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Definitions

• 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.
• HAMA human anti-mouse antibodies
• Rodent monoclonal antibodies for the treatment of graft rejection, which involves the temporary attenuation of the patient's immune response.
• antibodies may also be useful for treating certain lymphomas that involve immunodeficiencies.
• 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
• 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 is that while the highly immunogenic mouse Fc region is eliminated, the remaining mouse Fab sequences are still immunogenic
• the CDR grafting approach uses computer modeling to generate a completely artificial antibody in which the only mouse
• Somatic mutation has also been reported in a 15 kb mouse K gene construct in hyperimmunized transgenic mice
• mice Ig gene rearrangement, though studied in tissue culture cells, has not been extensively examined in transgenic mice. Only a handful of reports have been published describing rearrangement test constructs introduced into mice [Buchini, et al. (1987), Nature, 326, 409-411 (unrearranged chicken ⁇ transgene); Goodhart, et al. (1987) , Proc. Natl. Acad. Sci. USA, 84, 4229-4233) (unrearranged rabbit k gene); and
• transgenic nonhuman animals which are capable of producing a heterologous antibody, such as a human antibody.
• B-cells 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.
• 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.
• transgenic non-human animals contain rearranged, unrearranged or a combination of rearranged and unrearranged heterologous immunoglobulin heavy and light chain transgenes in the germline of the
• heterologous heavy and light chain immunoglobulin transgenes are found in the B-cells 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.
• 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 comprises DNA encoding at least one variable gene segment, one joining gene segment and one constant region gene segment.
• the gene segments 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 species not consisting of the transgenic non-human animal.
• 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 species not consisting of the transgenic non-human animal.
• transgene is constructed such that the individual gene segments are unrearranged, i.e., not rearranged so as to encode a functional immunoglobulin light or heavy chain.
• 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.
• 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.
• such fragments also
• variable gene segments comprise a substantial portion of the variable gene segments.
• the various regulatory sequences e.g. promoters, enhancers, class switch regions, recombination signals and the like, comprise corresponding sequences derived from the heterologous DNA.
• such regulatory sequences may be incorporated into the transgene from the same or a related species of the non-human animal used in the invention.
• human immunoglobulin gene segments may be combined in a transgene with a rodent immunoglobulin enhancer sequence for use in a transgenic mouse.
• a transgenic non-human animal containing germline unrearranged light and heavy
• D-cell differentiation - is contacted with an antigen to induce production of a heterologous antibody in a secondary repertoire B-cell.
• an antigen to induce production of a heterologous antibody in a secondary repertoire B-cell.
• Such induction causes somatic mutation in the
• 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, clones having sufficient affinity and
• specificity for a particular antigen are selected to provide a source of monoclonal antibody having low immunogenicity in the species from which the immunoglobulin gene segments of the transgenes are derived.
• vectors and methods to disrupt the endogenous immunoglobulin loci in the non-human animal to be used in the invention utilize a transgene, preferably positive-negative selection vector, which is constructed such that it targets the transgene, preferably positive-negative selection vector, which is constructed such that it targets the transgene, preferably positive-negative selection vector, which is constructed such that it targets the transgene, preferably positive-negative selection vector, which is constructed such that it targets the transgene, preferably positive-negative selection vector, which is constructed such that it targets 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.
• the resultant transgenic non-human animal is substantially incapable of mounting an immunoglobulin-mediated immune response as a result of homologous integration of the vector.
• 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 are 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
• each of the V segment DNAs encodes an immunoglobulin V segment and contains at each end a cleavage recognition site of a restriction endonuclease.
• 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 transgenes in the germline of the transgenic animal.
• Such animals contain primary repertoire B-cells that express such rearranged heavy and light transgenes.
• 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 animals 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.
• 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 transgenes.
• Such transgenic animals may be obtained from any of the aforementioned transgenic animals.
• the transgenic animal containing unrearranged heavy and light transgenes, the transgenic animal containing rearranged heavy and light transgenes or the animal containing one rearranged and one unrearranged transgene in the germline of the animal each contain primary repertoire B-cells having rearranged, heterologous heavy and light immunoglobulin transgenes.
• 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.
• the transgenic non-human animal is contacted,
• 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 plasmids, 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 ).
• transcription termination is located upstream of the cloning site so that transcripts originating at the promoter are terminated upstream of the cloning site.
• the cloning site is 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
• Fig. 1 depicts the complementarity determining regions
• Fig. 2 depicts the human ⁇ 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 disruption 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 antigens.
• Fig. 10 depicts a transgene construct containing a rearranged IgM gene ligated to a 25 kb fragment that contains human ⁇ 3 and ⁇ 1 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
• Fig. 19 depicts the construction of pHIG2 (D segment containing plasmid).
• Fig. 20 depicts the fragments covering the human Jk and human Ck gene segments used in constructing a transgene of the invention.
• Fig. 21 depicts the structure of pE ⁇ .
• 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 locus of mouse.
• Figs. 24A through 24C depict the construction of a positive-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 pCOR1.
• Fig. 30 depicts the transgene constructs for pIGM1, pHCl and pHC2.
• Fig. 31 depicts the structure of P ⁇ e2.
• Fig. 32 depicts the structure of pVGEl.
• Fig. 33 depicts the assay results of human Ig expression in a pHCl transgenic mouse.
• 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 H 49.8.
• 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 specific 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
• the transgene need not activate allelic exclusion.
• the transgene comprises a functionally rearranged heavy and/or light chain immunoglobulin gene
• the second criteria of functional gene rearrangement is
• Immunoglobulins also known as antibodies, are a group of glycoproteins present in the serum and tissue fluids of all mammals. They are produced in large amounts by plasma 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 is based 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.
• the immunoglobulin heavy chain comprises two regions designated 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 segment defines the class of immunoglobulin.
• the ⁇ constant region gene segments define the IgM class of antibody whereas the use of a ⁇ , ⁇ 2, ⁇ 3 or ⁇ 4 constant region gene segment defines the IgG class of antibodies as well as the IgG subclasses IgGl through IgG4.
• variable regions of the heavy and light immunoglobulin chains together contain the antigen binding domain of the antibody. Because of the need for diversity 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 families of specific variable region gene
• variable regions such families comprise variable (V) gene segments and joining (J) gene segments.
• V variable gene segments
• J joining gene segments
• 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 segment and one J gene segment, each
• the process for generating DNA encoding the heavy and light chain immunoglobulin genes occurs primarily in developing B-cells.
• V, D, J and constant (C) gene segments 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 segments in the precursors of primary repertoire B-cells.
• all of the gene segments for a heavy or light chain are located in relatively close proximity on a single chromosome.
• genomic DNA prior to recombination of the various immunoglobulin gene segments is referred to herein as
• RSS's recombination signal sequences
• the heptamer comprises the sequence CACAGTG or its analogue followed by a spacer of unconserved sequence and then a nonamer having the sequence 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
• the heptameric and nonameric sequences following a V L , V H or D segment are complementary to those preceding the J L , D or J H 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.
• variable recombination between the V and J segments 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.
• variation in the light chain typically occurs within the last codon of the V gene segment and the first 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.
• nucleotides may be inserted between the D and J H and between the V R and D gene segments which are not encoded by genomic DNA.
• the addition of these nucleotides is known as N-region diversity.
• RNA transcript which upon appropriate RNA splicing results in an mRNA which encodes a full length heavy or light immunoglobulin chain.
• 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 association with each other form an antibody molecule.
• variable region gene segments and variable recombination which may occur during such joining, is the production of a primary antibody repertoire.
• each B-cell which has
• allelic exclusion The process by which diploid B-cells maintain such mono-specificity is termed allelic exclusion.
• B-cells make up this initial response: 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 plasma cells in response to certain antigens.
• the other B-cells respond to initial exposure to antigen by entering a T-cell dependent maturation pathway. It is during this T-cell dependent
• B-cells that a second level of diversity is generated by a process termed somatic mutation (sometimes also referred to as hypermutation).
• somatic mutation sometimes also referred to as hypermutation.
• MHC major histocompatibility complex
• 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
• 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 (CDR1, CDR2 and CDR3) also referred tc as hypervariable regions 1, 2 and 3.
• CDR1, CDR2 and CDR3 also referred tc as hypervariable regions 1, 2 and 3.
• the CDRl and CDR2 are located within the variable gene segment whereas the CDR3 is largely the result of recombination between V and J gene segments 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 FR1, FR2, FR3 and FR4. See Fig. 1.
• 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 CDR1, 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 in one aspect of the invention are produced by introducing at least one of the immunoglobulin transgenes of the invention (discussed
• 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
• 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
• mice any non-human mammal which is capable of
• Such animals include non-human primates, such as 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.
• antibody refers to a glycoprotein comprising at least two identical light
• Each of the heavy and light polypeptide chains 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.
• a heterologous antibody is 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 consisting of the transgenic non-human animal.
• gene segments may be readily identified, e.g. by hybridization or DNA sequencing, as being from a species of organism other than the transgenic animal.
• 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 segments in the mouse genome. They may be readily detected in unrearranged form in the germ line and somatic cells not consisting of B-cells and in rearranged form in B-cells.
• the transgenes comprise rearranged heavy and/or light
• transgenes corresponding to functionally rearranged VDJ or VJ segments contain immunoglobulin DNA sequences which are also clearly distinguishable from the endogenous immunoglobulin gene segments in the mouse.
• human immunoglobulin amino acid sequences may be detected in the transgenic non-human animals of the invention with antibodies specific for immunoglobulin epitopes encoded by human immunoglobulin gene segments.
• Transgenic B-cells containing unrearranged transgenes from human or other species functionally recombine the
• substantially 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.
• isolated substantially pure and substantially homogenous are used interchangeably herein and describe a polypeptide protein which has been separated from components which naturally accompany it.
• a monomeric protein is substantially pure when at least about 60 to 75% of a sample exhibits a single polypeptide backbone. Minor variants or chemical modifications typically share the same polypeptide sequence.
• a substantially pure protein will typically comprise 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 as polyacrylamide gel electrophoresis of a protein sample,
• polyacrylamide gel upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized.
• a polypeptide is substantially free of naturally-associated components when it is separated from the native contaminants which- accompany it in its natural state.
• a polypeptide which is synthesized in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally-associated components.
• 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 is introduced.
