindlll fragment; Boer, P.H. , et al. , (1990) Biochemical Genetics, 28, 299-308) . This plasmid was used as the starting point for construction of the kappa targeting vector. The first step was to insert sequences homologous to the kappa locus 3' of the neo expression cassette. Mouse kappa chain εequenceε (Fig. 25a) were isolated from a genomic phage library derived from liver DNA using oligonucleotide probes εpecific for the Cc locuε: 5'- GGC TGA TGC TGC ACC AAC TGT ATC CAT CTT CCC ACC ATC CAG -3' and for the J/c5 gene εegment: 5'- CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA CGT AAG - An 8 kb Bglll/Sacl fragment extending 3 ' of the mouse C κ segment was isolated from a positive phage clone in two pieces, as a 1.2 kb Bglll/Sacl fragment and a 6.8 kb Sad fragment, and subcloned into Bglll/Sacl digested pGEM7 (KJl) to generate the plasmid pNE0-K3 ' (Fig. 25b) . A 1.2 kb EcoRI/Sphl fragment extending 5' of the J κ region was also isolated from a positive phage clone. An Sphl/Xbal/Bglll/EcoRI adaptor waε ligated to the Sphl εite of this fragment, and the resultin g EcoRI fra rτ ment waε li g ated into EcoRI digested pNE0-K3 ' , in the same 5 ' to 3 ' orientation as the neo gene and the downstream 3 ' kappa εequenceε, to generate pNE0-K5 '3 ' (Fig. 25c). The Herpeε Simplex Virus (HSV) thymidine kinase (TK) gene was then included in the construct in order to allow for enrichment of ES clones bearing homologouε reco binantε, aε described by Mansour et al. ((1988) Nature, 336, 348-352) . The HSV TK cassette waε obtained from the plasmid pGEM7 (TK) (M.A. Rudnicki) , which containε the εtructural εequenceε for the HSV TK gene bracketed by the mouεe pgk promoter and polyadenylation εequenceε as described above for pGEM7 (KJl) . The EcoRI site of pGEM7 (TK) was modified to a BamHI εite and the TK caεεette was then excised as a BamHI/Hindlll fragment and εubcloned into pGPlb to generate pGPlb-TK. This plasmid waε linearized at the Xhol εite and the Xhol fragment from pNEO-K5 ' 3 ' , containing the neo gene flanked by genomic εequenceε from 5' of J/c and 3' of Cc, was inserted into pGPlb-TK to generate the targeting vector J/C KI (Fig. 25d) . The putative structure of the genomic kappa locus following homologous recombination with J/C Kl is shown in Fig. 25e. Generation and analvsiε of ES cells with targeted inactivation of a kappa allele AB-1 ES cells were grown on mitotically inactive SNL76/7 cell feeder layers (McMahon, A.P. and Bradley,A. (1990) Cell. 62 , 1073-1085) essentially as described (Robertson, E.J. (1987) in Teratocarcinomas and Embryonic Stem Cellε: A Practical Approach. E.J. Robertson, ed. (Oxford: IRL Preεε) , p. 71-112) . The kappa chain inactivation vector J/C Kl waε digested with Notl and electroporated into AB-1 cells by the methods described (Hasty, P.R. , et al. (1991) Nature, 350 , 243-246) . Electroporated cells were plated onto 100 mm dishes at a density of 2-5 x 10 6 cells/dish. After 24 hours, G418 (200μg/ml of active component) and FIAU (0.5μM) were added to the medium, and drug-resistant clones were allowed to develop over 10-11 days. Clones were picked, trypsinized, divided into two p ortions, and further expanded. Half of the cells derived from each clone were then frozen and the other half analyzed for homologouε recombination between vector and target sequences. DNA analysis was carried out by Southern blot hybridization. DNA was isolated from the clones as described (Laird, P.W. et al. , (1991) Nucl. Acids Res.. 19, ) digested with Xbal and probed with the 800 bp EcoRI/Xbal fragment indicated in Fig. 25e aε the diagnostic probe. Thiε probe detectε a 3.7 kb Xbal fragment in the wild type locus, and a diagnostic 1.8 kb band in a locuε which haε homologouεly recombined with the targeting vector (εee Fig. 25a and e) . Of 358 G418 and FIAU reεiεtant clones εcreened by Southern blot analysis, 4 displayed the 1.8 kb Xbal band indicative of a homologouε recombination at the kappa locuε. Theεe 4 clones were further digested with the enzymes Bglll, Sad, and Pstl to verify that the vector integrated homologously into one of the kappa alleles. When probed with the diagnostic 800 bp EcoRI/Xbal fragment, Bglll, Sad, and Pstl digestε of wild type DNA produce fragmentε of 4.1, 5.4, and 7 kb, reεpectively, whereaε the preεence of a targeted kappa allele would be indicated by fragmentε of 2.4, 7.5, and 5.7 kb, reεpectively (see Fig. 25a and e) . All 4 positive cloneε detected by the Xbal digest showed the expected Bglll, Sad, and Pstl restriction fragments diagnostic of a homologous recombination at the kappa light chain. Generation of mice bearing the inactivated kappa chain The 4 targeted ES clones described in the previouε εection were injected into C57B1/6J blastocystε aε described (Bradley, A. (1987) in Teratocarcinomas and Embryonic Stem Cellε: A Practical Approach. E.J. Robertεon, ed. (Oxford: IRL Preεε) , p. 113-151) and transferred into the uteri of pseudopregnant females to generate chimeric mice repreεenting a mixture of cellε derived from the input ES cellε and the host blastocyst. Chimeric animals are visually identified by the presence of agouti coat coloration, derived from the ES cell line, on the black C57B1/6J background. The AB1 ES cells are an XY cell line, thus male chimeras are bred with C57BL/6J females and the offspring monitored for the presence of the dominant agouti coat color. Agouti offspring are indicative of germline transmission of the ES genome. The heterozygosity of agouti offspring for the kappa chain inactivation iε verified by Southern blot analysis of DNA from tail biopsieε uεing the diagnostic probe utilized in identifying targeted ES clones. Brother-sister matings of heterozygoteε are then carried out to generate mice homozygouε for the kappa chain mutation. EXAMPLE 12 Inactivation of the Mouεe Heavy Chain Gene by Homologouε Recombination Thiε example deεcribeε the inactivation of the endogenous murine immunoglobulin heavy chain locuε by homologouε recombination in embryonic stem (ES) cellε. The strategy is to delete the endogenous heavy chain J εegmentε by homologous recombination with a vector containing heavy chain εequenceε from which the J H region has been deleted and replaced by the gene for the selectable marker neo. Construction of a heavy chain targeting vector Mouse heavy chain sequenceε containing the J H region (Fig. 26a) were isolated from a genomic phage library derived from the D3 ES cell line (Gosεler, et al., (1986) Proc. Natl. Acad. Sci. U.S.A. , 83. 9065-9069) using a J H 4 specific oligonucleotide probe: 5'- ACT ATG CTA TGG ACT ACT GGG GTC AAG GAA CCT CAG TCA CCG -3' A 3.5 kb genomic Sacl/Stul fragment, spanning the J H region, was isolated from a positive phage clone and subcloned into Sacl/Smal digested puclδ. The resulting plaεmid was designated pucl8 J H . The neomycin reεiεtance gene (neo) , used for drug selection of transfected ES cells, was derived from the plasmid pGEM7 (KJl) . The Hindlll εite in pGEM7 (KJlj waε converted to a Sail site by addition of a synthetic adaptor, and the neo expression cassette excised by digeεtion with Xbal/Sall. The ends of the neo fragment were then blunted by treatment with the Klenow form of DNA poll, and the neo fragment was subcloned into the Nael site of pucis J generating the plasmid pucl8 J H -neo (Fig. 26b) . Further construction of the targeting vector waε carried out in a derivative of the plasmid pGPlb. pGPlb was digested with the restriction enzyme Notl and ligated with the following oligonucleotide as an adaptor: 5'- GGC CGC TCG ACG ATA GCC TCG AGG CTA TAA ATC TAG AAG AAT TCC AGC AAA GCT TTG GC -3 ' The resulting plasmid, called pGMT, was used to build the mouεe immunoglobulin heavy chain targeting conεtruct. The Herpes Simplex Virus (HSV) thymidine kinaεe (TK) gene waε included in the conεtruct in order to allow for enrichment of ES cloneε bearing homologous recombinantε, aε described by Mansour et al. ((1988) Nature 336, 348-352) . The HSV TK gene was obtained from the plasmid pGEM7 (TK) by digestion with EcoRI and Hindlll. The TK DNA fragment was subcloned between the EcoRI and Hindlll sites of pGMT, creating the plasmid pGMT-TK (Fig. 26c) . To provide an extensive region of homology to the target sequence, a 5.9 kb genomic Xbal/Xhol fragment, situated 5' of the J H region, was derived from a positive genomic phage clone by limit digestion of the DNA with Xhol, and partial digestion with Xbal. As noted in Fig. 26a and 26b, this Xbal site is not present in genomic DNA, but is rather derived from phage sequences immediately flanking the cloned genomic heavy chain insert in the positive phage clone. The fragment was subcloned into Xbal/Xhol digeεted pGMT-TK, to generate the plaεmid pGMT-TK-J H 5' (Fig. 26d) . The final εtep in the construction involved the excision of the 3 kb EcoRI fragment from puclδ J H ~neo which contained the neo gene and flanking genomic sequences. Thiε fragment was blunted by Klenow poly erase and subcloned into the similarly blunted Xhol site of pGMT-TK-J..5 ' . The reεulting construct, J H K01 (Fig. 26e) , contains 6.9 kb of genomic sequences flanking the J H locus, with a 2.3 kb deletion spanning the J H region into which has been inεerted the neo gene. Fig. 25f shows the structure of an endogenous heavy chain allele after homologous recombination with the targeting construct. EXAMPLE 13 Generation and analysis of targeted ES cells AB-1 ES cells (McMahon, A.P. and Bradley,A. (1990) Cell 62. 1073-1085) were grown on mitotically inactive SNL76/7 cell feeder layers essentially as described (Robertson, E.J. (1987) Teratocarcinomaε and Embryonic Stem Cellε: A Practical Approach. E.J. Robertson, ed. (Oxford: IRL Preεε) , pp. 71-112) . The heavy chain inactivation vector J H K01 waε digested with Notl and electroporated into AB-1 cells by the methods described (Hasty, P.R., et al. (1991) Nature 350, 243-246) . Electroporated cells were plated into 100 mm disheε at a density of 2-5 x 10 6 cells/dish. After 24 hours, G418 (200mg/ml of active component) and FIAU (0.5mM) were added to the medium, and drug-resistant clones were allowed to develop over 8-10 dayε. Cloneε were picked, trypεinized, divided into two portionε, and further expanded. Half of the cellε derived from each clone were then frozen and the other half analyzed for homologouε recombination between vector and target sequenceε. DNA analysis is carried out by Southern blot hybridization. DNA is isolated from the cloneε as described (Laird, P.W. et al., (1991) Nucl. Acids Res. , 19. ) digeεted with Hindlll and probed with the 500 bp EcoRI/StuI fragment designated aε the diagnostic probe in Fig. 26f. This probe detects a Hindlll fragment of 2.3 kb in the wild type locuε, whereas a 5.3 kb band is diagnostic of a targeted locus which has homologously recombined with the targeting vector (see Fig. 26a and f) . Additional digestε with the enzymeε Spel, Stul, and BamHI are carried out to verify the targeted diεruption of the heavy chain allele. EXAMPLE 14 Heavy Chain Minilocus Tranεqene A. Construction of plaεmid vectorε for cloning large DNA sequences 1. pGPla The plasmid pBR322 waε digeεted with EcoRI and Styl and ligated with the following oligonucleotideε: oligo-42 5'- caa gag ccc gcc taa tga gcg ggc ttt ttt ttg cat act gcg gcc get -3 ' oligo-43 5'- aat tag egg ccg cag tat gca aaa aaa age ccg etc att agg egg get -3 ' The reεulting plaεmid, pGPla, iε designed for cloning very large DNA constructs that can be excised by the rare cutting restriction enzyme Notl. It contains a Notl restriction εite downεtream (relative to the ampicillin reεiεtance gene, A pR) of a εtrong tranεcription termination signal derived from the trpA gene (Christie, G.E. et al. (1981) Proc. Natl. Acad. Sci. USA, 78, 4180) . This termination εignal reduceε the potential toxicity of coding sequences inεerted into the Notl site by eliminating readthrough tranεcription from the AmpR gene. In addition, thiε plaεmid is low copy relative to the pUC plaεmidε because it retains the pBR322 copy number control region. The low copy number further reduces the potential toxicity of inεert εequenceε and reduceε the selection against large insertε " due to DNA replication. 