Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Apr 15;90(8):3720–3724. doi: 10.1073/pnas.90.8.3720

Human immunoglobulin heavy-chain minilocus recombination in transgenic mice: gene-segment use in mu and gamma transcripts.

N Tuaillon 1, L D Taylor 1, N Lonberg 1, P W Tucker 1, J D Capra 1
PMCID: PMC46373  PMID: 8475122

Abstract

We (N.L. and L.D.T.) have introduced a human heavy-chain minilocus into mice transgenically. Constructs contain 2 heavy-chain variable (VH; psi VH3-105 and VH5-251), 10 diversity (D), 6 heavy-chain joining (JH), and either constant (C)mu or C mu and C gamma gene segments. Several founder lines were established and studied before immunization. Seventy heavy-chain transcripts were cloned and sequenced from murine splenic B lymphocytes, and gene-segment use was assessed before and after class-switching. In general, the repertoire was "fetal" in appearance with little evidence of somatic mutation in any gene segment. The two VH gene segments were found rearranged to mu- and gamma-chain C segments, with a preference of VH5-251. We observed a preponderance of the most-J-proximal D gene (DHQ52) segments among the mu transcripts (44%). The JH gene-segment use mimics most patterns seen in human antibodies. Diversification in CDR3 was extensive and included clear examples of D inversions and D-D fusions. These data suggest that a human immunoglobulin minilocus can undergo recombinatorial processes in a manner analogous to that seen in the human fetal/preimmune repertoire. This model, in addition to providing a potential source of human monoclonal antibodies, is ideal for the study of further questions concerning immunoglobulin gene-segment recombination.