• 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 segments from a species not consisting of the non-human animal into which said light chain transgene is introduced.
• Such heavy and light chain transgenes in this aspect of the invention contain the above-identified gene segments in an unrearranged form.
• interposed between the V, D and J segments in the heavy chain transgene and between the V and J segments on the light chain transgene are appropriate.
• transgenes also include appropriate RNA splicing signals to join a constant region gene segment with the VJ or VDJ
• 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.
• promoters In addition to promoters, other regulatory sequences which function primarily in B-lineage cells are used. Thus, for example, a light chain enhancer sequence situated
• 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
• regulatory gene segments 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
• 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 species 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
• transgenes will have an amino acid sequence and overall
• gene segments are derived from human beings.
• the transgenic constructs are derived from human beings.
• non-human animals harboring such heavy and light transgenes are capable of mounting an Ig-mediated immune response to a
• B-cells are produced within such an animal which are capable of producing heterologous human antibody.
• an appropriate monoclonal antibody e.g. a hybridoma
• a source of therapeutic human monoclonal antibody is provided.
• Such human Mabs have significantly reduced
• monoclonal antibodies for use in the veterinary sciences.
• the treatment of livestock and domestic animals with species-related monoclonal antibodies is also contemplated by the invention.
• Such antibodies may be similarly generated by using transgenes containing immunoglobulin gene segments from species such as bovine, ovine, porcine, equine, canine, feline and the like.
• ⁇ or ⁇ constant regions are largely determined by alternate splicing, permitting IgM and IgD to be coexpressed in a single cell.
• the other heavy chain isotypes ( ⁇ , ⁇ , and ⁇ ) are only expressed natively after a gene
• This gene rearrangement process deletes the C ⁇ and C ⁇ exons.
• This gene rearrangement process occurs by recombination between so called switch segments located immediately upstream of each heavy chain gene (except ⁇ ) .
• the individual switch segments are between 2 and 10 kb in length, and consist primarily of short repeated sequences. The exact point of recombination differs for individual class switching events.
• the ability of a transgene construction to switch 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 ⁇ heavy chain gene construct and found that in four independent mouse lines, a high proportion of the transgenic B-cells expressed the
• transgene-encoded variable region associated with IgG rather than IgM.
• isotype switching appears to have taken place between the transgene and the endogenous 7 constant region on another chromosome.
• switch sequence thus refers to those DNA sequences responsible for switch recombination.
• a "switch donor" sequence typically a ⁇ switch 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., ⁇ , ⁇ , etc.). As there is no specific site where recombination always occurs, the final gene sequence will typically not be predictable from the construct.
• the switch (S) region of the ⁇ gene, S ⁇ is located about 1 to 2 kb 5' to the coding sequence and is composed of numerous tandem repeats of sequences of the form
• GCGCT GCGCT n (GGGGT), where n is usually 2 to 5 but can range as high as 17.
• All the sequenced S regions include numerous occurrences of the pentamers GAGCT and GGGGT that are the basic repeated elements of the S ⁇ gene (T. Nikaido, et al. (1982): J. Biol. Chem., 257:7322-7329); in the other S regions these pentamers are not precisely tandemly repeated as in S ⁇ , but instead are embedded in larger repeat units.
• the S ⁇ 1 region has an additional higher-order structure: two direct repeat sequences flank each of two clusters of 49-bp tandem repeats.
• the switch machinery can apparently accommodate different alignments of the repeated homologous regions of germline S precursors and then join the sequences at different positions within the alignment.
• the switch machinery can apparently accommodate different alignments of the repeated homologous regions of germline S precursors and then join the sequences at different positions within the alignment.
• transgene constructs that are intended to undergo class switching should include all of the cis-acting sequences necessary to regulate these sterile transcripts.
• transgenic mice ⁇ and ⁇
• switching in transgenic mice involves the inclusion of the 400 bp direct repeat sequences that flank the human ⁇ gene (Yasui et al. 1989 Eur. J. Immunol., 19, 1399).
• Monoclonal antibodies can be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol., 6:511-519 (1976)).
• Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single 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 cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Various techniques useful in these arts are discussed, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York (1988) including: immunization of animals to produce immunoglobulins; production of monoclonal antibodies; labeling immunoglobulins for use as probes; immunoaffinity purification; and immunoassays.
• each locus consists 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 consist of three exons: a signal peptide exon, a variable region exon and a constant region exon.
• the rearranged heavy chain gene is somewhat more complex. It consists of a signal peptide exon, a variable region exon and a tandem array of multi-domain
• constant region regions each of which is encoded by several exons.
• Each of the constant region genes encode the constant portion of a different class of immunoglobulins.
• V region proximal constant regions are deleted leading to the expression of new heavy chain classes.
• alternative patterns of RNA, splicing give rise to both transmembrane and secreted immunoglobulins.
• the K light chain locus is spread out over three clusters on chromosome 2 (Fig. 3).
• the first two clusters covering 850 and 250 kb respectively contain only variable region gene segments.
• the third cluster covering about 1 Mb, contains approximately 40 V gene segments upstream of a cluster of 5 J segments followed by a single constant region gene segment.
• a total of 84 V gene segments have been identified, and approximately half of these are thought to be pseudogenes (Zachau (1989) in Immunoglobulin Genes, Academic Press, T.
• kde Approximately 25 kb downstream of the CK region there is a "k deleting element" (kde).
• the kde sequence recombines with upstream sequences, causing the deletion of the k constant region in ⁇ light chain expressing B-cells. This leads to isotopic exclusion in cells that successfully rearrange both K. and ⁇ genes.
• the human heavy chain locus is the largest and most diverse. It consists of approximately 200 V gene segments 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 families on the basis 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 4
• immunoglobulin heavy and light chain transgenes comprise unrearranged genomic DNA from humans.
• a preferred transgene comprises a Notl fragment having a length between 670 to 830 kb. The length of this fragment is ambiguous because the 3' restriction site has not been accurately mapped. It is known, however, to reside between the ⁇ 1 and ⁇ 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 ⁇ , ⁇ , ⁇ 3, ⁇ 1 and ⁇ 1 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 classes required for B-cell development as well as a large enough repertoire of variable regions to trigger a secondary response for most antigens. 2.
• a genomic fragment containing all of the necessary gene segments and regulatory sequences from a human light chain locus may be similarly constructed. Such a construct is described in the Examples.
• the 670-830 kb Notl fragment from the human immunoglobulin heavy chain locus may be formed in vivo in the non-human animal during transgenesis.
• Such in vivo transgene construction is produced by introducing two or more overlapping DNA fragments into an embryonic nucleus of the non-human animal.
• overlapping portions of the DNA fragments have DNA sequences which are substantially homologous.
• the overlapping DNA fragments homologously recombined in proper orientation to form the 670-830 kb Notl heavy chain fragment.
• in vivo transgene construction can be used to form any number of immunoglobulin transgenes which because of their size are otherwise difficult, or impossible, to make or manipulate by present technology.
• 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.
• in vivo homologous recombination may also be utilized to form "mini-locus" transgenes as described in the Examples.
• each overlapping DNA fragment In the preferred embodiments utilizing in vivo transgene construction, each overlapping DNA fragment
• portions of the DNA fragments preferably comprise about 500 bp to about 2000 bp, most preferably 1.0 kb to 2.0 kb. Homologous recombination of overlapping DNA fragments to form transgenes in vivo is further described in commonly assigned U.S. Patent Application entitled "Intracellular Generation of DNA by
• immunoglobulin minilocus refers to a DNA sequence (which may be within a longer
• V functional variable
• J functional joining
• C functional constant
• D functional diversity
• a light chain minilocus transgene will be at least 25 kb in length, typically 50 to 60 kb.
• a heavy chain will be at least 25 kb in length, typically 50 to 60 kb.
• transgene will typically be about 70 to 80 kb in length, preferably at least about 60 kb with two constant regions operably linked to switch regions, versus at least about 30 kb with a single constant region and incomplete switch regions.
• the individual elements of the minilocus are preferably in the germline configuration and capable of
• immunoglobulin heavy and light chain transgenes comprise one or more of each of the V, D, J and C gene segments. At least one of each appropriate type gene segment is incorporated into the
• the transgene contain at least one ⁇ gene segment and at least one other constant region gene segment, more preferably a ⁇ gene segment, and most preferably ⁇ 3 or ⁇ 1. This preference is to allow for class switching between IgM and IgG forms of the encoded
• immunoglobulin Other constant region gene segments 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 segments and three pseudo genes clustered in a 3 kb stretch of DNA. Given its relatively compact size and the ability to isolate these segments together with the ⁇ gene and the 5' portion of the ⁇ gene on a single 23 kb SFil/Spel fragment (Sado, et al. (1988), Biochem. Bioshys. Res. Comm., 154, 264271), it is preferred that all of the J region gene segments be used in the mini-locus construct.
• this fragment spans the region between the ⁇ and ⁇ genes, it is likely to contain all of the 3' cis-linked regulatory elements required for ⁇ expression. Furthermore, because this fragment includes the entire J region, it contains the heavy chain enhancer and the ⁇ switch region (Mills, et al. (1983), Nature, 306, 809; Yancopoulos and Alt (1986), Ann. Rev.
• 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 consists of 4 or 5 homologous 9 kb subregions, linked in tandem (Siebenlist, et al. (1981),
• Each subregion contains up to 10 individual D segments. Some of these segments have been mapped and are shown in Fig. 4.
• Two different strategies are used to generate a mini-locus D region. The first strategy involves using only those D segments located in a short contiguous stretch of DNA that includes one or two of the repeated D subregions.
• a candidate is a single 15 kb fragment that contains 12 individual D segments. This piece of DNA consists of 2 contiguous EcoRI fragments and has been completely
• 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 construct the heavy chain minilocus
• transgene A 10-15 kb piece of DNA containing one or two unrearranged V segments together with flanking sequences is isolated. A clone containing such DNA is selected using a probe generated from unique 5' sequences determined from the transcribed V region of a characterized human hybridoma such as that which produces anti-cytomegalovirus antibody (Newkirk et al. (1988) J. Clin. Invest., 81, 1511-1518). The 5'
• V segment 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.
• a V segment that is known to be incorporated in an antibody against a known antigen not only insures that this V segment is 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, discussed previously, to produce a mini-locus heavy chain trans ⁇ ene.