2. pGPlb pGPla waε digeεted with Notl and ligated with the following oligonucleotides: oligo-47 5'- ggc cgc aag ctt act get gga tec tta att aat cga tag tga tct cga ggc -3' oligo-48 5'- ggc cgc etc gag ate act ate gat taa tta agg ate cag cag taa get tgc -3 ' The reεulting plaεmid, pGPlb, contains a short polylinker region flanked by Notl siteε. Thiε facilitates the conεtruction of large inεertε that can be exciεed by Notl di g estion= 3. pGPe The following oligonucleotides: oligo-44 5'- etc cag gat cca gat ate agt ace tga aac agg get tgc -3 ' oligo-45 5'- etc gag cat gca cag gac ctg gag cac aca cag cct tec -3 ' were used to amplify the immunoglobulin heavy chain 3 ' enhancer (S. Petterson, et al. (1990) Nature, 344, 165-168) from rat liver DNA by the polymeraεe chain reaction technique. The amplified product waε digested with BamHI and Sphl and cloned into BamHI/Sphl digested pNN03 (pNN03 is a pUC derived plaεmid that contains a polylinker with the following restriction siteε, liεted in order: Notl, BamHI, Ncol, Clal, EcoRV, Xbal, Sacl, Xhol, Sphl, Pεtl, Bglll, EcoRI, Smal, Kpnl, Hindlll, and Notl) . The reεulting plaεmid, pRE3 , waε digeεted with BamHI and Hindlll, and the inεert containing the rat Ig heavy chain 3' enhancer cloned into BamHI/Hindlll digeεted pGPlb. The resulting plasmid, pGPe (Fig. 27 and Table 1) , contains several unique restriction siteε into which εequenceε can be cloned and subsequently excised together with the 3 ' enhancer by Notl digestion. AATTAGCggccgcctcgagatcactatcgattaattaaggatccagatatcag acctgaaacagggcttgctcacaac tctcrctctgtctctc gtct.ctgt.gt.gtgtσtctctctctgtctctgtctctctctgtctctcrgtctctgtgtr tctctctctgtctctctctctg^ctc_:ctgtctctctg^ctgtctctσtcΛctgtctctgtctctctctctc ctcr;=t tctctctctctctctctcacacacacacacacacacacacacacacctgccgagtgactcactctgtgcagggttggcc tcggggcacatgcaaatggatgtttgttccatgcagaaaaacatgtttctcattctctgagccaaaaatagcatcaa:_g ttcccccaccctgcagctgcaggttcaccccacctggccaggttgaccagctttggggatggggctgggggttccarga ccctaacggtgacattgaattcagtgttttcccatttatcgacactgctggaatctgaccctaggagggaatgacagga ataggcaaggtccaaacaccccagggaagtgggagagacaggaaggctgtgtgtgctccaggtcctgtgcatgct tctgaattcccgggtaccaagcttgcGGCCGCAGTATGCAAAAAAAAGCCCGCTCATTAGGCGGGCTCTTGGCAG. ATCCATCGCGTCCGCC_ATCTCCAGCAGCCGCACGCGGCGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCARG TCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGA CGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGC_-^CAACATGAATGGTCTTCGGTTTCCGTGTTTCGΓ AAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGQVTCGCAGGATGCTGCTGGCTACCCTGT G_^C^CCTACATCTGTAT AACGAAGCGCTGGCATTGACCCTGAGTGATT T CTCTGGTCCCGCCGCATCCATACCGC AGTTGTTTACCCTC__AC_AACGTTCCAGTAACCGGGCATGTT.^ CATCGGTATC^TTACCCCCATGAACAGAAATTCCCCCTTA^ RAAC^TGGCCCGCT TATCΛGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCA ACATCTGTGAATCGCT CACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGC3T CGGTGATGACGGTGAAAA TCTGACACATGCAGCTCCC3GAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCG TGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATA3 CATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCRCA TCAAAGGCGGTAATACGGTTATCCA(_AGAATCAGGGGATAACG(^GGAAAGAAC^^ CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGC CAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTC'RCC GTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCT TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGC CC TATCCGGTAACTATCGTCTTGAGTCC-TVACCCGGTAAGACIACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG ATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAG ATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACC CTGGTAGCGGTGGTTTT TTGTTTGC_AAGCAGCAGATTACGCGCAGAAAAAAAGG^^ TCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC'TTCA CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAA GCTTAATC_AGTGAGG(_ACCTATCTCAC-CGATCTGTCTATTTCGTTCATCC_ATAGTTGCCTGACTCCCCGTCGTGTAGAT ACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGARR ATCAGCAATAAACC_AGCC_AGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAAC TTATCCGCCTCCATCCAGTCTATΓ ATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGT GTGTC_ACGCTCGTCGTTTGGTATGGCT CATTCAGCTCCGGTTCCC_AACGATC_AAGGCGAGTTACATGATCCCCCATGR GTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTT TGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTT CTGTGACTGGTGAGTACTCAACCAAGTC TTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAA TTTAAAAGTGCT_ATCΛTTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCG TGTMCCCACTCGTGCACCC^CTGATCTTCAGCATCTTTTACTTTCACC^ CAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTC-ATACTCTTCCTTTTTCAAT^ CATTTATC_AGGGTTATTGTCTCATGAGCGGATA(-ΛTATTTGAATGT^^ CATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATC__ATGACATTAACCTATAAAAATAGGCGTATCAC AGGCCCTTCGTCTTCAAG Table 1 Sequence of vector pGPe. B. Construction of IgM expressing minilocus tranεgene, pIGMl 1. Isolation of J-u constant region cloneε and conεtruction of pJMl A human placental genomic DNA library cloned into the phage vector AEMBL3/SP6/T7 (Clonetech Laboratorieε, Inc., Palo Alto, CA) was screened with the human heavy chain J region specific oligonucleotide: oligo-1 5 1 - gga ctg tgt ccc tgt gtg atg ctt ttg atg tct ggg gcc aag -3 ' and the phage clone λl.3 isolated. A 6 kb Hindlll/Kpnl fragment from thiε clone, containing all εix J εegments as well aε D segment DHQ52 and the heavy chain J-μ intronic enhancer, was isolated. The εame library waε εcreened with the human μ εpecific oligonucleotide: oligo-2 5 1 - cac caa gtt gac ctg cct ggt cac aga cct gac cac eta tga -3 ' and the phage clone λ2.1 iεolated. A 10.5 kb Hindlll/Xhol fragment, containing the μ εwitch region and all of the μ conεtant region exonε, waε iεolated from thiε clone. Theεe twc fragmentε were ligated together with Kpnl/Xhol digeεted pNN03 to obtain the plasmid pJMl. 2. pJM2 A 4 kb Xhol fragment waε iεolated from phage clone A2.1 that contains sequences immediately downstream of the sequenceε in pJMl, including the εo called ∑μ element involved in μ deletion in certain IgD expressing B-cells (H. Yasui et al. (1989) Eur. J. Immunol. 19 , 1399) . Thiε fragment waε treated with the Klenow fragment of DNA polymerase I and ligated to Xhol cut, Klenow treated, pJMl. The resulting plaεmid, pJM2 (Fig. 28) , had loεt the internal Xhol εite but retained the 3 ' Xhol εite due to incomplete reaction by the Klenow enzyme. pJM2 containε the entire human J region, the heavy chain J-u intronic enhancer, the μ εwitch region and all of the μ conεtant region exonε, aε well aε the two 0.4 kb direct repeats, σμ and ∑μ, involved in μ deletion. 3. lεolation of D region clones and construction of pDHl The following human D region εpecific oligonucleotide: oligo-4 5'- tgg tat tac tat ggt teg ggg agt tat tat aac cac agt gtc -3 ' was used to εcreen the human placenta genomic library for D region cloneε. Phage cloneε λ4.1 and A4.3 were iεolated. A 5.5 kb Xhol fragment, that includeε the D elementε D κl , D N1 , and D M2 (Y. Ichihara et al. (1988) EMBO J. , 7, 4141) , waε iεolated from phage clone A4.1. An adjacent upstream 5.2 kb Xhol fragment, that includeε the D elementε D ι; D χpι , D χpιl , and D ftl , waε isolated from phage clone A4.3. Each of these D region Xhol fragmentε were cloned into the Sail εite of the plaεmid vector pSP72 (Promega, Madiεon, WI) so as to deεtroy the Xhol site linking the two sequences. The upstream fragment waε then excised with Xhol and Smal, and the downstream fragment with EcoRV and Xhol. The resulting isolated fragments were ligated together with Sail digeεted pSP72 to give the plasmid pDHl. pDHl contains a 10.6 kb insert that includes at least 7 D segments and can be excised with Xhol (5') and EcoRV (3') . 4. pCORl The plasmid pJM2 was digested with Asp718 (an iεoschizomer of Kpnl) and the overhang filled in with the Klenow fragment of DNA polymeraεe I. The resulting DNA waε then digeεted with Clal and the inεert iεolated. Thiε inεert was ligated to the XhoI/EcoRV insert of pDHl and Xhol/Clal digested pGPe to generate pCORl (Fig. 29) . 5. pVH251 A 10.3 kb genomic Hindlll fragment containing the two human heavy chain variable region εegmentε V R 251 and V κ 105 (C.G. Humphries et al. (1988) Nature 331, 446) waε subcloned into pSP72 to give the plasmid pVH251. 6. PlGMl The plasmid pCORl waε partially digeεted with Xhol and the isolated Xhol/Sall insert of pVH251 cloned into the upstream Xhol site to generate the plasmid pIGMl (Fig. 30) . pIGMl contains 2 functional human variable region εegmentε, at least 8 human D segmentε all 6 human J H εegmentε, the human J-μ enhancer, the human σμ element, the human μ switch region, all of the human μ coding exons, and the human ∑μ element, together with the rat heavy chain 3' enhancer, such that all of these sequence elements can be isolated on a single fragment, away from vector sequences, by digestion with Notl and microinjected into mouse embryo pronuclei to generate transgenic animalε. C. Conεtruction of IgM and IgG expreεεing minilocuε tranεgene, pHCl 1. Isolation of i constant region clones The following oligonucleotide, specific for human Ig g constant region genes: oligo-29 5'- cag cag gtg cac ace caa tgc cca tga gcc cag aca ctg gac -3 ' was used to screen the human genomic library. Phage cloneε 129.4 and A29.5 were isolated. A 4 kb Hindlll fragment of phage clone A29.4, containing a η switch region, was used to probe a human placenta genomic DNA library cloned into the phage vector lambda FIX™ II (Stratagene, La Jolla, CA) . Phage clone ASgl.13 was isolated. To determine the subclaεε of the different η cloneε, dideoxy sequencing reactions were carried out using subclones of each of the three phage clones aε templateε and the following oligonucleotide aε a primer: oligo-67 5 ' - tga gcc cag aca ctg gac -3 ' Phage cloneε A29.5 and λS rl.13 were both determined to be of the I εubclass. 2 . pηrel A 7.8 kb Hindlll fragment of phage clone A29.5, containin g the Ύ1 coding re g ion was cloned into ΏUCIS. The resulting plasmid, pLTl, was digested with Xhol, Klenow treated, and religated to destroy the internal Xhol εite. The resulting clone, pLTlxk, was digested with Hindlll and the insert isolated and cloned into pSP72 to generate the plasmid clone pLTlxks. Digestion of pLTlxks at a polylinker Xhol εite and a human sequence derived BamHI εite generates a 7.6 kb fragment containing the 71 constant region coding exons. This 7.6 kb XhoI/BamHI fragment was cloned together with an adjacent downstream 4.5 kb BamHI fragment from phage clone A29.5 into XhoI/BamHI digeεted pGPe to generate the plasmid clone P el. p7el containε all of the 7I conεtant region coding exonε, together with 5 kb of downεtrea sequenceε, linked to the rat heavy chain 3' enhancer. • P7e2 A 5.3 kb Hindlll fragment containing the 7I εwitch region and the firεt exon of the pre-εwitch εterile tranεcript (P. Sideras et al. (1989) International Immunol. 