Full text

PDF
3720

Images in this article

3723Fig. 4
on p.3723

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berman J. E., Humphries C. G., Barth J., Alt F. W., Tucker P. W. Structure and expression of human germline VH transcripts. J Exp Med. 1991 Jun 1;173(6):1529–1535. doi: 10.1084/jem.173.6.1529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brüggemann M., Caskey H. M., Teale C., Waldmann H., Williams G. T., Surani M. A., Neuberger M. S. A repertoire of monoclonal antibodies with human heavy chains from transgenic mice. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6709–6713. doi: 10.1073/pnas.86.17.6709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brüggemann M., Spicer C., Buluwela L., Rosewell I., Barton S., Surani M. A., Rabbitts T. H. Human antibody production in transgenic mice: expression from 100 kb of the human IgH locus. Eur J Immunol. 1991 May;21(5):1323–1326. doi: 10.1002/eji.1830210535. [DOI] [PubMed] [Google Scholar]
  4. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  5. Dersimonian H., McAdam K. P., Mackworth-Young C., Stollar B. D. The recurrent expression of variable region segments in human IgM anti-DNA autoantibodies. J Immunol. 1989 Jun 1;142(11):4027–4033. [PubMed] [Google Scholar]
  6. Feeney A. J. Lack of N regions in fetal and neonatal mouse immunoglobulin V-D-J junctional sequences. J Exp Med. 1990 Nov 1;172(5):1377–1390. doi: 10.1084/jem.172.5.1377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Guigou V., Guilbert B., Moinier D., Tonnelle C., Boubli L., Avrameas S., Fougereau M., Fumoux F. Ig repertoire of human polyspecific antibodies and B cell ontogeny. J Immunol. 1991 Feb 15;146(4):1368–1374. [PubMed] [Google Scholar]
  8. Huang C., Stewart A. K., Schwartz R. S., Stollar B. D. Immunoglobulin heavy chain gene expression in peripheral blood B lymphocytes. J Clin Invest. 1992 Apr;89(4):1331–1343. doi: 10.1172/JCI115719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hummel M., Berry J. K., Dunnick W. Switch region content of hybridomas: the two spleen cell Igh loci tend to rearrange to the same isotype. J Immunol. 1987 May 15;138(10):3539–3548. [PubMed] [Google Scholar]
  10. Humphries C. G., Shen A., Kuziel W. A., Capra J. D., Blattner F. R., Tucker P. W. A new human immunoglobulin VH family preferentially rearranged in immature B-cell tumours. Nature. 1988 Feb 4;331(6155):446–449. doi: 10.1038/331446a0. [DOI] [PubMed] [Google Scholar]
  11. Ichihara Y., Abe M., Yasui H., Matsuoka H., Kurosawa Y. At least five DH genes of human immunoglobulin heavy chains are encoded in 9-kilobase DNA fragments. Eur J Immunol. 1988 Apr;18(4):649–652. doi: 10.1002/eji.1830180426. [DOI] [PubMed] [Google Scholar]
  12. Ichihara Y., Matsuoka H., Kurosawa Y. Organization of human immunoglobulin heavy chain diversity gene loci. EMBO J. 1988 Dec 20;7(13):4141–4150. doi: 10.1002/j.1460-2075.1988.tb03309.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lutzker S., Alt F. W. Structure and expression of germ line immunoglobulin gamma 2b transcripts. Mol Cell Biol. 1988 Apr;8(4):1849–1852. doi: 10.1128/mcb.8.4.1849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lutzker S., Rothman P., Pollock R., Coffman R., Alt F. W. Mitogen- and IL-4-regulated expression of germ-line Ig gamma 2b transcripts: evidence for directed heavy chain class switching. Cell. 1988 Apr 22;53(2):177–184. doi: 10.1016/0092-8674(88)90379-0. [DOI] [PubMed] [Google Scholar]
  15. Meek K. D., Hasemann C. A., Capra J. D. Novel rearrangements at the immunoglobulin D locus. Inversions and fusions add to IgH somatic diversity. J Exp Med. 1989 Jul 1;170(1):39–57. doi: 10.1084/jem.170.1.39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nickerson K. G., Berman J., Glickman E., Chess L., Alt F. W. Early human IgH gene assembly in Epstein-Barr virus-transformed fetal B cell lines. Preferential utilization of the most JH-proximal D segment (DQ52) and two unusual VH-related rearrangements. J Exp Med. 1989 Apr 1;169(4):1391–1403. doi: 10.1084/jem.169.4.1391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pascual V., Capra J. D. Human immunoglobulin heavy-chain variable region genes: organization, polymorphism, and expression. Adv Immunol. 1991;49:1–74. doi: 10.1016/s0065-2776(08)60774-9. [DOI] [PubMed] [Google Scholar]
  18. Radbruch A., Müller W., Rajewsky K. Class switch recombination is IgG1 specific on active and inactive IgH loci of IgG1-secreting B-cell blasts. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3954–3957. doi: 10.1073/pnas.83.11.3954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  20. Sanz I., Casali P., Thomas J. W., Notkins A. L., Capra J. D. Nucleotide sequences of eight human natural autoantibody VH regions reveals apparent restricted use of VH families. J Immunol. 1989 Jun 1;142(11):4054–4061. [PubMed] [Google Scholar]
  21. Sanz I. Multiple mechanisms participate in the generation of diversity of human H chain CDR3 regions. J Immunol. 1991 Sep 1;147(5):1720–1729. [PubMed] [Google Scholar]
  22. Schroeder H. W., Jr, Hillson J. L., Perlmutter R. M. Early restriction of the human antibody repertoire. Science. 1987 Nov 6;238(4828):791–793. doi: 10.1126/science.3118465. [DOI] [PubMed] [Google Scholar]
  23. Schroeder H. W., Jr, Wang J. Y. Preferential utilization of conserved immunoglobulin heavy chain variable gene segments during human fetal life. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6146–6150. doi: 10.1073/pnas.87.16.6146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shen A., Humphries C., Tucker P., Blattner F. Human heavy-chain variable region gene family nonrandomly rearranged in familial chronic lymphocytic leukemia. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8563–8567. doi: 10.1073/pnas.84.23.8563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shimizu A., Honjo T. Immunoglobulin class switching. Cell. 1984 Apr;36(4):801–803. doi: 10.1016/0092-8674(84)90029-1. [DOI] [PubMed] [Google Scholar]
  26. Shimizu A., Nussenzweig M. C., Han H., Sanchez M., Honjo T. Trans-splicing as a possible molecular mechanism for the multiple isotype expression of the immunoglobulin gene. J Exp Med. 1991 Jun 1;173(6):1385–1393. doi: 10.1084/jem.173.6.1385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Siebenlist U., Ravetch J. V., Korsmeyer S., Waldmann T., Leder P. Human immunoglobulin D segments encoded in tandem multigenic families. Nature. 1981 Dec 17;294(5842):631–635. doi: 10.1038/294631a0. [DOI] [PubMed] [Google Scholar]
  28. Taylor L. D., Carmack C. E., Schramm S. R., Mashayekh R., Higgins K. M., Kuo C. C., Woodhouse C., Kay R. M., Lonberg N. A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins. Nucleic Acids Res. 1992 Dec 11;20(23):6287–6295. doi: 10.1093/nar/20.23.6287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Winter E., Krawinkel U., Radbruch A. Directed Ig class switch recombination in activated murine B cells. EMBO J. 1987 Jun;6(6):1663–1671. doi: 10.1002/j.1460-2075.1987.tb02415.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yamada M., Wasserman R., Reichard B. A., Shane S., Caton A. J., Rovera G. Preferential utilization of specific immunoglobulin heavy chain diversity and joining segments in adult human peripheral blood B lymphocytes. J Exp Med. 1991 Feb 1;173(2):395–407. doi: 10.1084/jem.173.2.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yaoita Y., Honjo T. Deletion of immunoglobulin heavy chain genes from expressed allelic chromosome. Nature. 1980 Aug 28;286(5776):850–853. doi: 10.1038/286850a0. [DOI] [PubMed] [Google Scholar]
  32. van der Heijden R. W., Bunschoten H., Pascual V., Uytdehaag F. G., Osterhaus D. M., Capra J. D. Nucleotide sequence of a human monoclonal anti-idiotypic antibody specific for a rabies virus-neutralizing monoclonal idiotypic antibody reveals extensive somatic variability suggestive of an antigen-driven immune response. J Immunol. 1990 Apr 1;144(7):2835–2839. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES