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Rapid cloning of any rearranged mouse immunoglobulin variable genes

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Abstract

Immunoglobulins (Ig) have been the focus of extensive study for several decades and have become an important research area for immunologists and molecular biologist. The use of polymerase chain reaction (PCR) technology has accelerated the cloning, sequencing, and characterization of genes of the immune system. However, cloning and sequencing the Ig variable (V) genes using the PCR technology has been a challenging task, primarily due to the very diverse nature of Ig V region genes. We have developed a simple, rapid, and reproducible PCR-based technique to clone any rearranged mouse Ig heavy or light chain genes. A close examination of all Ig heavy and light chain V gene families has resulted in the design of 5′ and 3′ universal primers from regions that are highly conserved across all heavy or light chain V gene families, and the joining or constant regions, respectively. We present our strategy for designing universal primers for Ig V gene families. These primers were able to rapidly amplify the rearranged Ig V genes, belonging to diverse Ig V gene families from very different cell lines, i.e., J558, MOPC-21, 36–60, and a chicken ovalbumin specific B-cell hybridoma. In addition, the present study provides the complete alignment of nucleotide sequences of all heavy and light chain variable gene families. This powerful method of cloning Ig V genes, therefore, allows rapid and precise analysis of B-cell hybridomas, B-cell repertoire, and B-cell ontogeny.

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References

  • Akolkar, P. N., Sikder, S. K., Bhattacharya, S. B., Liao, J., Gruezo, F., Morrison, S. L., and Kabat, E. A. Different V-L and V-H germ-line genes are used to produce similar combining sites with specificity for alpha (1,6) dextrans. J Immunol 138: 4472–4479, 1987

    Google Scholar 

  • Alt, F. W., Blackwell, T. K., and Yancopoulos, G. D.d Development of the primary antibody repertoire. Science 238: 1079–1087, 1987

    Google Scholar 

  • Bothwell, A. L., Paskind, M., Reth, M., Imanishi-Kari, T., Rajewsky, K., and Baltimore, D. Heavy chain variable region contribution to the NPb family of antibodies: Somatic mutation evident in a gamma-2a variable region. Cell 24: 625–637, 1981

    Google Scholar 

  • Bernard, O. D., Hozumi, N., and Tonegawa, S. Sequences of mouse immunoglobulin light chain genes before and after somatic changes. Cell 15: 1133–1144, 1978

    Google Scholar 

  • Brodeur, P.H. and Riblet, R. The immunoglobulin heavy chain variable region (Igh-V) locus in the mouse. I. One hundred Igh-V genes comprise seven families of homologous genes. Eur J Immunol 14: 922–930, 1984

    Google Scholar 

  • Brodeur, P. H., Osman, G. E., Mackle, J. J., and Lalor, T. M. The organization of the mouse Igh-V locus. Dispersion, interspersion, and evolution of V H gene family clusters. J Exp Med 168: 2261–2278, 1988

    Google Scholar 

  • Chaudhary, V. K., Batra, J. K., Gallo, M. G., Willingham, M. C., Fitzgerald, D. J., and Pastan, I. A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunoglobulins. Proc Natl Acad Sci USA 87: 1066–1070, 1990

    Google Scholar 

  • Chiang, Y. L., Sheng-Dong, R., Brow, M. A., and Larrick, J. W. Direct cDNA cloning of the rearranged immunoglobulin variable region. BioTechniques 7: 360–366, 1989

    Google Scholar 

  • Coloma, M. J., Larrick, J. W., Ayala, M., and Gavilondo-Cowley, J. V. Primer design for the cloning of immunoglobulin heavy-chain leader-variable regions from mouse hybridoma cells using the PCR. BioTechniques 11: 152–154, 1991

    Google Scholar 

  • Cory, S., Tyler, B. M., and Adams, J. M. Sets of immunoglobulin V kappa genes homologous to ten cloned V kappa sequences: implications for the number of germline V kappa genes. J Mol Appl Genet 1: 103–116, 1981

    Google Scholar 

  • D'Hoostelaere, L. and Klinman, D. Characterization of new mouse V kappa groups. J Immunol 145: 2706–2712, 1990

    Google Scholar 

  • Dildrop, R. A new classification of mouse V H sequences. Immunol Today 5: 85–86, 1984

    Google Scholar 

  • Eisen, H. N. and Reilly, E. B. Lambda chains and genes in inbred mice. Annu Rev Immunol 3: 337–365, 1985

    Google Scholar 

  • Embleton, M. J., Gorochov, G., Jones, P. T., and Winter, G. In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells. Nucleic Acids Res 20: 3831–3837, 1992

    Google Scholar 

  • Ferguson, S. E., Cancro, M. P., and Osborne, B. A. Analysis of anovel V (H)S107 haplotype in CLA-1 and WSA mice. Evidence for gene conversion among IgV(H) genes in outbred populations. J Exp Med 170: 1811–1823, 1989

    Google Scholar 

  • Gavilondo-Cowley, J. V., Coloma, M. J., Vazquez, J., Ayala, M., Macias, A., Fry, K. E., and Larrick, J. W. Specidic amplification of rearranged immunoglobulin variable region genes from mouse hybridoma cells. Hubridoma 9/(5): 407–417, 1990

    Google Scholar 

  • Gefter, M. L., Margolies, M. N., Near, R. I., and Wysocki, L. J. Analysis of the anti-azobenzenearsonate response at the molecular level. Ann Inst Pasteur Immunol 135: 17–30, 1984

    Google Scholar 

  • Hartman, A. B. and Rudikoff, S. VH genes encoding the immune response to beta-(1,6)-galactan: somatic mutation in IgM molecules. EMBO J 3: 3023–3030, 1984

    Google Scholar 

  • Hayashi, K. Manipulation of DNA by PCR. In K. B. Mullis, F. Ferre, and R. A. Gibbs. (eds.): The Polymerase Chain Reaction, pp. 3–13. Birkhauser Boston, Cambridge, 1994

    Google Scholar 

  • Heinrichs, A., Milstein, C., and Gherardi, E. A. Universal cloning and direct sequencing of rearranged antibody V genes using C region primers, biotin-captured cDNA and one-sided PCR. J Immunol Methods 178: 241–251, 1995

    Google Scholar 

  • Jones, S. T. and Bendig, M. M. Rapid PCR-cloning of full-length mouse immunoglobulin variable regions. Bio/Technology 9: 88–89, 1991

    Google Scholar 

  • Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C. Sequences of proteins of immunological interest. US Department of Health and Human Services, MD, 1991

    Google Scholar 

  • Kettleborough, C. A., Saldhana, J., Ansell, K. H., and Bendig, M. M. Optimization of primers for cloning libraries of mouse immunoglobulin genes using the polymerase chain reaction. Eur J Immunol 23: 206–211, 1993

    Google Scholar 

  • Kofler, R., Geley, S., Hofler, H., and Helmberg, A. Mouse variable-regions gene families: complexity, polymorphism and use in non-Autoimmune responses. Immunol Rev 128: 5–21, 1992

    Google Scholar 

  • Kurosawa, Y. and Tonegawa, S. Organization, structure, and assembly of Immunoglobulin heavy chain diversity DNA segments. J Exp Med 155: 201–218, 1982

    Google Scholar 

  • Kutemeier, G., Harloff, C., and Mocikat, R. Lapid isolation of immunoglobulin genes from cell lysates of rat hybridomas by polymerase chain reaction. Hybridoma 11: 23–32, 1992

    Google Scholar 

  • Larrick, J. M., Danielsson, L., Brenner, C., Abrahamson, M., Fry, K., and Borrebaeck, C. A. K. Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction. Biochem Biophys Res Commun 160: 1250–1256, 1989

    Google Scholar 

  • Larrick, J. W., Coloma, M. J., Del Valle, J., Fernandez, M. E., Fry, K. E., and Gavilondo-Cowley, J. V. Immunoglobulin V regions of a bactericidal anti-Neisseria meningitidis outer membrane protein monoclonal antibody. Scan J Immunol 32: 121–128, 1990

    Google Scholar 

  • Livant, D., Blatt, C., and Hood, L. One heavy chain variable region gene segment subfamily in BALB/c mouse contains 500–1000 or more members. Cell 47: 461–470, 1986

    Google Scholar 

  • Loh, E. Y., Elliot, J. D., Cwirla, S., Lanier, L. L., and Davis, M. M. Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain. Science 243: 217–220, 1989

    Google Scholar 

  • Matsuda, T. and Kabat, E. A. Variable region cDNA sequences and antigen binding specificity of mouse monoclonal antibodies to isomaltosyl oligosaccharides coupled to proteins: T-dependent analogues of alpha-(1–6)-dextran. J Immunol 142: 863–870, 1989

    Google Scholar 

  • Max, E. E., Maizel, J. V., and Leder, P. The nucleotide sequence of a 5.5-kilobase DNA segment containing the mouse kappa immunoglobulin J and C region genes. J Biol Chem 256: 5116–5120, 1981

    Google Scholar 

  • Meek, K., Rathbun, G., Reininger, L., Jaton, J. C., Kofler, R., Tucker, P., and Capra, J. D. Organization of murine immunoglobulin VH complex: placement of two new VH families (VH10 and VH11) and analysis of VH family clustering and interdigitation. Mol Immunol 27: 1073–1081, 1990

    Google Scholar 

  • Orlandi, R., Gussow, D. H., Jones, P. T., and Winter, G. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc Natl Acad Sci USA 86: 3833–3837, 1989

    Google Scholar 

  • Osman, G. E., Brodeur, P. H., Rosenberg, N., and Wortis, H. H. The Ig V H repertoire of fetal liver-derived pre-B cells is influenced by the expression of a gene liked to X-linked immune deficiency. J Immunol 148: 1928–1933, 1992

    Google Scholar 

  • Pennell, C. A., Mercolino, T. J., Grdina, T. A., Arnold, L. W., Houghton, G., and Clarke, S. Biased immunoglobulin variable region gene expression by Ly-1 B cells due to clonal selection. Eur J Immunol 19: 1289–1295, 1989