1, 631) was isolated from phage clone AS 7 1.13 and cloned into pSP72 with the polylinker Xhol site adjacent to the 5' end of the insert, to generate the plasmid clone PS7IS. The Xhol/Sall inεert of PS7IS was cloned into Xhol digested P7el to generate the plasmid clone P7e2 (Fig. 31) . P7e2 containε all of the 7 1 constant region coding exonε, and the upεtream εwitch region and sterile transcript exons, together with 5 kb of downstream sequences, linked to the rat heavy chain 3' enhancer. Thiε clone contains a unique Xhol site at the 5' end of the insert. The entire inεert, together with the Xhol εite and the 3' rat enhancer can be exciεed from vector εequenceε by digeεtion with Notl. 4. pHCl The plasmid pIGMl was digested with Xhol and the 43 kb insert isolated and cloned into Xhol digeεted pge2 to generate the plasmid pHCl (Fig. 30) . pHCl contains 2 functional human variable region segments, at least 8 human D segments all 6 human J H segmentε, the human J-μ enhancer, the human σμ element, the human μ switch region, all of the human μ coding exons, the human ∑μ element, and the human 71 constant region, including the associated switch region and sterile transcript associated exonε, together with the rat heavy chain 3* enhancer, such that all of these sequence elements can be isolated on a single fragment, away from vector εequenceε, by digestion with Notl and microinjected into mouse embryo pronuclei to generate tranεgenic animals. D. Construction of IgM and IgG expreεεing minilocuε tranεgene, PHC2 1. Iεolation of human heavy chain V region gene VH49.8 The human placental genomic DNA library lambda, FIX'" II, Stratagene, La Jolla, CA) waε εcreened with the following human VHl family specific oligonucleotide: oligo-49 5'- gtt aaa gag gat ttt att cac ccc tgt gtc etc tec aca ggt gtc -3 * Phage clone A49.8 waε isolated and a 6.1 kb Xbal fragment containing the variable εegment VH49.8 εubcloned into pNN03 (such that the polylinker Clal site iε downεtream of VH49.8 and the polylinker .Xhol εite iε upstream) to generate the plasmid pVH49.8. An 800 bp region of thiε insert waε sequenced, and VH49.8 found to have an open reading frame and intact splicing and recombination signalε, thuε indicating that the gene iε functional (Table 2) . TTCCTCAGGC AGGATTTAGG GCTTGGTCTC TCAGCATCCC ACACTTGTAC 5C A l -_ _.-V3_ lvαlv-_ι li_f-i. ijiυ_ _-____? -L_.__.c_r_.v_r Jυ CTGTGAAATA CCCTGCCTCA TGAATATGCA AATAATCTGA GGTCTTCTGA 150 GATAAATATA GATATATTGG TGCCCTGAGA GCATCACATA ACAACCAGAT 200 TCCTCCTCTA AAGAAGCCCC TGGGAGCACA GCTCATCACC ATGGACTGGA 25C ΞtAspTrpT CCTGGAGGTT CCTCTTTGTG GTGGCAGCAG CTACAGgtaa ggggcttcct 300 hrTrpArgPh eLeuPheVal ValAlaAlaA laThr agrc raagg cmgaggaagg gatcc ggtt ragttaaaga gga r ~at.r "cr 4 . nSerGIyAla GluValLysL ysProGIySe rSerValLys ValSerCysL AGGC__TCTGG AGGCACCTTC AGCAGCTATG CTATCAGC G GGTGCGACAG 50C ysAlaSerGl yGlvThrPhe SerSerlyrA lalleSerTr pValArgGlπ G(XCCTGGAC AAGGGCTTGA GTGGATGGGA AGGATCATCC CTATCCTTGG 55C AlaProGlyG InGlyLeuGl uTrpMetGly ArgllelleP roIleLeuGl TATAGCAAAC TACGCACAGA AGTTCCAGGG CAGAGTCACG ATTACCGCGG 600 vIleAlaAsn TyrAlaGlnL ysPheGlnGI yArgValT r IleThrAlaA soLvsSerT rSerT rAla Ar SerGlu J- i xΛJ __. AspThrAlaV alTvrTvrCv sAlaArα CTGΆGAG C Γ GAGAAAΓΓHΓ GAGGGAGAAG GCAGCTGTGC CGGGCTGAGG C AGATGACAGG GTTTATTAGG TTTAAGGCTG TTTACAAAAT G3GTTATATA 80C TTTGAGAAAA AA 812 Table 2 Sequence of. human Vj,I family gene V, 49.8 2. pV2 A 4 kb Xbal genomic fragment containing the human V H IV family gene V H 4-21 (I. Sanz et al. (1989) EMBO J.. 8 . , 3741) , subcloned into the plasmid pUCl2, was excised, with Smal and Hindlll, and treated with the Klenow fragment of polymerase I. The blunt ended fragment was then cloned into Clal digeεted, Klenow treated, pVH49.8. The reεulting plasmid, pV2, contains the human heavy chain gene VH49.8 linked upεtream of VH4-21 in the same orientation, with a unique Sail εite at the 3' end of the insert and a unique Xhol site at the 5' end. 3. PS-YI-5' A 0.7 kb Xbal/Hindlll fragment (representing εequenceε immediately upstream of, and adjacent to, the 5.3 kb 7I switch region containing fragment in the plaεmid P 7 e2) together with the neighboring upstream 3.1 kb Xbal fragment were iεolated from the phage clone ASgl.13 and cloned into Hindlll/Xbal digested pUC18 vector. The reεulting plasmid, PS 7 I-5', contains a 3.8 kb insert representing sequences upεtream of the initiation site of the sterile transcript found in B-cells prior to switching to the 71 iεotype (P. Sideraε et al. (1989) International Immunol. , 1, 631) . Becauεe the transcript iε implicated in the initiation of iεotype εwitching, and upεtream cis-acting sequences are often important for tranεcription regulation, these sequences are included in transgene constructs to promote correct expression of the sterile transcript and the associated switch recombination. 4. pVGEl The PS7I-5' insert was excised with Smal and Hindlll, treated with Klenow enzyme, and ligated with the following oligonucleotide linker: 5 ' - ccg gtc gac egg -3 ' The ligation product was digested with Sail and ligated to Sail digested pV2. The resulting plasmid, pVP, contains 3.8 kb of 7I switch 5' flanking sequences linked downstream of the two human variable gene segments VH49.8 and VH4-21 (see Table 2). The pVP insert is isolated by partial digestion with Sail and complete digestion with Xhol, followed by purification of the 15 kb f arτττ_ent on an agarose gel. The inεert iε then cloned into the Xhol εite of p7e2 to generate the plaεmid clone pVGEl (Fig. 32) . pVGEl contains two human heavy chain variable gene segments upstream of the human 7I constant gene and asεociated switch region. A unique Sail site between the variable and conεtant regionε can be used to clone in D, J, and μ gene segmentε. The rat heavy chain 3' enhancer iε linked to the 3' end of the 71 gene and the entire inεert is flanked by Notl siteε. 5. PHC2 The plaεmid clone pVGEl is digeεted with Sail and the Xhol insert of pIGMl is cloned into it. The resulting clone, pHC2 (Fig. 30) , containε 4 functional human variable region segments, at leaεt 8 human D segmentε all 6 human J H segmentε, the human J-m enhancer, the human σμ element, the human μ switch region, all of the human μ coding exons, the human ∑μ element, and the human 7 I constant region, including the asεociated switch region and sterile transcript asεociated exons, together with 4 kb flanking sequenceε upεtream of the sterile tranεcript initiation εite. Theεe human sequences are linked to the rat heavy chain 3' enhancer, such that all of the sequence elements can be isolated on a single fragment, away from vector sequenceε, by digestion with Notl and microinjected into mouse embryo pronuclei to generate transgenic animals. A unique Xhol site at the 5 ' end of the insert can be used to clone in additional human variable gene εegmentε to further expand the recombinational diversity of thiε heavy chain minilocus. E. Transgenic mice The Notl insertε of plas idε pIGMl and pHCl were isolated from vector seguenceε by agaroεe gel electrophoresis. The purified inserts were microinjected into the pronuclei cf fertilized (C57BL/6 x CBA)F2 mouse e bryoε and tranεferred the surviving embryos into pseudopregnant females as described by Hogan et al. (B. Hogan, F. Coεtantini, and E. Lacy, Methodε of Manipulating the Mouse Embryo, 1986, Cold Spring Harbor Laboratory. New York) . Mice that developed from injected embryos were analyzed for the presence of transgene sequenceε by Southern blot analysis of tail DNA. Transgene copy number was estimated by band intensity relative to control standardε containing known quantitieε of cloned DNA. At 3 to 8 weekε of age, serum waε isolated from these animals and assayed for the presence of transgene encoded human IgM and IgGl by ELISA aε described by Harlow and Lane (E. Harlow and D. Lane. Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Laboratory, New York) . Microtiter plate wellε were coated with mouse monoclonal antibodies specific for human IgM (clone AF6, #0285, AMAC, Inc. Weεtbrook, ME) and human IgGl (clone JL512, #0280, AMAC, Inc. Weεtbrook, ME) . Serum εampleε were serially diluted into the wells and the presence of εpecific i munoglobulinε detected with affinity iεolated alkaline phosphatase conjugated goat anti-human Ig (polyvalent) that had been pre-adsorbed to minimize cross-reactivity with mouse immunoglobulins. Fig. 33 showε the resultε of an ELISA assay for the presence of human IgM and IgGl in the serum of two animalε that developed from embryoε injected with the transgene insert of plaεmid pHCl. One of the animals (#18) waε negative for the transgene by Southern blot analysis, and showed no detectable levels of human IgM or IgGl. The second animal (#38) contained approximately 5 copies of the transgene, as assayed by Southern blotting, and showed detectable levelε of both human IgM and IgGl. The results of ELISA assays for 11 animals that developed from transgene injected embryoε iε summarized in the table below (Table 3) . Table 3. Detection of human IgM and IgGl in the serum of transgenic animals by ELISA assay approximate 5 injected transgene animal i transgene copy # (per ceil) human IgM human IgGl pIGMl 10 pIGMl 9 pIGMl 0 15 10 pIGMl 0 12 pIGMl 0 15 pIGMl 10 ' 20 18 pHCl 0 19 pHCl 1 25 21 pHCl <1 30 Table 3 shows a correlation between the presence of integrated transgene DNA and the presence of tranεgene encoded immunoglobulinε in the εerum. Two of the animalε that were found to contain the pHCl transgene did not express detectable levels of human immunoglobulins. These were both low copy animals and may not have contained complete copies of the transgenes, or the animalε may have been genetic moεaicε (indicated by the <1 copy per cell estimated for animal #21) , and the transgene containing cellε may not have populated the hematopoetic lineage. Alternatively, the tranεgenes may have integrated into genomic locationε that are not conducive to their expreεεion. The detection of human IgM in the serum of pIGMl transgenics, and human IgM and IgGl in pHCl transgenics, indicates that the transgene sequenceε function correctly in directing VDJ joining, tranεcription, and iεotype εwitching. EXAMPLE 15 Rearranged Heavy Chain Tranεgeneε A. Isolation of Rearranged Human Heavy Chain VDJ εegmentε. Two human leukocyte genomic DNA librarieε cloned into the phage vector 1EMBL3/SP6/T7 (Clonetech Laboratorieε, Inc., Palo Alto, CA) are εcreened with a 1 kb Pacl/Hindlll fragment of λl.3 containing the human heavy chain J-μ intronic enhancer. Poεitive cloneε are tested for hybridization with a mixture of the following V H specific oligonucleotideε: oligo-7 5'-tea gtg aag gtt tec tgc aag gca tct gga tac ace ttc acc-3 ' oligo-8 5 '-tec ctg aga etc tec tgt gca gcc tct gga ttc ace ttc agt-3 ' Cloneε that hybridized with both V and J-μ probeε are iεolated and the DNA εequence of the rearranged VDJ εegment determined. B. Construction of rearranged human heavy chain transgeneε Fragments containing functional VJ segmentε (open reading frame and εplice εignalε) are subcloned into the plasmid vector pSP72 such that the plasmid derived Xhol εite iε adjacent to the 5' end of the inεert sequence. A subclone containing a functional VDJ segment is digested with Xhol and Pad (Pad, a rare-cutting enzyme, recognizeε a site near the J-m intronic enhancer) , and the insert cloned into Xhol/Pacl digested pHC2 to generate a transgene construct with a functional VDJ segment, the J-μ intronic enhancer, the μ switch element, the μ conεtant region coding exonε, and the 71 conεtant region, including the εterile tranεcript aεεociated sequenceε, the 7I εwitch, and the coding exons. This tranεgene conεtruct iε excised with Notl and microinjected into the pronuclei of mouse embryos to generate transgenic animalε aε described above. EXAMPLE 16 Light Chain Tranεgeneε A. Construction of Plasmid vectorε 1. Plasmid vector pGPlc Plasmid vector pGPla is digested with Notl and the following oligonucleotides ligated in: oligo-81 5 ' -ggc cgc ate ccg ggt etc gag gtc gac aag ctt teg agg ate cgc-3 ' oligo-82 5 '-ggc cgc gga tec teg aaa get tgt cga cct cga gac ccg gga tgc-3 ' The resulting plaεmid, pGPlc, containε a polylinker with Xmal , Xhol, Sail, Hindlll, and BamHI reεtriction εiteε flanked by Notl siteε. 