• a large, contiguous stretch of DNA containing multiple V region segments is isolated from a YAC library. Different sized pieces of DNA, containing different numbers of V region segments, are tested for their ability to provide a human antibody repertoire in the minilocus transgene construct. It is also possible to build one large fragment from several non-contiguous V segment containing fragments using YAC vectors (Murray and Szostak (1983), Nature, 305, 189-193), F factor-based plasmids (O'Conner, et al. (1989),
• V region repertoire (described hereinafter) may be used.
• a minilocus light chain transgene may be similarly constructed from the human ⁇ or k immunoglobulin locus.
• Construction of a k light chain mini-locus is very similar to construction of the heavy chain mini-locus, except that it is much simpler because of its smaller size and lower complexity.
• the human k locus contains only one constant region segment; and this segment, together with 5' and 3' enhancers, and all 5 of the functional J segments, 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.
• an immunoglobulin heavy chain minilocus transgene construct e.g., of about 75 kb, encoding V, D, J and constant region sequences
• V, D, J and constant region sequences 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 constant region sequence, a D and J and constant region sequence or a constant region sequence, with each sequence being substantially homologous to human gene sequences.
• the sequences are operably linked to transcription regulatory sequences and are capable of undergoing rearrangement.
• constant region sequences e.g., ⁇ and 7
• switch regions switch recombination also occurs.
• An exemplary light chain transgene 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 regions, each fragment comprising either a V region sequence, J and constant region sequence or a constant region sequence.
• V gene segments generally far surpasses the number of corresponding gene segments for the D, J and C region gene segments.
• V region gene segments are utilized by a particular organism, such as the human being, when mounting an immunoglobulin-mediated immune response.
• a single V gene segment when combining with the J or DJ gene segments is capable of providing sufficient diversity at CDR3 for the generation of a primary repertoire which upon somatic mutation is able to provide further diversity
• variable region e.g. at CDR1 and CDR2 for the production of high affinity antibodies.
• methods and vectors are provided for determining which V gene segments are commonly utilized by an organism during an immune response. This method is based on determining which V segments are found in cDNA synthesized from B-cell polyA+ RNA. Such methods and vectors may also be used to facilitate the construction of a synthetic V segment repertoire. The outline of this strategy for identifying heavy chain V segments and for generating a synthetic V segment repertoire is depicted in Fig.s 5 and 6. It is similarly applicable for identifving light chain V segments with
• 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 sequences. This fragment is cloned into a plasmid such as pGPl or pGP2
• restriction sites, "x" and “y” (generally each about 6
• the second step is the synthesis of four sets of oligonucleotide primers, P1 through P4.
• P1 and P2 are
• non-unique oligomers having approximately 50 nucleotides each which are used to prime double stranded cDNA synthesis.
• P1 starts (going 5' to 3') with about 20 nucleotides of sequence homologous to the antisense strand of the recombination signal sequence in pVHl (including the recognition sequence of
• restriction enzyme "y" continues with approximately 30 nucleotides of antisense sequence hybridizing with about the last 30 nucleotides of the VH framework region 3 (FR3). Randor, bases are incorporated over about the last 30 nucleotides so as to generate a set of primers that hybridize with all of the different VH families.
• the second oligonucleotide, P2 is 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 segments.
• Oligonucleotides P3 and P4 are homologous to about the first 20 nucleotides of P1 and P2 respectively. These oligos 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 polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• primers P1 and P2 which are capable of hybridizing to and priming the synthesis of the variable segments of the heavy or light immunoglobulin locus may be readily determined by one skilled in the art.
• the nucleotide sequence for a number of human VH genes have been published, see e.g. Berman, J.E., et al.
• primers P1 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 various V segments are also conserved in the 3' portion of the P1 and P2 primers. For those nucleotide positions wherein variation is observed amongst variable segments, such nucleotide
• positions in the corresponding P1 and P2 primers are similarly varied to provide P1 and P2 primers which comprise a pool of primers which are capable of hybridizing to different VH or VL segments.
• the next step is to use these oligonucleotide primers to generate a library of human heavy-chain V-region cDNA sequences in the vector pHVl.
• P1 is used to prime first strand cDNA synthesis from human B-cell polyA+ RNA.
• the RNA is base hydrolyzed, and second strand synthesis primed with P2.
• Full length, double stranded cDNA is then purified on an acrylamide gel, electroeluted, and used as template for polymerase chain reaction (PCR) amplification using oligonucleotide primers P3 and P4.
• PCR polymerase chain reaction
• cDNA is first synthesized by PCR
• 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 offers three advantages over a conventional genomic library of variable segments.
• this library contains no pseudogenes, while a conventional library would contain up to 50% pseudogene
• the synthetic 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 because of differential expression.
• the two sources of bias are: (i) differential rates of V segment recombination, and (ii) differential selection of V segment expressing B-cell clones.
• the first source of bias is dealt with in two ways. First, fetal tissue is avoided as a source of B-cell RNA, as the bias is most pronounced in the fetal immunoglobulin repertoire. Second, the semi-random primers, P1 and P2, are divided into pools, each of which selectively cross-hybridizes with different V segment families. These primers are then used to generate 4 to 6 separate libraries, thus insuring that all of the V region families are
• RNA that includes the minimum fraction of antigen selected B-cells is used. Lymph nodes and spleen are avoided.
• Adult bone marrow is one source of unselected
• B-cells may contain a high proportion of
• RNA transcribed pseudogene sequences from pre-B-ceils.
• Another source of RNA is whole blood.
• Ninety percent of circulating B-cells are immature ⁇ or ⁇ , ⁇ expressing cells, and are recent bone marrow immigrants.
• the level of antigen selected IgG expressing cells can vary depending on the immune state of the individual. Therefore, isolated polyA+ RNA is checked for selected B-cell sequences by northern blot hybridization with ⁇ specific probes. If it is more practical to use spleen RNA, and if this RNA contains a high fraction of IgG sequences, a second approach is used to minimize selection bias.
• the first strand of cDNA synthesis is primed with about a 40 nucleotide constant-region exon 2 primer that is specific for IgM
• Second strand syntheses is then primed with P2, and a third round of synthesis primed with P1.
• the cDNA from this third round of synthesis provides the template for PCR amplification using P3 and P4.
• 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 as well as differential amplification by PCR methods.
• V segment characterization of the V segment library provides information as to the frequency and distribution of V segment utilization in a particular organism and as a consequence, the
• one or more predominant V. gene segments may be used in the above described mini-locus transgene construct. Further, selected clones from such a library may be used to identify genomic fragments containing frequently used V segments to facilitate identification of genomic fragments containing a particular desired V segment.
• V segment repertoire may be constructed by concatenation of the library sequences.
• 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
• 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 kb 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 segment repertoire. Inserts from the random sequence library are released with the enzymes "w” and “z”, and purified away from vector sequences. Inserts from the synthetic V segment library are isolated by cutting with "w” and "z”. Before purifying the V segment inserts, this DNA is treated with calf-intestinal phosphatase, to prevent self ligation. The V segment inserts are then ligated together with the random inserts to generate an alternating tandem array comprising a synthetic V segment repertoire. This ligation mixture is s.ize selected on a sucrose gradient, and the 50-100 kb fraction microinjected together with, for example, a D-J-constant mini-locus
• the synthetic V segment repertoire may be combined with a D-J-C minilocus to form a heavy chain transgene.
• a synthetic light chain immunoglobulin segment repertoire may be similarly constructed using appropriate primers for the light chain locus. Functional Disruption of
• immunoglobulin heavy and light transgenes is expected to have a dominant effect by suppressing the rearrangement of the
• endogenous immunoglobulin genes in the transgenic nonhuman animal.
• another way to generate a nonhuman that is devoid of endogenous antibodies is by mutating the endogenous immunoglobulin loci. Using embryonic stem cell technology and homologous recombination, the endogenous immunoglobulin
• this technology involves the inactivation of a gene, by homologous recombination, in a pluripotent cell line that is capable of differentiating into germ cell tissue.
• immunoglobulin gene is introduced into the nuclei of embryonic stem cells. In a portion of the cells, the introduced DNA recombines with the endogenous copy of the mouse gene,
• the mouse ⁇ locus contributes to only 5% of the immunoglobulins, inactivation of the heavy chain and/or ⁇ -light chain loci is sufficient. There are three ways to disrupt each of these loci, deletion of the J region, deletion of the J-C intron enhancer, and disruption of constant region coding sequences by the introduction of a stop codon. The last option is the most straightforward, in terms of DNA construct design. Elimination of the ⁇ gene disrupts B-cell maturation thereby preventing class switching to any of the functional heavy chain segments. The strategy for knocking out these loci is outlined below.
• neomycin resistance gene 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 cells. Therefore, neo can be used as 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 selection marker against random insertion events
• 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 screened 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.
• the heavy-chain locus is knocked out by disrupting the coding region of the ⁇ gene.
• This approach involves the same 15 kb Kpnl fragment used in the previous approach.
• the 1.1 kb insert from pMCIneo is inserted at a unique BamHI site in exon II, and the HSV tk gene added to the 3' Kpnl end. Double crossover events on either side of the neo insert, that eliminate the tk gene, are then selected for.
• PCR primers are derived from neo sequences and the other from mouse sequences outside of the targeting vector.
• the functional disruption of the mouse immunoglobulin loci is presented in the Examples.
• a premise underlying the previously discussed 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 locus.
• somatic hypermutation should be capable of delivering a high affinity antibody against that antigen.
• the antibody repertoire is determined by sequence diversity at CDR1, 2, and 3.
• the diversity at CDR1, 2, and 3 that gives rise to a complete antibody repertoire comes from three sources: recombinational diversity, junctional diversity, and somatic mutation. Recombinational diversity at CDR1, 2, and 3.
• CDR1 and 2 comes from the choice of different V segments containing different CDRl and 2 sequences.
• Recombinational diversity at CDR 3 comes from the choice of different D and J segments.
• Junctional diversity contributes only tc CDR3 diversity, while somatic mutation, acting across the entire V region, contributes to diversity at all three complimentarity determining regions. Recombinational and junctional diversity together constitute the diversity of the primary repertoire (Fig. 1).
• VDJ joining generates a set of IgM expressing primary B-cells.
• CDRl and 2 diversity is 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 antibodies for a large number of different antigens. Thus, methods and transgenic animals are provided for generating a broad antibody repertoire without primary diversity. Such diversity relies on somatic mutation for the generation of antibody diversity.
• a transgenic non-human animal such as a mouse
• 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
• transgene constructs are easy to generate; and second, the rearranged transgenes are capable of allelicly and isotypically excluding the rearrangement of the endogenous mouse genes, thus making it unnecessary to eliminate those genes by homologous recombination as previously described.
• the first step in this embodiment of the invention is the isolation of rearranged heavy and light chain genes from a human hybridoma that expresses 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.
• human hybridomas in existence, including several that react with promising antigens such as tetanus toxoid, pseudomonas, or gram negative bacteria (reviewed by James and Bourla (1987), J. Immunol.
• 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, is isolated on a second DNA fragment (about 20 kb). Each of these fragments are pieced together from clones
• the heavy chain construct (Fig. 10) consists of the 20 kb hybridoma fragment, containing the rearranged IgM gene, ligated to a 25 kb fragment that contains the human ⁇ 3 and ⁇ 1 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 construct consists of the intact 20 kb piece of DNA contaninng the rearranged k chain and 3' enhancer. These two constructs are coinjected so that they are integrated at a single site in the mouse genome.
• Transgenic mice are tested by Northern blot analysis for expression of the transgene mRNA. FACS analysis 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
• mice are tested for their ability to respond to a number of different antigens 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 antibodies directed against individual antigens.
• that antigen preferably is first coinjected together with the antigen associated with the hybridoma from which the genes were
• This hybridoma associated antigen is referred to as the co-antigen (sometimes as a second antigen), and the new antigen simply as the antigen (or first antigen). If possible, the second antigen is chemically cross-linked to the first antigen prior to injection. This causes the first antigen to be internalized and presented by the primary transgene
• a typical immunization schedule is as follows.
• Day l Mice are injected ip with first antigen mixed with, or cross-linked to, second antigen in complete Freunds adjuvant.
• Day 14 first antigen (without second antigen) is injected ip in incomplete Freunds adjuvant.
• Day 35 repeat injection with first antigen in incomplete Freunds.
• Day 45 Test for antibody response by ELISA on tail blood samples.
• Day 56 repeat injection of good responders with antigen in incomplete Freunds.
• Day 59 Fuse spleens of good responders.
• the antigen recognized by the hybridoma from which the Ig genes were isolated is used as an immunogen.
• New transgenic hybridomas are then isolated from the immunized animal that express somatically mutated versions of the original antibody. These new antibodies will have a higher affinity for the original antigen.
• This antibody "sharpening" procedure can also be applied to antibody genes generated by CDR grafting (E.P. Pub. No. 239400, published Sept. 30, 1987) or isolated from
• transgenic animals contain at least one rearranged and at least one unrearranged immunoglobulin transgene are produced by utilizing any of the aforementioned unrearranged and rearranged transgenes in combination to provide heavy and light transgenes in the transgenic animal.
• the unrearranged transgene may comprise a heavy or light genomic or mini-locus transgene construct with the rearranged transgene comprising an appropriate rearranged transgene.
• the appropriate other transgene is a fully rearranged heavy chain transgene.
• the rearranged transgene comprise a rearranged immunoglobulin light chain transgene and that the unrearranged transgene comprise an immunoglobulin heavy chain genomic or mini-locus transgene, most preferably an unrearranged heavy chain transgene with associated A and y constant regions.
• the combination of rearranged and unrearranged transgene provides an intermediate level of diversity within the primary repertoire B-cells.
• the primary diversity at CD1 , CD2 and CD3 in the rearranged transgene is fixed in the primary repertoire B-cell, the primary diversity at the CDR1, CDR2 and CDR3 produced by the rearrangement of the unrearranged transgene provides a population of primary
• nucleic acids the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98 to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective 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 substantially pure form.
• a nucleic acid is "isolated” or “rendered substantially 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 electrophoresis 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).
• nucleic acid compositions of the present invention while often in a native sequence (except for
• modified restriction sites and the like from either cDNA, genomic or mixtures may be mutated, thereof in accordance with standard techniques to provide gene sequences.
• these mutations may affect amino acid sequence as desired.
• DNA sequences substantially homologous to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where
• a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
• a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
• operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
• operably linked indicates that the sequences are capable of effecting switch recombination.
• a preferred embodiment of the invention is 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. Animals are bred to homozygosity for each of these three traits.
• Such animals have the following genotype: a single copy (per haploid set of chromosomes) 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 segments (described in Examples 9 and 12).
• Such animals are bred with mice that are homozygous for the deletion of the JH segments (Examples 9 and 12) to produce offspring that are homozygous for the JH deletion and hemizygous for the human heavy and light chain constructs.
• 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 monospecific with regards 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 as described in Example 9 and 12.
• 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 isotypically exclude the rearrangement of the endogenous mouse k and lambda chain genes in a significant fraction of B-cells.
• the total immunoglobulin levels will range from about 0.1 to 10 mg/ml of serum, preferably 0.5 to 5 mg/ml, ideally at least about 1.0 mg/ml.
• the adult mouse ratio of serum IgG to IgM is preferably about 10:1.
• 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
• immunoglobulins will typically recognize about one-half or more of highly antigenic proteins, including, but not limited to: pigeon cytochrome C, chicken lysozyme, pokeweed mitogen, bovine serum albumin, keyhole limpit
• immunoglobulins will exhibit an affinity for preselected antigens of at least about 10 -7 M- 1 , preferably 10 -8 M -1 to 10 -9 M -1 or greater.
• transgenic animals containing an unrearranged heavy and rearranged light immunoglobulin transgene are defined by the disclosure herein and more particularly by the transgenes described in the Examples.
• Four categories of transgenic animal may be defined: I. Transgenic animals containing an unrearranged heavy and rearranged light immunoglobulin transgene.
• Example 5 H, I or J or 14, 17 or 21 animal bred with Example 7 or 16 animal.
• Example 5 H, I or J or 14, 17 or 21 animal bred with Example 6(B, C or D) or 16 animal.
• Example 6(B) or 15 construct coinjected with Example 8 or 15 construct Example 6(B) or 15 construct coinjected with Example 8 or 15 construct.
• Transgenic mice are derived according to Hogan, et al., "Manipulating the Mouse Embryo: A Laboratory Manual”.
• 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; Zjilstra, et al. (1989), Nature, 342, 435-438; and Schwartzberg, P., et al. (1989), Science, 246, 799-803).
• Oligonucleotides are synthesized on an Applied Bio Systems oligonucleotide synthesizer according to specifications 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).
• Nuclei are isolated from fresh human placental tissue as described by Marzluff, W.F., et al. (1985), "Transcription and Translation: A Practical Approach", B.D. Hammes and
• the isolated nuclei (or PBS washed human spermatocytes) are
• Fractions enriched for the Notl fragment are assayed by Southern hybridization to detect one or more of the sequences encoded by this fragment.
• sequences include the heavy chain D segments, J segments, ⁇ and 71 constant regions together with representatives 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 are pooled and cloned into the Notl site of the vector pYACNN in Yeast cells.
• Plasmid 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'.
• the cloned Notl insert is isolated from high molecular weight yeast DNA by pulse 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 embryos previously described.
• a 110 kb Spel fragment of human genomic DNA containing VH6, D segments, J segments, the ⁇ constant region and part of the 7 constant region 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 segments.) The two fragments are coinjected into the nucleus of a mouse single cell embryo as described in Example 1.
• DNA is isolated from resultant transgenic animals and those animals found to be containing both transgenes by Southern blot hybridization
• a 450 kb Xhol to Notl fragment that includes all of Ck, the 3' enhancer, all J segments, and at least five
• V segments (a) is isolated and microinjected into the nucleus of single cell embryos as described in Example 1.
• a 750 kb Mlul to Notl fragment that includes all of the above plus at least 20 more V segments (b) is isolated 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 BssHII fragment (c) have sequence overlap defined by the BssHII and Xhol restriction sites shown in Fig. 11. Homologous 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).
• pBR322 is digested with EcoRI and Styl and ligated with the following oligonucleotides to generate pGPl which contains a 147 base pair insert containing the restriction sites shown in Fig. 13. The general overlapping of these oligos is also shown in Fig. 13.
• oligonucleotides are:
• AAA AGC CCG CTC ATT AGG CGG GCT - 3'
• This plasmid contains a large polylinker flanked by rare cutting Notl sites for building large inserts that can be isolated from vector sequences for microinjection.
• the plasmid is based on pBR322 which is relatively low copy compared to the pUC based plasmids (pGP1 retains the pBR322 copy number control region near the origin of replication). Low copy number reduces the potential toxicity of insert sequences.
• pGPl contains a strong transcription terminator sequence derived from trpA (Christie, G.E., et al. (1981),
• Plasmid pGP2 is derived from pGP1 to introduce an additional restriction site (Sfil) in the polylinker.
• pGP1 is digested with Mlul and Spel to cut the recognition sequences in the polylinker portion of the plasmid.
• a 3' pGP2 is identical to pGP1 except that it contains an additional Sfi I site located between the Mlul and Spel sites. This allows inserts to be completely excised with Sfil as well as with Notl.
• the heavy chain region 3' enhancer described by S. Pettersson, et al. (1990), Nature, 344, 165-168) is isolated and cloned.
• the rat IGH 3' enhancer sequence is PCR amplified by using the following oligonucleotides:
• 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').
• a 6.3 kb BamHI/Hindlll fragment that includes all human J segments is isolated from human genomic DNA library using the oligonucleotide GGA CTG TGT CCC TGT GTG ATG CTT TTG ATG TCT GGG GCC AAG.
• pGP1 is digested with BamHI and Bglll followed by treatment with calf intestinal alkaline phosphatase.
• Fragments (a) and (b) from Fig. 14 are cloned in the digested pGP1.
• a clone is then isolated which is oriented such that 5' BamHI site is destroyed by BamHI/Bgl fusion. It is identified as pMU (see Fig. 15).
• pMU is digested with BamHI and fragment (c) from Fig. 14 is inserted. The orientation is checked with Hindlll digest.
• the resultant plasmid pHIGl (Fig. 15) contains an 18 kb insert encoding J and C ⁇ segments. D. Cloning of C ⁇ Region
• pGP1 is digested with BamHI and Hindlll is followed by treatment with calf intestinal alkaline phosphatase (Fig. 14).
• pHIGl contains J segments, switch and ⁇ sequences in its 18 kb insert with an Sfil 3' site and a Spel
• pCON1 5' site in a polylinker flanked by Notl sites, will be used for rearranged VDJ segments.
• pCON1 is identical except that it lacks the J region and contains only a 12 kb insert. The use of pCON1 in the construction of fragment containing rearranged
• Phage clones containing the ⁇ -1 region are identified and isolated using the following oligonucleotide which is specific for the third exon of 7-l (CH3).
• Fig. 16 is 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 is confirmed by BamHI/Spel digestion.
• pHIGl contains human J segments and the C ⁇ constant region exons.
• pHIGl was digested with Sfil (Fig. 15).
• the plasmid pREG2 was also digested with Sfil to produce a 13.5 kb insert containing human C ⁇ exons and the rat 3' enhancer sequence.
• a second plasmid encoding human C ⁇ and human C ⁇ 1 without J segments is constructed by digesting pCON1 with Sfil and combining that with the Sfil fragment containing the human C ⁇ region and the rat 3' enhancer by digesting pREG2 with Sfil.
• the resultant plasmid, pCON (Fig. 17) contains a 26 kb
• Notl/Spel insert containing human C ⁇ , human 7I and the rat 3' enhancer sequence containing human C ⁇ , human 7I and the rat 3' enhancer sequence.
• Fig. 18 Phage clones from the human genomic library containing D segments are identified and isolated using probes specific for diversity region sequences (Y. Ichihara, et al. (1988), EMBO J., 7 , 4141-4150). The following
• oligonucleotides are used: DXP1: 5' - TGG TAT TAC TAT GGT TCG GGG AGT TAT TAT
• DXP4 5' - GCC TGA AAT GGA GCC TCA GGG CAC AGT GGG
• DN4 5' - GCA GGG AGG ACA TGT TTA GGA TCT GAG GCC
• a 5.2 kb Xhol fragment (fragment (b) in Fig. 18) containing DLR1, DXP1, DXP'1, and DA1 is isolated from a phage clone identified with oligo DXP1.
• a 3.2 kb Xbal fragment (fragment (c) in Fig. 18) containing DXP4 , DA4 and DK4 is isolated from a phage clone identified with oligo DXP4.
• Fragments (b), (c) and (d) from Fig. 18 are combined and cloned into the Xbal/Xhol site 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 pGP1 digested with Xhol and Xbal. The
• This plasmid contains diversity segments cloned into the polylinker with a unique 5' Sfil site and unique 3' Spel site. The entire polylinker is flanked by Notl sites.
• a restriction map of the unrearranged V segment is determined to identify unique restriction sites 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 sequences will include promoter and other regulatory sequences whereas the 3' flanking sequence provides recombination sequences necessary for V-DJ joining.
• This approximately 3.0 kb V segment insert is cloned into the polylinker of pGB2 to form pVH1.
• pHIG5 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 plus the size of the V segment insert.
• pHIG5 The insert from pHIG5 is excised by digestion with Notl and Spel and isolated.
• pHIG3 ' which contains J, C ⁇ and C ⁇ 1 segments is digested with Spel and Notl and the 3' kb fragment containing such sequences and the rat 3' enhancer sequence is isolated. These two fragments are combined and ligated into Notl digested pGP1 to produce pHIG which contains insert encoding a V segment, nine D segments, six functional J segments, C ⁇ , C ⁇ and the rat 3' enhancer.
• the size of this insert is approximately 43 kb plus the size of the V segment insert.
• the insert of pHIG is approximately 43 to 45 kb when a single V segment is employed. This insert size is at or near the limit of that which may be readily cloned into plasmid vectors.
• the following describes in vivo homologous recombination of
• the resultant is plasmid designated pHIG5 '0 (overlap).
• the insert contained in this Plasmid contains human V D and J segments. When the single V segment from pVH1 is used, the size of this insert is approximately 17 kb plus 2 kb.
• This insert is isolated and combined with the insert from pHIG3' which contains the human J, C ⁇ , 71 and rat 3' enhancer
• Both inserts contain human J segments which provide for approximately 6.3 kb of overlap between the two DNA
• This approach provides for the addition of a multiplicity of V segments into the transgene formed in vivo.
• a multiplicity of V segments contained on (1) isolated genomic DNA, (2) ligated DNA derived from genomic DNA, or (3) DNA encoding a synthetic V segment repertoire is cloned into pHIG2 at the Sfil site to generate pHIG5' V N .
• the J segments fragment (a) of Fig. 14 is then cloned into pHIG5' V N and the insert isolated.
• This insert now contains a multiplicity of V segments and J segments which overlap with the J segments contained on the insert isolated from pHIG3'.
• Synthetic V H region fragments are generated and isolated as previously described. These fragments are
• transgenic animals coinjected with the purified Notl insert of plasmid pHIG (or a version of pHIG that does not contain any V segments).
• the coinjected DNA fragments are inserted into a single site in the chromosome.
• Some of the resulting transgenic animals 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).
• pE ⁇ l The construction of pE ⁇ l 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 using oligo:
• This E ⁇ fragment is cloned into EcoRV/Xbal digested pGPl by blunt end filling in EcoRI site.
• the resultant plasmid is designated pEmul.
• the K construct contains at least one human V,.
• the k enhancer in mouse is 9 kb downstream from C ⁇ . However, it is as yet unidentified in the human.
• the construct contains a copy of the mouse heavy chain J-C ⁇
• the minilocus is constructed from four component fragments:
• Human placental DNA is digested with Smal and fractionated on agarose gel by electrophoresis. Similarly, human placental DNAis 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
• Smal fraction destroys the Smal sites and lases Xhol sites in tact.
• pKapl resultant plasmid was designated pKapl. See Fig. 22.
• the above Ck specific oligonucleotide is used to probe the ⁇ EMBL3/BamHI library to identify an 11 kb clone
• Vk segments are thereafter subcloned into the Mlul site of pKap2 to yield the plasmid pKapH which encodes the human Vk segments, the human Jk segments, the human Ck segments and the human E ⁇ enhancer.
• This insert is excised by digesting pKapH with Notl and purified by agarose gel
• the thus purified insert is microinjected into the pronucleus of a mouse zygote as previously described.
• 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 site.
• the resultant plasmid is designated pKAPint.
• One or more Vk segments is inserted into the polylinker between the BamHI and Spel sites in pKAPint to form pKapHV.
• the insert of pKapHV is excised by digestion with Notl and purified.
• the insert from pKap2 is excised by digestion with Notl and
• Each of these fragments contain regions of homology in that the fragment from pKapHV contains a 5 kb sequence of DNA that include the J ⁇ segments which is substantially
• these inserts are capable of homologously recombining when microinjected into a mouse zygote to form a transgene encoding V ⁇ , J ⁇ and C ⁇ .
• Synthetic Vk, region fragments are generated and isolated as previously described. These DNA fragments are coinjected with the purified Notl insert of plasmid pKap2 or plasmid pKapH. The coinjected DNA fragments are inserted into a single site in the chromosome.
• transgenics will contain transgene inserts that have synthetic V regions located adjacent and upstream of the sequences in the pKap2 or pKapH construct. These animals will have a larger human k light chain primary repertoire than those described in Example 6 (B).
• This example describes the cloning of immunoglobulin k light chain genes from cultured cells that express an
• Such cells may contain multiple alleles of a given immunoglobulin gene.
• a given immunoglobulin gene For example, a
• hybridoma might contain four copies of the k light chain gene, two copies from the fusion partner cell line and two copies from the original B-cell expressing the immunoglobulin of interest. Of these four copies, only one encodes the
• RNA 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 isolation of polyA+ RNA.
• the RNA is then used for the synthesis of oligo dT primed cDNA using the enzyme reverse transcriptase.
• the single stranded cDNA is then isolated and G residues are added to the 3' end using the enzyme polynucleotide terminal transferase.
• the Gtailed single-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:
• 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 expressed
• the double stranded cDNA described in part A is denatured and used as a template for a third round of DNA synthesis using the following oligonucleotide primer:
• This primer contains sequences specific for the constant portion of the k light chain message (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 synthesized DNA strand (GTA CGC CAT ATC AGC TGG ATG AAG).
• the sequence is amplified by PCR using the following two oligonucleotide primers:
• PCR amplified sequence is then purified by gel electrophoresis and used as template for dideoxy sequencing reactions using the following oligonucleotide as a primer:
• DNA from the Ig expressing cell line is then cut with Smal and second enzyme (or BamHI or Kpnl if there is Smal site inside V segment) . Any resulting non-blunted ends are treated with the enzyme T4 DNA polymerase to give blunt ended DNA molecules. Then add restriction site encoding linkers (BamHI, EcoRI or Xhol depending on what site does not exist in
• V segment containing clones are then cut with the corresponding linker enzyme to give DNA fragments with BamHI, EcoRI or Xhol ends.
• the DNA is then size fractionated by agarose gel electrophoresis, and the fraction including the DNA fragment covering the expressed V segment is cloned into lambda EMBL3 or Lambda FIX (Stratagene, La Jolla, California). V segment containing clones are
• DNA is isolated from positive clones and subcloned into the polylinker of pKapl.
• the resulting clone is called pRKL.
• This example describes the cloning of immunoglobulin heavy chain ⁇ genes from cultured cells of expressed and immunoglobulin of interest. The procedure described in this example allows for the selective cloning of the expressed copy of a ⁇ heavy chain gene.
• Double-stranded cDNA is prepared and isolated as described in part A of Example 7.
• the double-stranded cDNA is denatured and used as a template for a third round of DNA synthesis using the following oligonucleotide primer:
• This primer contains sequences specific for the constant portion of the ⁇ heavy chain message (ACA GGA GAC GAG GGG GAA AAG GGT TGG GGC GGA TGC) as well as unique sequences 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'
• Mlul is a rare cutting enzyme that cleaves between the J segment and mu CH1
• Mlul is a rare cutting enzyme that cleaves between the J segment and mu CH1
• a unique restriction endonuclease site is identified upstream of the rearranged V segment.
• DNA from the IG expressing cell line is 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 is then size fractionated by agarose gel electrophoresis, and the fraction including the DNA fragment covering the expressed V segment is cloned directly into the plasmid pGPl.
• V segment containing clones are isolated using the unique probe o-mu, and the insert is subcloned into Mlul or Mlul/Spel cut plasmid pCON2. The resulting plasmid is called pRMGH.
• This example describes the deletion of the endogenous mouse heavy chain gene by homologous recombination in embryonic stem (ES) cells (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 mouse (Teratocarcinomas and embryonic stem cells: a practical approach, E.J. Robertson, ed., IRL press, Washington, D.C., 1987).
• ES embryonic stem
• Plasmid pGPl is digested with the restriction endonucleases BamHI and Bglll and religated to form the plasmid pGPldl. This plasmid is then used to build the so-called gene knockout construct.
• mouse genomic clones are isolated from a phage library derived from non-lymphoid tissue (such as liver) using the J H specific oligonucleotide probe:
• 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 pMKO1.
• Neomycin resistance (Neo) and Herpes Simplex Virus thymidine k inase (TK) genes for drug selection of recombinants (M. Capecchi (1989), Science, 244, 1288-1292) are then isolated 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 pMKO1 using the following oligonucleotide as an adapter:
• the resulting. plasmid is designated pMKO2.
• This plasmid contains the neomycin resistance gene flanked by sequences that flank the mouse J H segments. This plasmid alone can be used for deletion of the heavy chain gene.
• Herpes TK gene can be added to the construct to improve the frequency of homologous recombination events in Neo resistant clones (M. Capecchi (1989), Science, 244,
• the resulting plasmid is designated pMKO3.
• This 12 kb fragment includes the C ⁇ coding exons, or a substantial portion of that fragment which includes the 5'
• This example describes the deletion of the endogenous mouse light chain gene by homologous recombination in embryonic stem cells (see previous Example).
• a DNA sequence that homologously recombines into the mouse chromosome to delete the k light chain constant region exon is constructed.
• the design of this construct is outlined below.
• the resulting plasmid is designated pKKO1.
• mouse genomic clones are isolated from a phage library derived from non-lymphoid tissue (such as liver) using the mouse /c light chain specific oligo designated o-MKC given below:
• probe o-MK3 DNA is isolated from positive clone and a 2.3 kb Bglll fragment (P.S. Neumaier and H.G. Zachau (1983), Nucl. Acids Res., 11, 3631-3656) that hybridizes with probe o-MK3 is isolated.
• the sequence of probe o-MK3 is as follows:
• This 2.3 kb Bglll fragment is subcloned into BamHI digested pKKO1 such that the 3' end of the fragment is adjacent to the polylinker Sfil site.
• the resulting plasmid is
• the 4 kb Sphl to Hpal DNA fragment that hybridizes with oligonucleotide o-MKC is isolated from positive phage clone and subcloned into EcoRV to Sphl digested plasmid pKKO2. The resulting plasmid is designated pKKO3.
• a 2 kb Sail to EcoRI fragment of pGEM7 (KJ1) Sal (M.A. Rudnicki, 3/15/89) is isolated and cloned into the BssHII site of plasmid pKKO3 using linker adapters. This is carried out by first ligating a mixture of the following three
• the ligation mixture is then digested with the enzyme BssHII and ligated to BssHII digested plasmid pKKO3.
• the resulting plasmid is designated pKKO4.
• the insert of pKK04 is isolated by digesting with Notl and electroporated into ES cells. Homologous recombinant clones are isolated and used to generate a C ⁇ deleted mouse as described by Zjilstra, et al. (1989), Nature, 342, 435-438.
• This example describes the inactivation of the mouse endogenous kappa locus by homologous 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 embryos (blastocysts).
• the strategy is to delete J ⁇ and C ⁇ by homologous recombination with a vector containing DNA sequences homologous to the mouse kappa locus in which a 4.5 kb segment of the locus, snanning the J k gene and C k segments, is deleted and replaced by the selectable marker neo.
• the plasmid pGEM7 (KJ1) (M. A. Rudnicki, Whitehead Institute) contains the neomycin resistance gene (neo), used for drug selection of transfected ES cells, under the
• the plasmid also includes a heterologous polyadenylation site for the neo gene, derived from the 3' region of the mouse 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 sequences (Fig. 25a) were isolated from a genomic phage library derived from liver DNA using oligonucleotide probes specific for the Ck locus: 5'- GGC TGA TGC TGC ACC AAC TGT ATC CAT CTT CCC ACC ATC CAG -3' and for the Jk5 gene segment: 5'- CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA CGT AAG -
• Sphl/Xbal/Bglll/EcoRI adaptor was ligated to the Sphl site of this fragment, and the resulting EcoRI fragment was ligated into EcoRI digested pNEO-K3', in the same 5' to 3' orientation as the neo gene and the downstream 3' kappa sequences, to generate pNEO-K5'3' (Fig. 25c).
• HSV Herpes Simplex Virus
• TK thymidine kinase gene
• the HSV TK cassette was obtained from the plasmid pGEM7 (TK) (M.A. Rudnicki), which contains the structural sequences for the HSV TK gene bracketed by the mouse pgk promoter and polyadenylation sequences as described above for pGEM7 (KJ1).
• TK The EcoRI site of pGEM7 (TK) was modified to a BamHI site and the TK cassette was then excised as a BamHI/Hindlll fragment and subcloned into pGP1b to generate pGP1b-TK.
• This plasmid was linearized at the Xhol site and the Xhol fragment from pNEO-K5'3', containing the neo gene flanked by genomic sequences from 5' of Jk and 3' of
• the kappa chain inactivation vector J/C K1 was 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 (200 ⁇ g/ml of active component) and FIAU (0.5 ⁇ M) were added to the medium, and drug-resistant clones were allowed to develop over 10-11 days. Clones were picked, trypsinized, divided into two portions, and further expanded. Half of the cells derived from each clone were then frozen and the other half analyzed for homologous recombination between vector and target
• 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 as the diagnostic probe. This probe detects a 3.7 kb Xbal fragment in the wild type locus, and a diagnostic 1.8 kb band in a locus which has homologously recombined with the targeting vector (see Fig. 25a and e).
• restriction fragments diagnostic of a homologous recombination at the kappa light chain.
• pseudopregnant females to generate chimeric mice representing a mixture of cells derived from the input ES cells 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 is verified by Southern blot analysis of DNA from tail biopsies using the diagnostic probe utilized in identifying targeted ES clones.
• This example describes the inactivation of the endogenous murine immunoglobulin heavy chain locus by
• the strategy is to delete the endogenous heavy chain J segments by homologous recombination with a vector containing heavy chain sequences from which the J H region has been deleted and
• the resulting plasmid was designated pucl8 J H .
• the neomycin resistance gene (neo) used for drug selection of transfected ES cells, was derived from the plasmid pGEM7 (KJ1).
• the Hindlll site in pGEM7 (KJ1) was converted to a Sall site by addition of a synthetic adaptor, and the neo expression cassette excised by digestion 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 puc18 J H ,
• pGPlb was digested with the restriction enzyme Notl and ligated with the following oligonucleotide as an adaptor:
• pGMT mouse immunoglobulin heavy chain targeting construct
• HSV Herpes Simplex Virus
• TK thymidine kinase
• 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.
• 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 digested pGMT-TK, to generate the plasmid pGMT-TK-J H 5' (Fig. 26d).
• the final step in the construction involved the excision of the 3 kb EcoRI fragment from pucl ⁇ J H -neo which contained the neo gene and flanking genomic sequences. This fragment was blunted by Klenow polymerase and subcloned into the similarly blunted Xhol site of pGMT-TK-J H 5'.
• the resulting construct, J H KO1 (Fig. 26e), contains 6.9 kb of genomic
• Fig. 25f shows the structure of an endogenous heavy chain allele after homologous recombination with the targeting construct.
• the heavy chain inactivation vector J H KO1 was 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 dishes at a density of 2-5 x 10 6 cells/dish. After 24 hours, G418
• DNA analysis is carried out by Southern blot hybridization.
• DNA is isolated from the clones as described (Laird, P.W. et al., (1991) Nucl. Acids Res., 19.) digested with Hindlll and probed with the 500 bp EcoRI/StuI fragment designated as the diagnostic probe in Fig. 26f.
• This probe detects a Hindlll fragment of 2.3 kb in the wild type locus, 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 digests with the enzymes Spel, Stul, and BamHI are carried out to verify the targeted
• the plasmid pBR322 was digested with EcoRI and Styl and ligated with the following oligonucleotides: oligo-42 5'- caa gag ccc gcc taa tga gcg ggc ttt ttttg cat act gcg gcc get -3' oligo-43 5'- aat tag egg ccg cag tat gca aaaaaa age ccg etc att agg egg get -3'
• the resulting plasmid, pGP1a is designed for cloning very large DNA constructs that can be excised by the rare cutting restriction enzyme Notl. It contains a Notl
• AmpR resistance gene of a strong transcription termination signal derived from the trpA gene (Christie, G.E. et al. (1981) Proc. Natl. Acad. Sci. USA, 78, 4180).
• This termination signal reduces the potential toxicity of coding sequences inserted into the Notl site by eliminating readthrough transcription from the AmpR gene.
• this plasmid is low copy relative to the pUC plasmids because it retains the pBR322 copy number control region. The low copy number further reduces the potential toxicity of insert sequences and reduces the
• pGPla was digested 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 atc gat taa tta agg atc cag cag taa get tgc -3'
• the resulting plasmid, pGPlb contains a short
• oligo-44 5'- etc cag gat cca gat ate agt ace tga aac agg gct 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 polymerase chain reaction technique.
• pNNO3 is a pUC derived plasmid that contains a polylinker with the following restriction sites, listed in order: Notl, BamHI, Ncol, Clal, EcoRV, Xbal, Sacl, Xhol, Sphl, Pstl, Bglll, EcoRI, Smal, Kpnl, Hindlll, and Notl).
• the resulting plasmid, pRE3 was digested with BamHI and Hindlll, and the insert containing the rat Ig heavy chain 3' enhancer cloned into BamHI/Hindlll digested pGPlb.
• the resulting plasmid, pGPe (Fig. 27 and Table 1), contains several unique restriction sites into which sequences can be cloned and subsequently excised together with the 3' enhancer by Notl digestion.
• a 4 kb Xhol fragment was isolated from phage clone A2.1 that contains sequences immediately downstream of the sequences in pJM1, including the so called ⁇ element involved in ⁇ deletion in certain IgD expressing B-cells (H. Yasui et al. (1989) Eur. J. Immunol. 19, 1399).
• This fragment was treated with the Klenow fragment of DNA polymerase I and ligated to Xhol cut, Klenow treated, pJMl.
• the resulting plasmid, pJM2 (Fig. 28), had lost the internal Xhol site but retained the 3' Xhol site due to incomplete reaction by the Klenow enzyme.
• pJM2 contains the entire human J region, the heavy chain J- ⁇ intronic enhancer, the ⁇ switch region and all of the ⁇ constant region exons, as well as the two 0.4 kb direct repeats, ⁇ and ⁇ , involved in ⁇ deletion.
• oligo-4 5'- tgg tat tac tat ggt teg ggg agt tat tat aac cac agt gtc -3' was used to screen the human placenta genomic library for D region clones. Phage clones ⁇ 4.1 and A4.3 were isolated. A 5.5 kb Xhol fragment, that includes the D elements D k1 , D N1 , and D M2 (Y. Ichihara et al. (1988) EMBO J., 7, 4141), was isolated from phage clone A4.1.
• An adjacent upstream 5.2 kb Xhol fragment that includes the D elements D LR1 , D XP1 , D XP 1 , and D A1 , was isolated from phage clone ⁇ 4.3. Each of these D region Xhol fragments were cloned into the Sail site of the plasmid vector pSP72 (Promega, Madison, WI) so as to destroy the Xhol site linking the two sequences. The upstream fragment was then excised with Xhol and Smal, and the downstream fragment with EcoRV and Xhol. The resulting isolated fragments were ligated together with Sall digested pSP72 to give the plasmid pDH1.
• pDH1 contains a 10.6 kb insert that includes at least 7 D segments and can be excised with Xhol (5') and EcoRV (3').
• the plasmid pJM2 was digested with Asp718 (an isoschizomer of Kpnl) and the overhang filled in with the
• the plasmid pCOR1 was partially digested with Xhol and the isolated Xhol/Sall insert of pVH251 cloned into the
• pIGMl contains 2 functional human variable region segments, at least 8 human D segments all 6 human J H segments, the human J- ⁇ enhancer, the human ⁇ element, the human ⁇ switch region, all of the human ⁇ coding exons, and the human ⁇ 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 animals.
• 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 clones 129.4 and A29.5 were isolated. A 4 kb Hindlll fragment of phage clone A29.4, containing a ⁇ switch region, was used to probe a human placenta genomic DNA library cloned into the phage vector lambda FIXTM II (Stratagene, La Jolla, CA). Phage clone ⁇ Sgl.13 was isolated.
• dideoxy sequencing reactions were carried out using subclones of each of the three phage clones as templates and the following oligonucleotide as a primer: oligo-67 5'- tga gcc cag aca ctg gac -3'
• Phage clones ⁇ 29.5 and ⁇ S ⁇ r1.13 were both determined to be of the ⁇ 1 subclass. 2 . p ⁇ el
• 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 site and a human sequence derived BamHI site generates a 7.6 kb fragment containing the 71 constant region coding exons.
• p ⁇ e1 contains all of the ⁇ 1 constant region coding exons, together with 5 kb of downstream sequences, linked to the rat heavy chain 3' enhancer.
• p ⁇ e2 contains all of the ⁇ 1 constant region coding exons, and the upstream switch region and sterile transcript exons, together with 5 kb of downstream sequences, linked to the rat heavy chain 3' enhancer.
• This clone contains a unique Xhol site at the 5' end of the insert. The entire insert, together with the Xhol site and the 3' rat enhancer can be excised from vector sequences by digestion with Notl.
• pHCl contains 2 functional human variable region segments, at least 8 human D segments all 6 human J H segments, the human J- ⁇ enhancer, the human ⁇
• the human ⁇ switch region all of the human ⁇ coding exons, the human ⁇ element, and the human 71 constant region, including the associated switch region and sterile transcript associated exons, together with the rat heavy chain 3'
• pronuclei to generate transgenic animals.
• Phage clone A49.8 was isolated and a 6.1 kb Xbal fragment containing the variable segment VH49.8 subcloned into pNN03 (such that the polylinker Clal site is downstream of
• An 800 bp region of this insert was sequenced, and VH49.8 found to have an open reading frame and intact splicing and recombination signals, thus indicating that the gene is functional (Table 2). 2.
• 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 digested, Klenow treated, pVH49.8. The resulting plasmid, pV2, contains the human heavy chain gene VH49.8 linked upstream of VH4-21 in the same orientation, with a unique Sail site at the 3' end of the insert and a unique Xhol site at the 5' end.
• a 0.7 kb Xbal/Hindlll fragment (representing sequences immediately upstream of, and adjacent to, the 5.3 kb ⁇ 1 switch region containing fragment in the plasmid p ⁇ e2) together with the neighboring upstream 3.1 kb Xbal fragment were isolated from the phage clone ⁇ Sgl.13 and cloned into Hindlll/Xbal digested pUC18 vector. The resulting plasmid, pS ⁇ 1-5',
• the pS ⁇ 1-5' insert was excised with Smal and Hindlll, treated with Klenow enzyme, and ligated with the following oligonucleotide linker:
• the ligation product was digested with Sall and ligated to Sall 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 fragment on an agarose gel.
• the insert is then cloned into the Xhol site of p ⁇ e2 to generate the plasmid clone pVGE1 (Fig. 32).
• pVGE1 contains two human heavy chain variable gene segments upstream of the human ⁇ 1 constant gene and associated switch region.
• a unique Sail site between the variable and constant regions can be used to clone in D, J, and ⁇ gene segments.
• the rat heavy chain 3' enhancer is linked to the 3' end of the 71 gene and the entire insert is flanked by Notl sites.
• the plasmid clone pVGE1 is digested with Sall and the
• Xhol insert of pIGMl is cloned into it.
• the resulting clone, pHC2 (Fig. 30), contains 4 functional human variable region segments, at least 8 human D segments all 6 human J H segments, the human J-m enhancer, the human ⁇ element, the human ⁇ switch region, all of the human ⁇ coding exons, the human ⁇ element, and the human ⁇ 1 constant region, including the associated switch region and sterile transcript associated exons, together with 4 kb flanking sequences upstream of the sterile transcript initiation site.
• rat heavy chain 3' enhancer 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 sequences, 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 segments to further expand the recombinational diversity of this heavy chain minilocus.
• the Notl inserts of plasmids pIGMl and pHCl were isolated from vector seguences by agarose gel electrophoresis.
• the purified inserts were microinjected into the pronuclei of fertilized (C57BL/6 x CBA) F2 mouse embryos and transferred the surviving embryos into pseudopregnant females as described by Hogan et al. (B. Hogan, F. Costantini, and E. Lacy, Methods of Manipulating the Mouse Embryo, 1986, Cold Spring Harbor
• mice that developed from injected embryos were analyzed for the presence of transgene sequences by Southern blot analysis of tail DNA. Transgene copy number was estimated by band intensity relative to control standards containing known quantities of cloned DNA. At 3 to 8 weeks of age, serum was isolated from these animals and assayed for the presence of transgene encoded human IgM and IgGl by ELISA as described by Harlow and Lane (E. Harlow and D. Lane.
• Microtiter plate wells were coated with mouse monoclonal antibodies specific for human IgM (clone AF6, #0285, AMAC, Inc. Westbrook, ME) and human IgGl (clone JL512, #0280, AMAC, Inc. Westbrook, ME). Serum samples were serially diluted into the wells and the presence of specific IgM (clone AF6, #0285, AMAC, Inc. Westbrook, ME) and human IgGl (clone JL512, #0280, AMAC, Inc. Westbrook, ME). Serum samples were serially diluted into the wells and the presence of specific IgM (clone AF6, #0285, AMAC, Inc. Westbrook, ME) and human IgGl (clone JL512, #0280, AMAC, Inc. Westbrook, ME). Serum samples were serially diluted into the wells and the presence of specific IgM (clone AF6, #0285, AMAC, Inc. Westbrook, ME) and human Ig
• FIG. 33 shows the results of an ELISA assay for the presence of human IgM and IgGl in the serum of two animals that developed from embryos injected with the transgene insert of plasmid pHCl.
• One of the animals (#18) was negative for the transgene by Southern blot analysis, and showed no detectable levels of human IgM or IgG1.
• the second animal (#38) contained approximately 5 copies of the transgene, as assayed by Southern blotting, and showed detectable levels of both human IgM and IgGl.
• Table 3 shows a correlation between the presence of integrated transgene DNA and the presence of transgene encoded immunoglobulins in the serum.
• Two of the animals 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 animals may have been genetic mosaics
• the transgene containing cells may not have populated the hematopoetic lineage.
• the transgenes may have integrated into genomic locations that are not conducive to their expression.
• the detection of human IgM in the serum of pIGMl transgenics, and human IgM and IgGl in pHCl transgenics, indicates that the transgene sequences function correctly in directing VDJ joining, transcription, and isotype switching.
• oligo-7 5'-tea gtg aag gtt tec tgc aag gca tct gga tac ace ttc acc-3' oligo-8 5'-tec ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttc agt-3'
• VDJ segment the J- ⁇ intronic enhancer, the ⁇ switch element, the ⁇ constant region coding exons, and the ⁇ 1
• This transgene construct is excised with Notl and microinjected into the pronuclei of mouse embryos to generate transgenic animals as described above.
• Plasmid vector pGPla is digested with Notl and the following oligonucleotides ligated in: oligo-81 5'-ggc cgc ate ccg ggt ctc gag gtc gac aag ctt tcg agg atc cgc-3'
• the resulting plasmid, pGPlc contains a polylinker with Xmal, Xhol, Sall, Hindlll, and BamHI restriction sites flanked by Notl sites.
• Plasmid vector pGPla is digested with Notl and the following oligonucleotides ligated in: oligo-87 5'-ggc cgc tgt cga caa get tat cga tgg atc ctc gag tgc -3' oligo-88 5'-ggc cgc act cga gga tcc atc gat aag ctt gtc gac agc
• the resulting plasmid, pGPld contains a polylinker with Sall, Hindlll, Clal, BamHI, and Xhol restriction sites flanked by Notl sites.