    Google Scholar 

  • Potter, M., Newell, J. B., Rudikoff, S., and Haber, E. Classification of mouse V kappa groups based on the partial amino acid sequence to the first invariant tryptophan: impact of 14 new sequences from IgG myeloma proteins. Mol Immunol 12: 1619–1630, 1982

    Google Scholar 

  • Ratech, H. Rapid cloning of rearranged immunoglobulin heavy chain genes from human B-cell lines using anchored polymerase chain reation. Biochem Biophys Res Commun 182: 1260–1263, 1992

    Google Scholar 

  • Reininger, L., Kaushik, A., Izui, S., and Jaton, J. C. A member of a new V H gene family encodes anti-bromelinized mouse red blood cell autoantibodies. Eur J Immunol 18: 1521–1526, 1988

    Google Scholar 

  • Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491, 1988

    Google Scholar 

  • Sakano, H., Maki, R., Kurosawa, Y., Roeder, W., and Tonegawa, S. Two types of somatic recombination are necessary for the generation of complete immunoglobulin heavy-chain genes. Nature 286: 676–683, 1980

    Google Scholar 

  • Sanchez, P., Marche, P. N., Rueff-Juy, D., and Cazenave, P. A. Mouse V-lambda-X gene sequence generates no junctional diversity and is conserved in mammalian species. J Immunol 144: 286–2820, 1990

    Google Scholar 

  • Shapiro, M. A. and Weigert, M. How immunoglobulin V kappa genes rearrange. J Immunol 139: 3834–3839, 1987

    Google Scholar 

  • Shefner, R., Mayer, R., Kaushik, A., D'Eustachio, P., Bona, C., and Diamond, B. Identification of a new V kappa gene family that is highly expressed in hybridomas from an autoimmune mouse strain. J Immunol 145: 1609–1614, 1990

    Google Scholar 

  • Seidman, J. G., Max, E. E., and Leder, P. A. A kapa-immunoglobulin gene is formed by site-specific recombination without further somatic mutation. Nature 280: 370–375, 1979

    Google Scholar 

  • Songsivilai, S., Bye, J. M., Marks, J. D., and Hughes-Jones, N. C. Cloning and sequencing of human lambda immunoglobulin genes by polymerase chain reaction. Eur J Immunol 20: 2661–2666, 1990

    Google Scholar 

  • Strohal, R., Helmberg, A., Kroemer, G., and Kofler, R. Mouse V kappa gene classification by nucleic acid sequence similarity. Immunogenetics 30: 475–493, 1989

    Google Scholar 

  • Tonegawa, S., Maxam, A. M., Tizard, R., Bernard, O. D., and Gilbert, W. Sequence of a mouse germ-line gene for a variable region of an immunoglobulin light chain. Proc Natl Acad Sci USA 75: 1485–1489, 1978

    Google Scholar 

  • Tutter, A., Brodeur, P. H. Shlomchik, M., and Riblet, R. Structure, map position, and evolution of twonewly diverged mouse Ig V H gene families. J Immunol 147: 3215–3223, 1991

    Google Scholar 

  • Valiante, N. M. and Caton, A. J. A new Igk-V gene family in the mouse. Immunogenetics 32: 345–350, 1990

    Google Scholar 

  • Weiss, S. and Wu, G. E. Somatic point mutations in unrearranged immunoglobulin gene segments encoding the variable region of lambda light chains. EMBO J 6: 927–932, 1987

    Google Scholar 

  • Williams, J. F. Optimization strategies for the polymerase chain reation. BioTechniques 7: 762–769, 1989

    Google Scholar 

  • Winter, E., Radbruch, A., and Krawinkel, U. Members of novel VH gene families are found in VDJ regions of polyclonally activated B-lymphocytes. EMBO J 4: 2861–2867, 1985

    Google Scholar 

  • Zhou, H., Fisher, R. J., and Papas, T. S. Optimization of primer sequences for mouse scFv repertoire display library construction. Nucleic Acids Res 22: 888–889, 1994

    Google Scholar 

  • Zwickl, M., Zaninetta, D., McMaster, G. K., and Hardman, N. Selective cloning of B cell hybridoma-specific rearranged immunoglobulin gene loci using the polymerase chain reaction. J Immunol Methods 130: 49–55, 1990

    Google Scholar 

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Authors and Affiliations

  1. Center for Bioengineering, University of Washington, 98195, Seattle, WA, USA

    Anupam K. Dattamajumdar

  2. Department of Molecular Biotechnology, University of Washington, School of Medicine, 98195, Seattle, WA, USA

    David P. Jacobson, Leroy E. Hood & Gamal E. Osman

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  1. Anupam K. Dattamajumdar
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  2. David P. Jacobson
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  3. Leroy E. Hood
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  4. Gamal E. Osman
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Additional information

The nucloetide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence database and have been assigned the accession number U32111

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Dattamajumdar, A.K., Jacobson, D.P., Hood, L.E. et al. Rapid cloning of any rearranged mouse immunoglobulin variable genes. Immunogenetics 43, 141–151 (1996). https://doi.org/10.1007/BF00176675

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  • DOI: https://doi.org/10.1007/BF00176675

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