2. Plasmid vector pGPld Plasmid vector pGPla is digeεted with Notl and the following oligonucleotideε ligated in: oligo-87 5 '-ggc cgc tgt cga caa get tat cga tgg ate etc gag tgc -3' oligo-88 5'-ggc cgc act cga gga tec ate gat aag ctt gtc- gac age -3' The resulting plasmid, pGPld, contains a polylinker with Sail, Hindlll, Clal, BamHI, and Xhol restriction εiteε flanked by Notl sites. B. Isolation of J/c and Cc cloneε A human placental genomic DNA library cloned into the phage vector AEMBL3/SP6/T7 (Clonetech Laboratorieε, Inc., Palo Alto, CA) waε screened with the human kappa light chain J region εpecific oligonucleotide: oligo-36 5'- cac ctt egg cca agg gac acg act gga gat taa acg taa gca -3 ' and the phage cloneε 136.2 and 136.5 iεolated. A 7.4 kb Xhol fragment that includes the J l segment waε iεolated from 136.2 and subcloned into the plasmid pNN03 to generate the plasmid clone p36.2. A neighboring 13 kb Xhol fragment that includeε Jk segments 2 through 5 together with the C/c gene segment waε isolated from phage clone 136.5 and subcloned into the plasmid pNN03 to generate the plasmid clone p36.5. Together these two clones span the region beginning 7.2 kb upstream of J/cl and ending 9 kb downstream of C/c. C. Construction of rearranged light chain tranεgeneε 1. pCKl, a Cc vector for expressing rearranged variable segments The 13 kb Xhol insert of plasmid clone p36.5 containing the C/c gene, together with 9 kb of downstream sequences, is cloned into the Sail site of plasmid vector pGPlc with the 5' end of the insert adjacent to the plasmid Xhol site. The resulting clone, pCKl can accept cloned fragments containing rearranged VJ/c segments into the unique 5 ' Xhcl site. The transgene can then be excised with Notl and purified from vector sequences by gel electrophoresiε. The resulting transgene construct will contain the human J-C/c intronic enhancer and may contain the human 3' /c enhancer. 2. pCK2, a C/c vector with heavy chain enhancers for expresεing rearranged variable segments A 0.9 kb Xbal fragment of mouse genomic DNA containing the mouse heavy chain J-μ intronic enhancer (J. Banerji et al. (1983) Cell 33, 729-740) was subcloned into pUC18 to generate the plasmid pJH22.1. This plasmid waε linearized with Sphl and the endε filled in with klenow enzyme. The klenow treated DNA waε then digeεted with Hindlll and a 1.4 kb Mlul(klenow)/Hindlll fragment of phage clone λl.3 (previous example) , containing the human heavy chain J-μ intronic enhancer (A. Hayday et al. (1984) Nature 307, 334-340), to it. The resulting plaεmid, pMHEl, conεiεtε of the mouεe and human heavy chain J-μ intronic enhancers ligated together into pUClδ such that they are excised on a single BamHI/Hindlll fragment. This 2.3 kb fragment is isolated and cloned into pGPlc to generate pMHE2. pMHE2 is digested with Sail and the 13 kb Xhol insert of p36.5 cloned in. The resulting plaεmid, pCK2, is identical to pCKl, except that the mouse and human heavy chain J-μ intronic enhancerε are fused to the 3 ' end of the tranεgene insert. To modulate expression of the final transgene, analogous constructs can be generated with different enhancers, i.e. the mouse or rat 3' kappa or heavy chain enhancer (K. Meyer and M.S. Neuberger, (1989) EMBO J. , 8 , 1959-1964; S. Petterson, et al. (1990) Nature. 344, 165-168). 2. Isolation of rearranged kappa light chain variable segmentε Two human leukocyte genomic DNA librarieε cloned into the phage vector AEMBL3/SP6/T7 (Clonetech Laboratorieε, Inc. , Palo Alto, CA) were εcreened with the human kappa light chain J region containing 3.5 kb Xhol/Smal fragment of p36.5. Positive clones were tested for hybridization with the following V/c specific oligonucleotide: oligo-65 5'-agg ttc agt ggc agt ggg tct ggg aca gac ttc act etc ace ate agc-3 ' Clones that hybridized with both V and J probes are iεolated and the DNA εequence of the rearranged VJ/c segment determined. 3. Generation of tranεgenic mice .containing rearranged human light chain constructs. Fragments containing functional VJ segments (open reading frame and splice signals) are subcloned into the unique Xhol sites of vectors pCKl and pCK2 to generate rearranged kappa light chain transgenes. The tranεgene constructε are isolated from vector sequenceε by digeεtion with Notl. Agaroεe gel purified insert is microinjected into mouεe embryo pronuclei to generate tranεgenic animalε. Animalε expreεεing human kappa chain are bred with heavy chain minilocuε containing tranεgenic animalε (EXAMPLE 14) to generate mice expressing fully human antibodies. Becauεe not all VJ/c combinationε may be capable of forming stable heavy-light chain complexes with a broad spectrum of different heavy chain VDJ combinationε, several different light chain transgene constructε are generated, each using a different rearranged VJk clone, and tranεgenic mice that result from these constructs are bred with heavy chain minilocus transgene expressing mice. Peripheral blood, spleen, and lymph node lymphocytes are isolated from double tranεgenic (both heavy and light chain constructs) animals, stained with fluorescent antibodies specific for human and mouse heavy and light chain immunoglobulins (Pharmingen, San Diego, CA) and analyzed by flow cytometry using a FACScan analyzer (Becton Dickinson, San Jose, CA) . Rearranged light chain transgeneε constructs that result in the highest level of human heavy/light chain complexes on the surface of the higheεt number of B cellε, and do not adversely affect the immune cell compartment (aε aεεayed by flow cytometric analyεiε with B and T cell εubset specific antibodies) , are εelected for the generation of human monoclonal antibodieε. D. Construction of unrearranged light chain minilocus transgenes 1. pJCKl, a J/c, C/c containing vector for constructing minilocus transgenes The 13 kb Cc containing Xhol insert of p36.5 iε treated with klenow enzyme and cloned into Hindlll digested, klenow treated, plasmid pGPld. A plaεmid clone iε selected such that the 5' end of the insert is adjacent to the vector derived Clal site. The resulting plasmid, p36.5-ld, iε digested with Clal and klenow treated. The Jcl containing 7.4 kb Xhol insert of p36.2 is then klenow treated and cloned into the Clal, klenow treated p36.5-ld. A clone iε selected in which the p36.2 insert is in the same orientation as the p36.5 insert. This clone, pJCKl (Fig. 34) , containε the entire human J/c region and Cc, together with 7.2 kb of upstream εequenceε and 9 kb of downεtream εequenceε. The inεert alεo containε the human J-Cc intronic enhancer and may contain a human 3 ' K enhancer. The insert iε flanked by a unique 3' Sail εite for the purpose of cloning additional 3 ' flanking sequences such as heavy chain or light chain enhancers. A unique Xhol site is located at the 5 ' end of the insert for the purpose of cloning in unrearranged V/c gene segmentε. The unique Sail and Xhol εites are in turn flanked by Notl εiteε that are uεed to isolate the completed transgene construct away from vector sequences. 2. Isolation of unrearranged V/c gene segments and generation of transgenic animals expressing human Ig light chain protein The V/c specific oligonucleotide, oligo-65 (diεcusse above) , is used to probe a human placental genomic DNA library cloned into the phage vector 1EMBL3/SP6/T7 (Clonetech Laboratories, Inc. , Palo Alto, CA) . Variable gene segmentε from the resulting cloneε are sequenced, and clones that appear functional are selected. Criteria for judging functionality include: open reading frames, intact splice acceptor and donor sequenceε, and intact recombination εequence. DNA fragmentε containing εelected variable gene segments are cloned into the unique Xhol site of plasmid pJCKl to generate minilocus constructs. The resulting clones are digested with Notl and the inserts isolated and injected into mouεe embryo pronuclei to generate transgenic animals. The transgenes of these animals will undergo V to J joining in developing B—cellε. Animals expressing human kappa chain are bred with heavy chain minilocus containing transgenic animals (EXAMPLE 14) to generate mice expressing fully human antibodieε. EXAMPLE 17 Synthetic Heavy Chain Variable Region Thiε example iε outlined in Fig. 35. A. Conεtruction of Cloning Vector pVHf 1. pGPlf The plasmid pGPla (previous example) is digested with Notl and the following oligonucleotideε are ligated to it: oligo-"a" 5 '-ggc cgc atg eta etc gag tgc aag ctt ggc cat cca-3 ' oligo-"b" 5'-ggc ctg gat ggc caa get tgc act cga gta gca tgc-3 ' The reεulting plasmid, pGPlf, containε Sphl, Xhol, and Hindlll sites flanked by Notl and Sfil sites. 2. pVHf The human V H -V family variable gene segment V H 251 (C.G. Humphries et al. (1988) Nature. 331, 446) together with approximately 2.4 kb of 5' flanking sequences and approximately 1.4 kb of 3' flanking sequences was isolated on a 4.2 kb Sphl/Hindlll fragment from the plasmid clone pVH251 (previous example) and cloned into the plasmid vector pSelect m -l (Promega Corp., Madison, WI) . The 5' flanking sequenceε, together with the promoter, first exon and first intron of V H 251, are amplified by polymerase chain reaction (PCR) from this template using the following oligonucleotides: oligo-83 5*-cag etc gag etc ggc aca ggc gcc tgt ggg-3 ' oligo-84 5'-etc tag agt cga cct gca ggc-3 ' The 3' flanking sequenceε are amplified by PCR uεing the following oligonucleotideε: O l r_rr_-.R ~_ t. I -arrr <-•+ • _ ■ ■ ι~tr* G τ*ι—> rrt- i-. +- -_ _ _ —• _z_ r* r>r< J r nr> ~. -> = /-• -_. • . I oligo-86 5 '-ggt gac act ata gaa tac tea agc-3 * The amplified 5' sequences are digested with Sphl and Xhol, and the 3' sequences digested with Hindlll and Xhol. The resulting fragments are cloned together into the plasmid pGPlf to generate plasmid pVHf. Plaεmid pVHf contains the ciε acting regulatory elements that control transcription of v H 251, together with the signal sequence encoding firεt exon. pVHf iε uεed aε an expression cassette for heavy chain variable sequences. Such sequenceε are cloned into the Kaεl/Xhol digeεted plasmid aε deεcribed below. B. Isolation of Variable Gene Coding Sequenceε 1. Amplification of expreεεed V H gene cDNA εequenceε Poly (A) * RNA iε iεolated from human peripheral blood lymphocytes (PBL) . First strand cDNA iε synthesized with reverse transcriptase, using oligo- (dT) as a primer. The first strand cDNA is isolated and tailed with, oligo (dG) using terminal transferase. The 5 sequenceε of IgM transcripts are then specifically amplified by a modification of the method of Frohman et al. (1988, Proc. Natl. Acad. Sci. USA, 85 , 8998) . Oligo-(dC) 13 and the following oligonucleotide: oligo-69 5 '-gga att etc aca gga gac gag-3 are used as 5' and 3' primers, respectively, in a polymerase chain reaction with dG-tailed firεt strand PBL cDNA. Oligo-69 is complimentary to sequences encoding amino acidε 11-17 of the IgM constant domain. Therefore these primerε will amplify DNA fragmentε of approximately 0.6 kb that include expresεed V H gene εequenceε. 