• a 7.4 kb Xhol fragment that includes the J/cl segment was isolated from 136.2 and subcloned into the plasmid pNN03 to generate the plasmid clone p36.2.
• a neighboring 13 kb Xhol fragment that includes Jk segments 2 through 5 together with the Ck gene segment was isolated from phage clone 136.5 and subcloned into the plasmid pNNO3 to generate the plasmid clone p36.5. Together these two clones span the region beginning 7.2 kb upstream of Jk1 and ending 9 kb downstream of Ck.
• pCK1 a Ck 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, pCK1 can accept cloned fragments containing rearranged VJ/c segments into the unique 5' Xhol site.
• the transgene can then be excised with Notl and purified from vector sequences by gel electrophoresis.
• the resulting transgene construct will contain the human J-Ck intronic enhancer and may contain the human 3' k enhancer.
• pCK2 a Ck vector with heavy chain enhancers for expressing rearranged variable segments
• the resulting plasmid, pMHE1 consists of the mouse and human heavy chain J- ⁇ intronic enhancers ligated together into pUC18 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 plasmid, pCK2 is identical to pCK1, except that the mouse and human heavy chain J- ⁇ intronic enhancers are fused to the 3' end of the transgene 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.
• VJ segments containing functional VJ segments are subcloned into the unique Xhol sites of vectors pCK1 and pCK2 to generate rearranged kappa light chain transgenes.
• the transgene constructs are isolated from vector sequences by digestion with Notl. Agarose gel purified insert is microinjected into mouse embryo
• mice expressing human kappa chain are bred with heavy chain minilocus
• VJk combinations may be capable of forming stable heavy-light chain complexes with a broad
• the 13 kb Ck containing Xhol insert of p36.5 is treated with klenow enzyme and cloned into Hindlll digested, klenow treated, plasmid pGPld.
• a plasmid clone is selected such that the 5' end of the insert is adjacent to the vector derived Clal site.
• the resulting plasmid, p36.5-ld is digested with Clal and klenow treated.
• the J/cl 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 is selected in which the p36.2 insert is in the same orientation as the p36.5 insert.
• This clone, pJCK1 (Fig. 34), contains the entire human Jk region and Ck, together with 7.2 kb of upstream sequences and 9 kb of downstream sequences.
• the insert also contains the human J-Ck intronic enhancer and may contain a human 3' k enhancer.
• the insert is flanked by a unique 3' Sail site 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 Vk gene segments. The unique Sail and Xhol sites are in turn flanked by Notl sites that are used to isolate the completed transgene construct away from vector sequences.
• Vk specific oligonucleotide oligo-65 (discussed above), is used to probe a human placental genomic DNA library cloned into the phage vector 1EMBL3/SP6/T7 (Clonetech).
• Variable gene segments from the resulting clones are sequenced, and clones that appear functional are selected. Criteria for judging functionality include: open reading frames, intact splice acceptor and donor sequences, and intact recombination sequence.
• DNA fragments containing selected 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 mouse embryo pronuclei to generate transgenic animals. The transgenes of these animals will undergo V to J joining in developing B-cells.
• mice expressing fully human antibodies generate mice expressing fully human antibodies.
• the plasmid pGPla (previous example) is digested with
• oligonucleotides are ligated to it: oligo-"a" 5'-ggc cgc atg cta ctc 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 resulting plasmid, pGPlf contains Sphl, Xhol, and Hindlll sites flanked by Notl and Sfil sites.
• PCR polymerase chain reaction
• oligo-84 5'-ctc tag agt cga cct gca ggc-3'
• the 3' flanking sequences are amplified by PCR using the following oligonucleotides:
• 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
• Plasmid pVHf contains the cis acting regulatory elements that control transcription of V H 251, together with the signal sequence encoding first exon.
• pVHf is used as an expression cassette for heavy chain variable
• sequences are cloned into the Kasl/Xhol digested plasmid as described below.
• Poly (A) + RNA is isolated from human peripheral blood lymphocytes (PBL).
• First strand cDNA is 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' sequences 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-"c" 5'-ctg acg act ctg tat ggc gcc (ct)a(cg) t(cg)(ct)
• Oligo-"c includes a 21 nucleotide nondegenerate sequence 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 segments (Genbank; Los Alamos, NM).
• the primer is extended with DNA polymerase and the product isolated from unused primer by size fractionation. The product is then denatured and annealed to the following oligonucleotide: oligo-"d" 5'-ggg ctc gag gct ggt ttc tct cac tgt gtg t(cgt)t
• Oligo-"d includes a 30 nucleotide nondegenerate sequence 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 sequence encoding the last seven amino acids 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 synthesis followed by removal of primers are carried out to ensure the sequence integrity of individual variable gene fragments.
• oligo-"d primer extension
• oligo-"e 5'-ctg acg act ctg tat ggc gcc-3'
• oligo-"f 5'-ggg ctc gag gct ggt ttc tct-3'
• the resulting 0.36 kb PCR product is purified by gel
• the entire library of synthetic germline-configuration V H genes is grown up together and plasmid DNA isolated.
• the medium copy plasmid pVHf which includes a strong transcription terminator between the ampicillin resistance gene and the cloning site, is designed to minimize the expansion of
• Plasmid DNA is digested with Sfil, treated with calf intestinal phosphatase to remove 5' phosphate groups, and then digested with Notl. The calf intestinal phosphatase is removed prior to Notl digestion so that only the Sfil ends are dephosphorylated. The digested DNA is then isolated from vector sequences by agarose gel
• oligo-"g" 5'-ggc cta act gag cgt ccc ata ttg aga acc tcc -3' oligo-"h" 5'-ggt tct caa tat ggg acg ctc agt ta-3' O1igo-"h" is 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.
• V segments will concatenate in the same orientation such that each V segment is separated by a single oligonucleotide spacer unit from the next V segment.
• Large concatomers are sized by electrophoresis and isolated from agarose gels. The size fractionated concatomers are then directly coinjected into mouse embryo pronucleitogether with D-J-C containing DNA fragments (such as the pHC1 or pHC2 inserts) to generate transgenic animals with large primary repertoires. Alternatively, the concatomers are cloned into a plasmid vector such as pGPf.
• mice with xenogeneic (i.e. human) immunoglobulins (B-cell receptors) or T-cell receptors leads predominantly to the generation.
• B-cell receptors immunoglobulins
• T-cell receptors T-cell receptors
• transgenic mouse expressing human immunoglobulins will be immunologically tolerant of those shared B-cell epitopes and will therefore be useful for generating antibodies that
• mice 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.
• Human immunoglobulin expressing transgenic mice are inoculated with immunoglobulins isolated from a healthy donor or from a patient with a B-cell malignancy expressing a high 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.
• TCR T-cell receptor
• TCR a and ⁇ chain cDNA clones are inserted into transgene
• TCR a and ⁇ chain cDNA transgene constructs are coinjected into mouse embryo pronuclei to generate transgenic animals. Ectopic expression 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,
• T-cell receptor a and ⁇ chain transgenic mice are bred with human immunoglobulin expressing transgenic mice to
• mice that are useful for generating human monoclonal antibodies that recognize specific subsets of human T-cells.
• Such mice are inoculated with T-cell derived proteins isolated 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 hybridomas that secrete human antibodies that specifically recognize B-cell subsets are selected.
• This Example describes the cloning of a human genomic heavy chain immunoglobulin transgene which is then introduced into the murine germline via microinjection into zygotes or integration in ES cells.
• Nuclei are isolated from fresh human placental tissue asdescribed by Marzluff, W.F., et al. (1985), Transcription and Translation: A Practical Approach, B.D. Hammes and S.J.
• the isolated nuclei (or PBS washed human spermatocytes) are embedded in 0.5% low melting point agarose blocks and lysed with 1 mg/ml
• the proteinase K is inactivated by incubating the blocks in 40 ⁇ g/ml PMSF in TE for 30 minutes 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
• Notl digested DNA is then fractionated by pulsed field gel electrophoresis as described by Anand, R. et al.
• Plasmid pYACNN is prepared by digestion of pYACneo (Clontech) 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 as described by Traver et al. (1989), Proc. Natl. Acad. Sci. USA, 86, 5898-5902.
• the cloned Notl insert is isolated from high molecular weight yeast DNA by pulse field gel electrophoresis as described by M. Finney, op. cit.
• the DNA is condensed by the addition of 1 mM spermine and
• the DNA is isolated by pulsed field gel electrophoresis and introduced into ES cells by lipofection (Gnirke et al. (1991), EMBO J., 10, 1629-1634), or the YAC is introduced into ES cells by spheroplast fusion.
• Example 1 Typically, coinjection of two different DNA fragments result in the integration of both fragments at the same
• 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 sequences from both smaller YACs.
• 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.
• the insert is recloned in the vector using unique restriction sites 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
• a map of the human k light chain has 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 least five different V segments (a), or a 750kb Mlul to Notl fragment that includes all of the above plus at least 20 more V segments (b) is isolated and introduced into zygotes or ES cells as described in Example 1.
• the 750kb Mlul to Notl fragment is 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 BssHII fragment (c) have sequence overlap defined by the
• An antigen of interest is used to immunize (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York (1988)) a mouse with the following genetic traits:
• the spleen is removed, and spleen cells used to generate hybridomas.
• Cells from an individual hybridoma clone that secretes antibodies 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.
• fragments in the 2-10 kb range one of which contains the single copy of the rearranged human heavy chain VDJ sequences and one of which contains the single copy of the rearranged human light chain VJ sequence. These two fragments are size fractionated on agarose gel and cloned directly into pUC18.
• the cloned inserts are then subcloned respectively into heavy and light chain expression cassettes that contain constant region sequences.
• the plasmid clone P7el (Example 14) is used as a heavy chain expression cassette and rearranged VDJ sequences are cloned into the Xhol site.
• the plasmid clone pCK1 is used as a light chain expression cassette and rearranged VJ sequences are cloned into the Xhol site.
• the resulting clones are used together to transfect SP 0 cells to produce antibodies that react with the antigen of interest (M.S. Co. et al. (1991)
• 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.

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