2. Back-conversion of cDNA sequences into germline form The following oligonucleotide: oligo-"c" 5'-ctg acg act ctg tat ggc gcc (ct)a(cg) t(cg) (ct) (cg)ag (ag)t(cg) ca(ag) ct(gt) gtg (cg)a(ag) tc(gt) gg(gt)-3 « is annealed to denatured, PCR amplified, IgM 5' sequenceε. Oligo-"c" includeε a 21 nucleotide nondegenerate εequence that includes a Kasl site, followed by a 30 nucleotide degenerate sequence that is homologous to the 5 ' end of the second exon of many human V H segmentε (Genbank; Loε Ala oε, NM) . The primer is extended with DNA polymerase and the product isolated from unused primer by size fractionation. The product iε then denatured and annealed to the following oligonucleotide: oligo-"d" 5 '-ggg etc gag get ggt ttc tct cac tgt gtg t(cgt)t (acgt) (ag) (ct) aca gta ata ca(ct) (ag)g(ct)-3' Oligo-"d" includes a 30 nucleotide nondegenerate εequence that includes an Xhol site and part of the V to DJ recombination sequence, followed by a 21 nucleotide degenerate sequence that is complimentary to the the εequence encoding the laεt εeven amino acidε in framework region three of many human variable gene segments. The annealed oligonucleotide is then extended with DNA polymerase and the product isolated from unused primer by size fractionation. Single rounds of DNA εyntheεiε followed by removal of primerε are carried out to enεure the sequence integrity of individual variable gene fragments. The product of oligo-"d" primer extension is amplified by PCR using the following two oligonucleotides as primers: oligo-"e" 5 '-ctg acg act ctg tat ggc gcc-3 ' oligo-"f" 5 '-ggg etc gag get ggt ttc tct-3 ' The reεulting 0.36 kb PCR product iε purified by gel electrophoresis and digested with the restriction enzymes Kaε. and Xhol. Digestion productε are then cloned into Kaεl/Xhol digested pVHf to generate a library of expressed variable gene sequences in germline configuration. Ligation into the Kasl site of pVHf recreates the εplice acceptor εite at the 5' end of the secon exon, while ligation into the Xhol site recreates the recombination signal at the 3' end of the variable gene segment. Alternative versions of degenerate oligonucleotides "c" and "d" are used to amplify different populations of variable genes, and generate germline-configuration libraries representing those different populations (Genbank; Los Alamos, NN) . C. Construction of Synthetic Locuε The entire library of εynthetic germline-configuration V H genes is grown up together and plasmid DNA isolated. The medium copy plasmid pVHf, which includes a strong tranεcription terminator between the a picillin reεiεtance gene and the cloning εite, iε deεigned to minimize the expanεion of particular cloneε within the library. Plasmid DNA is digested with Sfil, treated with calf intestinal phosphatase to remove 5' phosphate groups, and then digested with Notl. The calf intestinal phosphataεe is removed prior to Notl digeεtion εo that only the Sfil endε are dephoεphorylated. The digeεted DNA iε then isolated from vector sequenceε by agarose gel electrophoresiε and ligated to the following oligonucleotideε: oligo-"g" 5'-ggc eta act gag cgt ccc ata ttg aga ace tec -3' oligo-"h" 5'-ggt tct caa tat ggg acg etc agt ta-3 ' 01igo-"h" iε kinased while oligo-"g" is left unphosphorylated. The ligation reaction is carried out with a large molar excess of oligonucleotides so that all of the V gene fragment Notl ends will be ligated to oligonucleotides and not other V region fragments. Because the Sfil endε are not self compatible, the V εegmentε will concatenate in the same orientation εuch that each V segment is separated by a single oligonucleotide spacer unit from the next V segment. Large concatomers are sized by electrophoresiε and isolated from agarose gels. The size fractionated concatomers are then directly coinjected into mouεe embryo pronuclei toσ<-."r , h_-»r vi t-V. n— J— -t-he r-iWP . or pHC2 inserts) to generate transgenic animalε with large primary repertoires. Alternatively, the concatomers are cloned into a plasmid vector such as pGPf. EXAMPLE 18 Generation of Lymphoid Cell Receptor Subset Specific Antibodies. The inoculation of mice with xenogeneic (i.e. human) immunoglobulins (B-cell receptors) or T-cell receptors leads predominantly to the generation.of mouse antibodies directed againεt particular epitopeε (dominant epitopes) that εhared by all or most immunoglobulins or T-cell receptors of a given εpecies, but differ between specieε. It iε therefore difficult to isolate antibodies that distinguiεh particular εubεetε of B or T cell receptors (e.g., idiotypes or variable region families) . However, the transgenic mouse expressing human immunoglobulins (described in the above examples) will be immunologically tolerant of those εhared B-cell epitopeε and will therefore be useful for generating antibodieε that distinguish subsets of human immunoglobulins. This concept is extended by generating transgenic mice expressing human T-cell receptor coding sequences and breeding these mice with the human immunoglobulin transgenic mice. Such mice are inoculated with isolates containing human T-cell receptor proteins and monoclonal antibodies are generated that recognize T-cell receptor subsets. Studies have demonstrated that there iε a limited variability of T cell antigen receptorε involved in certain autoimmune diseases (T.F. Davies et al. (1991) New England J. Med. , 325, 238) . Because of this limited variability, it iε possible to generate human monoclonal antibodieε that specifically recognize that subset of human T cells which iε auto-reactive. A. Generation of B-cell subset εpecific antibodieε Human immunoglobulin expressing transgenic mice are inoculated with immunoglobulins isolated from a healthy donor or from a patient with a B—cell mali τ nan r ' r e p ressin rτ a hi rf h level of a single immunoglobulin type (Miller et al. (1982) New Eng. J. Med. 306, 517-522) . Monoclonal antibody secreting hybridomas are generated as described by Harlow and Lane (E. Harlow and D. Lane. Antibodies: A Laboratory Manual. 1988. Cold Spring Harbor Laboratory, New York) . Individual hybridomas that secrete human antibodies that specifically recognize B-cell subsets are selected. B. Tranεgenic mice expresεing human T-celi receptor sequences. DNA fragments containing intact and fully rearranged human T-cell receptor (TCR) α and β geneε are coinjected into mouεe embryo pronuclei to generate tranεgenic animalε. Tranεgenic animalε are aεεayed by FACS analyεiε for the expreεεion of both tranεgeneε on the εurface of their T-cellε. Animals are selected that expresε only low levelε of the human α and β TCR chainε on a fraction of their T-cells. Only low level expression is required to obtain immunological tolerance, and high level expression will disturb the animal's immune system and interfere with the ability to mount an immune response required for the generation monoclonal antibodies. Alternatively, because correct tissue or cell type specific expression is not required to obtain immunologic tolerance, TCR a and β chain cDNA clones are inserted into transgene expression cassettes (T. Choi et al. (1991) Mol. Cell. Biol. , 11, 3070-3074) under the control of non-TCR tranεcription signals. TCR and β chain cDNA transgene conεtructε are coinjected into mouse embryo pronuclei to generate transgenic animals. Ectopic expreεsion of the TCR chains will not result in cell surface expression because the TCR is a multichain complex (H. Clevers et al. 1988 Ann. Rev. Immunol., 6_, 629-662) ; however, cell surface expression is not required for antigen presentation (Townsend et al. (1986) Nature, 324 , 575-577) and tolerance induction. T-cell receptor a and β chain transgenic mice are bred with human immunoglobulin expressing transgenic mice to generate mice that are useful for generating human monoclonal antibodies that reco g nize specific subsets of human T—cellε. Such mice are inoculated with T-cell derived proteinε iεolated from a healthy donor or from a patient with a T-cell malignancy expressing a single TCR type. Monoclonal antibody secreting hybridomas are generated and individual hybridomaε that εecrete human antibodieε that specifically recognize B-cell εubεetε are selected. EXAMPLE 19 Genomic Heavy Chain Human Ig Transgene This Example describeε the cloning of a human genomic heavy chain immunoglobulin tranεgene which iε then introduced into the murine germline via microinjection into zygoteε or integration in ES cellε. Nuclei are iεolated from fresh human placental tiεεue asdescribed by Marzluff, W.F., et al. (1985), Transcription and Translation: A Practical Approach, B.D. Hammes and S.J. Higgins, eds., pp. 89-129, IRL Presε, Oxford) . The iεolated nuclei (or PBS washed human spermatocytes) are embedded in 0.5% low melting point agarose blockε and lyεed with 1 mg/ml proteinase K in 500mM EDTA, 1% SDS for nuclei, or with lmg/ml proteinase K in 500mM EDTA, 1% SDS, lOmM DTT for εpermatocyteε at 50 β C for 18 hourε. The proteinaεe K is inactivated by incubating the blocks in 40μg/ml PMSF in TE for 30 minuteε at 50*C, and then washing extensively with TE. The DNA is then digested in the agarose with the restriction enzyme Notl as described by M. Finney in Current Protocols in Molecular Biology (F. Ausubel et al., eds. John Wiley & Sons, Supp. 4, 1988, e.g., Section 2.5.1) . The Notl digested DNA is then fractionated by pulsed field gel electrophoresis as described by Anand, R. et al. (1989) , Nuc. Acids Res. , 17 , 3425-3433. Fractions enriched for the Notl fragment are assayed by Southern hybridization to detect one or more of the sequences encoded by thiε fragment. Such sequences include the heavy chain D segmentε, J εegmentε, and 7 I constant regions together with representatives of all 6 V H families (although this fragment is identified as 670 kb fragment from HeLa cellε by Berman et al. (1988), εupra., we have found it to be an 830 kb fragment from human placental and sperm DNA) . Those fractions containing this Notl fragment (see Fig. 4) are ligated into the Notl cloning εite of the vector pYACNN aε described (McCormick, M. et al. (1990), Technique 2_, 65-71) . Plasmid pYACNN is prepared by digeεtion of pYACneo (Clontech) with EcoRI and ligation in the preεence of the oligonucleotide 5' - AAT TGC GGC CGC - 3'. YAC cloneε containing the heavy chain Notl fragment are isolated as described by Traver et al. (1989), Proc. Natl. Acad. Sci. USA, 86, 5898-5902. The cloned Notl insert iε isolated from high molecular weight yeaεt DNA by pulεe field gel electrophoresis as described by M. Finney, op. cit. The DNA is condenεed by the addition of 1 mM εpermine and microinjected directly into the nucleuε of single cell embryoε previously described. Alternatively, the DNA iε iεolated by pulεed field gel electrophoreεiε and introduced into ES cellε by lipofection (Gnirke et al. (1991), EMBO J. , 10, 1629-1634), or the YAC iε introduced into ES cellε by spheroplaεt fuεion. EXAMPLE 20 Diεcontinuous Genomic Heavy Chain Ig Transgene An 85 kb Spel fragment of human genomic DNA, containing V H 6, D segments, J segments, the μ constant region and part of the 7 constant region (see Fig. 4) , has been isolated by YAC cloning essentially as described in Example 1. A YAC carrying a fragment from the germline variable region, such as a 570 kb Notl fragment upstream of the 670-830 kb Notl fragment described above containing multiple copies of V through V 5 iε isolated as described. (Berman et al. (1988) , supra, detected two 570 kb Notl fragments, each containing multiple V segmentε.) The two fragmentε are coinjected into the nucleuε of a mouεe εingle cell embryo aε deεcribed in Example 1. Typically, coinjection of two different DNA fragments result in the integration of both fragments at the same insertion site within the chromosome. Therefore, approximately 50% of the resulting transgenic animals that contain at least one copy of each of the two fragments will have the V segment fragment inserted upstream of the constant region containing fragment. Of these animals, about 50% will carry out V to DJ joining by DNA inversion and about 50% by deletion, depending on the orientation of the 570 kb Notl fragment relative to the position of the 85 kb Spel fragment. DNA is isolated from resultant transgenic animals and those animals found to be containing both transgenes by Southern blot hybridization (specifically, those animals containing both multiple human V segments and human constant region genes) are tested for their ability to express human immunoglobulin molecules in accordance with standard techniques. EXAMPLE 21 Joining Overlapping YAC Fragments Two YACs carrying a region of overlap are joined in yeast by meiotic recombination as described by Silverman et al. (1990), Proc. Natl. Acad. Sci. USA. 87, 9913-9917, to derive a single, large YAC carrying sequenceε from both εmaller YACε. The two YACs are aligned with respect to the arms, such that the joined YAC will contain one centromeric vector arm and one non-centromeric vector arm. If necessary, the insert is recloned in the vector using unique restriction εiteε at the ends of the insert. If the insert is not a unique restriction fragment, unique sites are inserted into the vector arms by oligonucleotide transformation of yeast, as described by Guthrie and Fink, op. cit. To join YACs carrying noncontiguous sequences which do not overlap, an overlap is created as follows. The 3 ' terminal region of the 5' YAC and the 5' terminal region of the 3' YAC are subcloned, joined in vitro to create a junction fragment, and reintroduced into one or both YACs by homologous recombination (Guthrie and Fink, op cit) . The two YACs are then meiotically recombined as described by Silverman et al., op cit). The joined YAC iε introduced into mice, e.g., as in Example 1. EXAMPLE 22 Genomic /c Light Chain Human Ig Transgene A map of the human /c light chain has been described in Lorenz, W. et al. (1987), Nucl. Acids Reε. , 15, 9667-9677 and is depicted in Fig. 11. A 450 kb Xhol to Notl fragment that includes all of Cc, the 3' enhancer, all J εegments, and at least five different V segments (a) , or a 750kb Mlul to Notl fragment that includes all of the above pluε at leaεt 20 more V segments (b) is isolated and introduced into zygotes or ES cellε aε deεcribed in Example 1. EXAMPLE 23 Genomic /c Light Chain Human Ig Tranεgene Formed by In Vivo Homologouε Recombination The 750kb Mlul to Notl fragment iε digeεted with BεsHII to produce a fragment of about 400 kb (c) . The 450 kb Xhol to Notl fragment (a) plus the approximately 400 kb Mlul to BssHII fragment (c) have sequence overlap defined by the BssHII and Xhol restriction siteε εhown in Fig. 11. Homologouε recombination of theεe two fragments upon microinjection of a mouse zygote resultε in a tranεgene containing at least an additional 15-20 V segments over that found in the 450 kb Xhol/Notl fragment (Example 22) . EXAMPLE 24 Identification of functionally rearranged variable region sequences in transgenic B cells An antigen of interest is used to immunize (εee Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York (1988)) a mouse with the following genetic traits: homozygosity at the endogenous having chain locus for a deletion of J H (Examples 9 and 12) ; hemizygouε for a εingle copy of unrearranged human heavy chain minilocuε tranεgene (exampleε 5 and 14) ; and hemizygouε for a εingle copy of a rearranged human kappa light chain transgene (Examples 7 and 16) . Following the schedule of immunization, the spleen is removed, and spleen cellε used to generate hybridomaε. Cells from an individual hybridoma clone that secreteε antibodieε reactive with the antigen of interest are used to prepare genomic DNA. A sample of the genomic DNA is digested with several different restriction enzymes that recognize unique six base pair sequences, and fractionated on an agarose gel. Southern blot hybridization is used to identify two DNA fragments in the 2-10 kb range, one of which contains the single copy of the rearranged human heavy chain VDJ sequenceε and one of which contains the εingle copy of the rearranged human light chain VJ εequence. Theεe two fragmentε are εize fractionated on agarose gel and cloned directly into pUClδ . The cloned inserts are then εubcloned reεpectively into heavy and light chain expreεsion casεetteε that contain conεtant region sequences. The plasmid clone P7el (Example 14) is used aε a heavy chain expression cassette and rearranged VDJ sequences are cloned into the Xhol site. The plasmid clone pCKl is used aε a light chain expreεεion caεεette and rearranged VJ εequenceε are cloned into the Xhol site. The resulting clones are used together to transfect SP 0 cells to produce antibodieε that react with the antigen of interest (M.S. Co. et al. (1991) Proc. Natl. Acad. Sci. USA JL8:2869) . Alternatively, mRNA is isolated from the cloned hybridoma cells described above, and used to synthesize cDNA. The expressed human heavy and light chain VDJ and VJ sequence are then amplified by PCR and cloned (J.W. Larrich et al. (1989) Biol. Technology, 2:934-938). After the nucleotide sequence of theεe clones haε been determined, oligonucleotides are synthesized that encode the εame polypeptideε, and synthetic expression vectors generated as deεcribed by C. Queen et al. (1989) Proc. Natl. Acad. Sci. USA.. 84:5454-5458. The foregoing deεcription of the preferred embodiments of the preεent invention haε been presented for purpoεeε cf illustration and description. They are not intended to be exhaustive or to limit the invention to the precise form disclosed, and many modifications and variations are posεible in light of the above teaching. A_ __ ications h rein are incorporated by reference to the same extent aε if each individual publication or patent application waε specifically and individually indicated to be incorporated by reference. Such modifications and variations which may be apparent to a person skilled in the art are intended to be within the scope of this invention.

Claims: WHAT IS CLAIMED IS: 1. An isolated immunoglobulin heavy-chain transgene comr>risin<τ DNA encoding at least one variable gene sefrne t, one diversity gene segment, one joining gene segment, and one constant region gene segment, wherein each of said gene segments is derived from DNA corresponding to immunoglobulin heavy chain gene segments from the εame εpecieε or an individual of said specieε and wherein the εegments are capable of undergoing gene rearrangement in vivo to form a rearranged gene encoding a heavy chain polypeptide. 2. An immunoglobulin heavy-chain transgene of Claim 1 wherein the length of said tranεgene iε leεε than the length of the corresponding genomic DNA containing all of the gene segments contained on said heavy-chain tranεgene. 3. The tranεgene of Claim 1 wherein εaid at leaεt one constant region gene segment comprises at least two constant region gene segments. 4. The transgene of Claim 3 wherein εaid one conεtant region gene εegmentε compriεeε a μ and a conεtant region gene εegment. 5. The tranεgene of Claim 1 wherein εaid at leaεt one constant region gene segment comprises a 7 constant region gene segment. 6. The transgene of Claim 1 wherein said at least one variable gene segment comprises DNA corresponding to a first functional D-proximal variable region gene εegment of said species or said individual. 7. The transgene of Claim 1 wherein the relative poεitions of each of said gene εegmentε iε the εame aε the relative poεition of the correεponding gene εegmentε in the genome of said species. The transgene of Claim 1 wherein εaid species is human. 9. The transgene of Claim 1 wherein εaid tranεgene is unrearranged and capable of undergoing rearrangement to generate V region diversity, when introduced into a B-cell of a non-human transgenic animal. 10. An isolated immunoglobulin light-chain transgene comprising DNA encoding at least one variable gene segment, one joining gene segment, and one constant region gene εegment, wherein each of said gene εegmentε iε derived from genomic DNA corresponding to immunoglobulin light chain gene εegmentε from the same species or an individual of said species and wherein the εegmentε are capable of undergoing gene rearrangement in vivo to form a rearranged gene encoding a light chain polypeptide. 11. An immunoglobulin light-chain tranεgene of Claim 9 wherein the length of εaid tranεgene iε leεε than the length of the corresponding genomic DNA containing all of the gene segmentε contained on εaid light-chain tranεgene. 12. The transgene of Claim 11 wherein said at least one variable region gene segment comprises DNA corresponding to a first functional J-proximal variable gene segment of said species or said individual. 13. The transgene of Claim 11 wherein the relative positionε of said gene segments iε the εame as the relative positions of the corresponding gene segments in the genome of said species or said individual. 14. The transgene of Claim 9 wherein εaid εpecies or individual iε human. 15. The transgene of Claim 10 wherein said transgene is unrearranged and capable of undergoing rearrangement to generate V region diversity, when introduced into a B-cell of a non—human trans g enic animal. 16. A transgenic non-human animal containing heterologous immunoglobulin heavy and light chain transgenes in the germline of said animal. 17. The transgenic non-human animal of Claim 16 wherein said heavy and εaid light chain tranεgeneε are rearranged. 18. The tranεgenic non-human animal of Claim 16 wherein εaid heavy and said light chain tranεgeneε are unrearranged. 19. The tranεgenic non-human animal of Claim 16 wherein one of said heavy and said light chain tranεgeneε iε rearranged and the other of εaid heavy and said light transgeneε is unrearranged. 20. The transgenic non-human animal of Claim 19 wherein said heavy chain transgene iε unrearranged and εaid light chain transgene is rearranged. 21. The tranεgenic non-human animal of Claim 16 wherein said transgenes are functionally rearranged in B-cells of said animal. 22. The transgenic non-human animal of Claim 21 wherein said B-cells produce a heterologouε antibody. 23. The transgenic non-human animal of Claim 16 wherein said heterologouε heavy and light chain geneε are. human. 24. The tranεgenic non-human animal of Claim 16 wherein said non-human animal is a rodent. 25. A transgenic non—human animal ca p able of producing a heterologous antibody in at least one cell of εaid transgenic animal containing an immunoglobulin heavy-chain transgene and an immunoglobulin light-chain transgene, wherein said heavy chain transgene compriseε DNA encoding at leaεt one variable gene εegment, one diverεity gene εegment, one joining gene εegment, and one conεtant region gene εegment, and εaid immunoglobulin light-chain tranεgene compriεeε DNA encoding at leaεt one variable gene εegment, one joining gene εegment, and one conεtant region gene segment, wherein each of εaid gene εegments of εaid heavy and light chain tranεgeneε iε iεolated from or correεpondε to DNA encoding immunoglobulin heavy and light chain gene εegmentε from a εpecieε not conεiεting of εaid non-human animal or an individual of said species. 26. The transgenic animal of Claim 25 wherein said transgenic non-human animal is a rodent. 27. The transgenic non-human animal of Claim 25 wherein said transgeneε encode human antibody gene segmentε. 28. The tranεgenic non-human animal of claim 25 wherein at least one of the endogenous immunoglobulin loci of said transgenic animal is functionally disrupted. 29. The transgenic non-human animal of Claim 28 wherein the functional disruption of εaid endogenouε loci is produced by disrupting endogenous immunoglobulin gene segments encoding heavy or light immunoglobulin chains, said gene segments being selected from the group consisting of diversity, joining and constant gene εegmentε. 30. The tranεgenic non-human animal of Claim 29 wherein εaid endogenous immunoglobulin gene segmentε are joining region gene segments encoding said heavy and light immunoglobulin chains. 31= The transgenic non—human animal of Claim 29 wherein said disruption iε by deletion of εubεtantially all of said selected gene segments. 32. The transgenic non-human animal of Claim 25 wherein said heavy and said light chain transgeneε are unrearranged. 33. The tranεgenic non-human animal of Claim 25 wherein said heavy and said light chain transgeneε are rearranged. 34. The tranεgenic non-human animal of Claim 25 wherein one of said heavy and said light chain tranεgeneε iε rearranged and the other of εaid heavy and εaid light chain transgenes is unrearranged. 35. The tranεgenic non-human animal of Claim 34 wherein said heavy chain transgenes is unrearranged and εaid light chain transgenes is rearranged. 36. A non-human B-cell derived from a tranεgenic non- human animal, said B-cell being capable of producing a heterologous antibody. 37. The non-human B-cell of Claim 36 wherein said B- cell contains a functionally rearranged heterologous heavy chain immunoglobulin transgene and a functionally rearranged heterologous light chain immunoglobulin tranεgene. 38. The non-human B-cell of Claim 37 wherein each of said functionally rearranged heavy and light chain immunoglobulin transgeneε are of human origin. 39. The non-human B-cell of Claim 36 wherein εaid B-cell doeε not produce a homologouε antibody. 40- A hvbridoϊP.a comπrising a mveloma cell fused to a non-human B-cell derived from a transgenic non-human animal, said hybridoma capable of producing a monoclonal antibody heterologous to said B-cell. 41. The hybridoma of Claim 40 wherein genomic material of said hybridoma derived from εaid non-human B-cell containε a functionally rearranged heavy chain immunoglobulin transgene and a functionally rearranged light chain immunoglobulin transgene, each of εaid functionally rearranged transgenes being heterologous to εaid B-cell. 42. The hybridoma of Claim 41 wherein said tranεgenes are of human origin. 43. The hybridoma of Claim 40 wherein εaid monoclonal antibody iε a human antibody. 44. The hybridoma of Claim 40 wherein said B-cell iε of rodent origin. 45. The hybridoma of Claim 40 wherein said myeloma cell iε of murine origin. 46. A method of producing a transgenic non-human animal wherein at least one of the endogenous immunoglobulin gene loci of said animal has been functionally diεrupted, εaid method comprising the stepε of: contacting at leaεt one embryonic εte cell of a non-human animal with a transgene targeting the functional disruption of a family of gene segments encoding an endogenous heavy or light immunoglobulin chain, said family of gene segments being selected from the group consiεting of functional endogenous diversity, joining and constant gene εegment familieε; and selecting at least one embryonic stem cell wherein said transgene haε integrated into the genome of εaid non-human animal by homologous recombination. 47. The method of Claim 46 wherein said tranεgene comprises a positive-negative selection vector. 48. The method of Claim 46 wherein said functional diεruption comprises the deletion of all or part of εaid family of gene segments. 49. The method of Claim 48 wherein εaid deletion iε of the family of joining gene εegmentε. 50. The method of Claim 46 wherein εaid functional diεruption comprises the introduction of a tranεcription or tranεlation εtop εequence. 51. The method of Claim 46 wherein εaid functional diεruption iε of a conεtant region gene segment. 52. The method of Claim 51 wherein said gene segment iε εelected from the group conεiεting of the μ heavy chain constant region gene segment, the K light chain conεtant region gene εegment and the group of functional A light chain conεtruct region gene segments." 53. A method for producing a heterologous antibody in a transgenic non-human animal having at least one B-cell containing at least one rearranged heterologouε immunoglobulin heavy chain transgene and at least one rearranged heterologouε immunoglobulin light chain tranεgene, εaid antibody being capable of binding an antigen, εaid method compriεing the εtep of: contacting εaid tranεgenic animal with εaid antigen to induce the production of εaid heterologouε antibody. 54. The method of Claim 53 wherein said contacting causes somatic mutation of at least one of said transgeneε. 55. The method of Claim 53 further cornerisin rf the step of immortalizing at leaεt one of the B-cellε producing said heterologous antibody to provide a source of monoclonal antibody corresponding to said heterologous antibody. 56. The method of Claim 55 wherein εaid immortalizing iε by fuεion of said B-cell with a myeloma cell line to form a hybridoma. 57. A monoclonal antibody made according to the method of Claim 55. 58. The monoclonal antibody of Claim 57 wherein εaid heavy and light chain tranεgeneε compriεe human immunoglobulin gene εegmentε and said antibody is a human antibody. 59. A method for producing a first heterologous antibody in a transgenic non-human animal having at least one B-cell containing at least one rearranged heterologous immunoglobulin heavy chain transgene and at least one rearranged heterologous immunoglobulin light chain transgene, wherein said first heterologouε antibody iε capable of binding a first antigen and said heavy and light transgeneε are capable of undergoing somatic mutation in response to a known εecond antigen to produce a second heterologous antibody, εaid method comprising the εtep of: contacting εaid transgenic animal with at leaεt said first antigen to induce the somatic mutation of at least one of said heavy or light chain transgeneε to produce a B-cell capable of producing εaid first heterologouε antibody. 60. The method of Claim 59 wherein εaid contacting compriεeε the sequential or simultaneous contacting of said firεt antigen and said εecond known antigen to produce firεt and second B-cells capable of producing said firεt and εaid second heterologous antibodies respectively. 61= The method of Claim 59 further comprising the step of immortalizing at least one of said B-cells producing said first heterologous antibody to provide a εource of monoclonal antibody corresponding to said first heterologous antibody. 62. The method of Claim 61 wherein said immortalizing is by fusion of said B-cell with a myeloma cell line to form a hybridoma. 63. A monoclonal antibody made according to the method of Claim 61. 64. The monoclonal antibody of Claim 63 wherein εaid heavy and light chain rearranged tranεgenes compriεe human immunoglobulin gene εegments and εaid antibody iε a human antibody. 65. A method for producing a εynthetic immunoglobulin V segment repertoire comprising the εtepε of: (a) generating a population of immunoglobulin V segment DNAS, each of said V segment DNAε encoding an immunoglobulin V segment and containing at each end a first cleavage recognition εite of a first restriction endonuclease; and (b) concatenating said population of immunoglobulin V segment DNAs to form a εynthetic immunoglobulin V segment repertoire. 66. The method of Claim 65 wherein εaid population of V εegment DNAε iε derived from genomic DNA and εaid generating is by PCR amplification uεing Pl and P2 primerε, εaid Pl primer comprising a mixture of primers encoding, from 5' to 3', said cleavage recognition εite and sequences capable of hybridizing to one strand of the C-terminal portion of a multiplicity of immunoglobulin V segmentε in said genomic DNA, and said P2 primer comprising a mixture of primers encoding, from 5' to 3', εaid cleavage recognition εite and sequenceε capable of hybridizing to the complementarv strand of the N-terminal portion of said multiplicity of immunoglobulin V segments in said genomic DNA. 67. The method of Claim 65 wherein said population of immunoglobulin V segment DNAs is derived from B-cell mRNA and said generating comprises the steps of: (i) priming syntheεiε of εingle εtranded cDNA from said mRNA with primer Pl comprising a mixture of primerε encoding, from 5' to 3', said cleavage recognition site and sequences capable of hybridizing to the coding strand of the C-terminal portion of a multiplicity of immunoglobulin V segments in the genomic DNA from which said mRNA is transcribed; (ii) priming synthesiε of double εtranded cDNA from said εingle stranded cDNA with primer P2 compriεing a mixture of primers encoding, from 5' to 3', εaid cleavage recognition εite and εequenceε capable of hybridizing to the antiεenεe strand of the N-terminal portion of εaid multiplicity of immunoglobulin V εegmentε in εaid genomic DNA. 68. The method of Claim 67 wherein each of εaid immunoglobulin V segment DNAs contains a first cleavage recognition site at one end of said V εegment DNA and a εecond different cleavage recognition site at the other end of said V segment DNA and said method further compriεeε the step of: amplifying said double stranded cDNA uεing P3 and P4 primerε, said P3 primer comprising DNA encoding said first cleavage recognition εite and said P4 primer compriεing DNA encoding said second cleavage recognition site. 69. The method of Claim 65 wherein said immunoglobulin V segmentε comprise DNA encoding the first signal sequence exon and second exon of more than one immunoglobulin V segment encoded by genomic DNA. 70. The method of Claim 65 wherein said population immunoglobulin V segment DNAs comprise DNA encoding the εecond exon of more than one immunoglobulin V εegment encoded by genomic DNA. 71. The method of Claim 70 wherein each of the members of εaid population iε ligated into an expresεion vector to form an expression cassette comprising, in order, a second cleavage site of a second restriction endonuclease, an immunoglobulin promoter, an immunoglobulin secretion signal εequence, a third cleavage recognition εite of a third reεtriction endonuclease, a recombination εignal εequence and a fourth cleavage recognition εite of a fourth reεtriction endonuclease, said ligation being into said third cleavage recognition site to form εaid expression cassette between εaid εecond and said fourth cleavage recognition site. 72. The method of Claim 71 wherein said concatenation iε of the expression cassette by digesting said expreεεion vector with εaid second and said fourth restriction endonucleaseε. 73. The synthetic V segment repertoire made according to the method of Claim 65. 74. A method for producing an expressible V segment comprising the steps of: (a) generating at least one V segment DNA encoding the second exon of a V segment and containing at each end thereof a first cleavage recognition site of a first restriction endonuclease; and (b) ligating said V segment DNA into an expression vector, said expression vector comprising, in order, an immunoglobulin promoter, an immunoglobulin secretion signal sequence, a second cleavage recognition εite of a second reεtriction endonuclease capable of ligating said V segment DNA when said vector is cleaved with said second restriction endonuclease and a recombination sequence. 75. An immunool obuT in ,Iσ. hoa chain m iniloeuε transgene construct formed from a plurality of DNA fragments, the construct comprising DNA sequences that encode human variable (V) , diversity (D) , joining (J) and constant regionε of a human Ig protein, which εequenceε are operably linked to transcription regulatory sequences and capable of undergoing gene rearrangement in vivo to produce a rearranged gene encoding a human heavy chain polypeptide; wherein each of at least three of the DNA fragments comprises: a V region εequence, a D region sequence, a J and conεtant region εequence, a D and J and conεtant region εequence, or a conεtant region εequence; wherein all of the Ig protein coding εequenceε are substantially homologous to human gene sequences. 76. The minilocuε transgene construct of claim 75 wherein the minilocus transgene construct iε about 75 kb in length. 77. The minilocuε transgene construct of claim 75 wherein the constant region gene sequenceε encode two different immunoglobulin isotypes. 78. The minilocuε tranεgene construct of claim 77 wherein the conεtant region gene sequenceε are capable of undergoing switch recombination. 79. The minilocus transgene construct of claim 77 wherein the isotypeε are mu and gamma. 80. The minilocuε transgene construct of claim 75 wherein constant region gene sequences encode a heavy chain gamma isotype. 81. The minilocus transgene conεtruct of ciaim 75 wherein the Ig coding sequences are from a single individual. 82. An Ig minilocus transgene construction of claim 75 wherein the constant region sequence compriseε a μ constant region 5' from a second constant region. 83. An Ig minilocus transgene construction of claim 82, wherein the construct further comprises a switch donor region 5 ' from the μ conεtant region and a εwitch acceptor region between the μ constant region and the second conεtant region, which εwitch regionε are operably linked to effect switching in vivo. 84. An Ig minilocus transgene construction of claim 83, wherein the switch donor region iε a human μ εwitch region. 85. An Ig minilocus tranεgene conεtruction of claim 83, wherein the switch acceptor region iε a human η χ εwitch region. 86. An Ig minilocuε tranεgene construction of claim 83, wherein the second constant region is a ~ι 1 constant region. 87. An immunoglobulin (Ig) light chain minilocus transgene construct formed from a plurality of DNA fragments, the construct comprising DNA sequences that encode human variable (V) , joining (J) and constant regions of a human Ig protein, which sequences are operably linked to transcription regulating sequences and are capable of undergoing gene rearrangement in vivo to produce a rearranged gene encoding a human light chain polypeptide; wherein each of at least three of the DNA fragments comprises: a V region sequence, a J and constant region sequence, or a constant region εequence; wherein all of the Ig protein coding sequences are substantially homologous to human gene sequences. 88. The minilocus transgene construct of claim 87 wherein the gene sequences are from a single individual. 89. The minilocus transgene conεtruct of claim 87 wherein the minilocuε tranεgene construct conεistε of about 50 kb. 90. The minilocus tranεgene conεtruct of claim 87 wherein one variable gene iε operably joined to the joining gene sequence. 91. The minilocuε tranεgene conεtruct of claim 87 wherein the relative poεition of each region iε the εame aε that of a correεponding region in a germline light chain gene. 92. A yeaεt artificial chromosome (YAC) containing an immunoglobulin gene insert comprising: an immunoglobulin variable gene εequence, a joining gene εequence and a conεtant region εequence which are capable of effecting rearrangement in vivo to form a rearranged gene of the εequenceε, which gene encodeε an immunoglobulin polypeptide. 93. The YAC of claim 92 wherein the chromosome further comprises a diversity gene sequence. 94. The YAC of claim 92 wherein the chromosome further compriseε a switch donor region and a switch acceptor region which are operably linked to effect isotype εwitching in vivo. 95. The YAC of claim 92 wherein the gene sequences are from the same individual. 96. The YAC of claim 92 wherein the insert is about 85 kb. 97. The YAC of claim 92 further comprising transcription regulatory sequences operably linked to the insert. 98. A method of producing a modified genome in a transgenic non-human mammal, the method comprising: condensing two yeast artificial chromosomeε having overlapping sequences, which chromosomes together encode a functional immunoglobulin locus; and inserting the yeast artificial chromosomes into the mammal genome. 99. The method of claim 98 wherein the yeast artificial chromosome is condensed with polyamineε. 100. The method of claim 98, wherein the inεerting step iε by transfecting or lipofecting. 101. The method of claim 98 wherein the yeast artificial chromosome is integrated into the genome. 102. A method of inactivating an endogenouε immunoglobulin gene in a non-human mammal, the method comprising introducing into the genome of the mammal a DNA fragment comprising a nucleotide sequence homologous to the endogenous immunoglobulin gene, whereby the nucleotide fragment incorporates into the genome by homologous recombination and thereby disrupts the endogenous gene. 103. The method of claim 102 wherein the step of introducing the DNA fragment into the genome iε carried out by transforming an embryonic stem cell. 104. The method of claim 102 wherein the diεruption compriseε deletion of joining gene εegmentε. 105. The method of claim 102 wherein the disruption comprises deletion of an enhancer or of a kappa constant region. 106. A non-human mammal produced by the method of claim 102. 107. An iεolated immunoglobulin chain minilocuε tranεgene construct of claim 75 or 87, wherein the V region sequence comprises VH251. 108. A transgenic non-human mammal capable of producing a rearranged human immunoglobulin and having an endogenous immunoglobulin gene with a heterologouε nucleotide εequence inεert. 109. A mammal of claim 108 wherein the heterologouε nucleotide sequence decreaseε expreεsion of endogenouε immunoglobulin polypeptideε. 110. A mammal of claim 108 wherein the heterologouε nucleotide sequence decreases expresεion of endogenouε μ heavy chain polypeptides. 111. A mammal of claim 108 wherein the heterologous nucleotide sequence decreases expresεion of endogenouε light chain polypeptides. 112. A mammal of claim 111 wherein the light chain polypeptides are kappa or lambda. 113. A mammal of claim 108 wherein the heterologouε nucleotide sequence introduces a transcription or translation stop sequence. 114. A mammal of claim 108 wherein the mammal is a mouse. 115. A mammal of claim 108 wherein the immunoglobulin is expressed from an immunoglobulin minilocus transgene construct having a variable gene segment, a joining gene segment,- and. a constant region gene segment. 116. A mammal of claim 115 wherein the minilocus transgene construct is a heavy chain gene. 117. A mammal of claim 108 comprising a rearranged immunoglobulin transgene construct. 118. A mammal of claim 117 wherein the rearranged transgene construct encodes a kappa light chain. 119. A mammal of claim 117 wherein the rearranged transgene construct encodeε a heavy chain. 120. A method for producing an immunoglobulin from a transgenic non-human mammal, the mammal having a rearranged immunoglobulin transgene construct that recognizes a εelected antigen, the method comprising: immunizing the transgenic mammal with the εelected antigen; and εcreening for B cells from the tranεgenic mammal that produce immunoglobulinε that bind the εelected antigen. 121. The method of claim 120 wherein the εtep of screening includes immortalizing B cells isolated from the transgenic mammal. 122. An immortalized cell produced according to the method of claim 121, wherein the cell is a hybridoma formed by fusing the selected B cell with a myeloma cell. 123. A monoclonal antibody produced by the hybridoma of claim 120. 124. A method according to claim 120, further compriεing the steps of: immunizing the transgenic animal with a second antigen r.τ- .1-vτ* +T. -t*Vιc_ c -• ■ >-_____r. . rsrr c.4-___τ.. ar». screening for B cells that produce immunoglobulins that bind the εecond antigen. 125. The method of claim 120, wherein one of the antigenε is a human immunoglobulin. 126. The method of claim 120, wherein the mouse comprises T-cell receptor transgenes and one of the antigenε is a human ' T-cell receptor. 127. A transgenic mouse having a genome comprising: (i) a heavy chain human transgene compriεing a variable gene εequence, a diverεity gene εequence, a joining gene εequence, and two constant region sequenceε operably linked to a εwitch donor εequence and a εwitch acceptor sequence; (ii) a light chain transgene comprising a variable gene sequence, a joining gene εequence and a conεtant gene εequence; and (iii) tranεcription regulating εequenceε operably linked to each transgene; wherein the gene sequenceε and regulatory εequenceε effect production of developmentally regulated diversity in vivo to form a plurality of human immunoglobulins. 128. The transgenic mouεe of claim 127 which produceε about a mg of human immunoglobulinε per ml serum 129. The transgenic mouse of claim 127 wherein greater than about 10% of the human immunoglobulins are IgG. 130. A transgenic mouεe of claim 127 wherein the human immunoglobulinε exhibit an affinity for preεelected antigenε of greater than about 10 "7 M _1 . 131. The tranεgenic mouεe of claim 127 which mountε an immune response when immunized against foreign antigens, which immune res p onse comτ-rises producing human lmmuno"lobulins that are capable of binding to at leaεt about 10% of antigenε bound by endogenouε immunoglobulinε of a non-tranεgenic mouεe immunized with the antigenε. 132. The transgenic mouse of claim 131 which is capable of producing greater than about 1000 different human immunoglobulins. 133. The tranεgenic mouεe of claim 131 which iε capable of producing greater than about 1000 different human IgG immunoglobulins. 134. A method of producing human immunoglobulinε compriεing immortalizing B cellε from a tranεgenic mouεe of claim 131 immunized with an antigen; and growing εelected immortalized B-cellε εecreting immunoglobulinε reactive with the antigen. 135. A human immunoglobulin (Ig) produced according to claim 134 which Ig is isolated with respect to human proteinε natively asεociated with human immunoglobulinε. 136. A human immunoglobulin of claim 135 which haε an affinity for the antigen of greater than 10 "7 M -1 . 137. A transgenic mouse having a genome compriεing a human immunoglobulin heavy chain minilocuε tranεgene, the tranεgene being capable of rearrangement and εwitching in the mouεe, whereby two or more rearranged human heavy chain iεotypeε are produced. 138. The tranεgenic mouεe of claim 137, wherein the iεotypeε are μ and 7. 139. An isolated human monoclonal antibody produced by a mouse of claim 137. 140= A human immunoglobulin specifically reactive with a lymphoid cell subεet. 141. A human immunoglobulin of claim 140, wherein the subset iε a human T-cell εubεet. 142. A human immunoglobulin of claim 141, wherein the εubεet iε autoreactive T-cellε. 143. A plasmid vector useful in cloning immunoglobulin DNA fragmentε, the vector comprising (i) an origin of replication; (ii) a copy control region; (iii) a cloning site flanked by rare reεtriction enzyme εiteε; (iv) a tranεcriptional terminator located downstream of a plasmid-derived promoter and upstream of the cloning site, whereby a tranεcript originating at the promoter is terminated upεtream of the cloning εite. 144. A vector according to claim 143, wherein the rare restriction enzyme sites are selected from Notl, Sfil, and Pa . 145. A vector according to claim 143, selected from the group consisting of pGPlb, pGPlc, pGPld, pGPlf and pGPe. 146. A composition consisting esεentially of a human VDJ rearranged gene fragment iεolated from a B cell developed within a transgenic mouεe of claim 16, 25, 108, 127, or 137. 147. A method for producing a human immunoglobulin from a transgenic rodent, the rodent having a genome comprising germline copies of human heavy and light chain immunoglobulin transgeneε, the method compriεing: immunizing the rodent with an antigen; and screening for B cells from the rodent that produce immunoglobulins that bind the antigen. 148. The method of claim 147, wherein the light chain transgene is rearranged in the germline. 149. The method of claim 148, wherein the heavy chain transgene is unrearranged in the rodent germline. 150. An isolated human immunoglobulin produced according to the method of claims 40, 53, 59, 120, or 147.

Inventors: KAY ROBERT M, LONBERG NILS, KAY, ROBERT, M., LONBERG, NILS
Applicants: GENPHARM INT, GENPHARM INTERNATIONAL, INC.
Application year: 1991
Main classification: A01H1
IPC codes: A01K67, A61K38, A61K39, C07K14, C07K16, C12N5, C12N15, C12P21