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. 1985 Oct;82(19):6600–6604. doi: 10.1073/pnas.82.19.6600

Retroviral insertional mutagenesis of a target allele in a heterozygous murine cell line.

W Frankel, T A Potter, N Rosenberg, J Lenz, T V Rajan
PMCID: PMC391257  PMID: 2995973

Abstract

A clonal murine cell line that is heterozygous at the beta 2-microglobulin locus (B2m) was obtained by Abelson murine leukemia virus (Ab-MuLV) transformation of liver cells from (C57BL/6 X BALB/c) F1 fetuses. To obtain proviral insertional mutants, we superinfected a subclone of these cells, which does not express the env surface protein of the Moloney leukemia virus (Mo-MuLV, the helper virus that was used to transmit the defective Ab-MuLV genome during transformation), with Mo-MuLV. Mutant clones that fail to express the C57BL/6 allele of B2m (B2mb) were then immunoselected by using a monoclonal antibody that specifically recognizes the B2mb gene product and not that of the B2ma allele. Of 22 independent clones obtained, one contains a proviral insertion that is near or in the first exon of the B2mb gene. Surprisingly, the insertion is of the Ab-MuLV genome and not of replication-competent Mo-MuLV. This indicates that superinfection with Mo-MuLV "rescued" the defective Ab-MuLV genome, which then inserted into the B2mb gene. We conclude that when an allele-specific selection procedure exists, proviral insertion is a potential method for obtaining mutations in heterozygous mammalian cells. This approach may thereby provide a method for molecular cloning of such selectable genes, using a retroviral hybridization probe.

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Selected References

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

  1. Bingham P. M., Levis R., Rubin G. M. Cloning of DNA sequences from the white locus of D. melanogaster by a novel and general method. Cell. 1981 Sep;25(3):693–704. doi: 10.1016/0092-8674(81)90176-8. [DOI] [PubMed] [Google Scholar]
  2. Copeland N. G., Jenkins N. A., Lee B. K. Association of the lethal yellow (Ay) coat color mutation with an ecotropic murine leukemia virus genome. Proc Natl Acad Sci U S A. 1983 Jan;80(1):247–249. doi: 10.1073/pnas.80.1.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Goff S. P., Gilboa E., Witte O. N., Baltimore D. Structure of the Abelson murine leukemia virus genome and the homologous cellular gene: studies with cloned viral DNA. Cell. 1980 Dec;22(3):777–785. doi: 10.1016/0092-8674(80)90554-1. [DOI] [PubMed] [Google Scholar]
  4. Hyman R., Trowbridge I. Analysis of lymphocyte surface antigen expression by the use of variant cell lines. Cold Spring Harb Symp Quant Biol. 1977;41(Pt 1):407–415. doi: 10.1101/sqb.1977.041.01.049. [DOI] [PubMed] [Google Scholar]
  5. Jaenisch R., Harbers K., Schnieke A., Löhler J., Chumakov I., Jähner D., Grotkopp D., Hoffmann E. Germline integration of moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death. Cell. 1983 Jan;32(1):209–216. doi: 10.1016/0092-8674(83)90511-1. [DOI] [PubMed] [Google Scholar]
  6. Jenkins N. A., Copeland N. G., Taylor B. A., Lee B. K. Dilute (d) coat colour mutation of DBA/2J mice is associated with the site of integration of an ecotropic MuLV genome. Nature. 1981 Oct 1;293(5831):370–374. doi: 10.1038/293370a0. [DOI] [PubMed] [Google Scholar]
  7. King W., Patel M. D., Lobel L. I., Goff S. P., Nguyen-Huu M. C. Insertion mutagenesis of embryonal carcinoma cells by retroviruses. Science. 1985 May 3;228(4699):554–558. doi: 10.1126/science.3838595. [DOI] [PubMed] [Google Scholar]
  8. Kuff E. L., Feenstra A., Lueders K., Smith L., Hawley R., Hozumi N., Shulman M. Intracisternal A-particle genes as movable elements in the mouse genome. Proc Natl Acad Sci U S A. 1983 Apr;80(7):1992–1996. doi: 10.1073/pnas.80.7.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Michaelson J. Genetics of beta-2 microglobulin in the mouse. Immunogenetics. 1983;17(3):219–260. doi: 10.1007/BF00364409. [DOI] [PubMed] [Google Scholar]
  10. Parnes J. R., Seidman J. G. Structure of wild-type and mutant mouse beta 2-microglobulin genes. Cell. 1982 Jun;29(2):661–669. doi: 10.1016/0092-8674(82)90182-9. [DOI] [PubMed] [Google Scholar]
  11. Rajan T. V., Halay E. D., Potter T. A., Evans G. A., Seidman J. G., Margulies D. H. H-2 hemizygous mutants from a heterozygous cell line: role of mitotic recombination. EMBO J. 1983;2(9):1537–1542. doi: 10.1002/j.1460-2075.1983.tb01620.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Reddy E. P., Smith M. J., Srinivasan A. Nucleotide sequence of Abelson murine leukemia virus genome: structural similarity of its transforming gene product to other onc gene products with tyrosine-specific kinase activity. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3623–3627. doi: 10.1073/pnas.80.12.3623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Roeder G. S., Fink G. R. DNA rearrangements associated with a transposable element in yeast. Cell. 1980 Aug;21(1):239–249. doi: 10.1016/0092-8674(80)90131-2. [DOI] [PubMed] [Google Scholar]
  14. Rosenberg N. E., Clark D. R., Witte O. N. Abelson murine leukemia virus mutants deficient in kinase activity and lymphoid cell transformation. J Virol. 1980 Dec;36(3):766–774. doi: 10.1128/jvi.36.3.766-774.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rosenberg N., Baltimore D. A quantitative assay for transformation of bone marrow cells by Abelson murine leukemia virus. J Exp Med. 1976 Jun 1;143(6):1453–1463. doi: 10.1084/jem.143.6.1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rosenberg N., Baltimore D. The effect of helper virus on Abelson virus-induced transformation of lymphoid cells. J Exp Med. 1978 Apr 1;147(4):1126–1141. doi: 10.1084/jem.147.4.1126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schnieke A., Harbers K., Jaenisch R. Embryonic lethal mutation in mice induced by retrovirus insertion into the alpha 1(I) collagen gene. 1983 Jul 28-Aug 3Nature. 304(5924):315–320. doi: 10.1038/304315a0. [DOI] [PubMed] [Google Scholar]
  18. Searles L. L., Jokerst R. S., Bingham P. M., Voelker R. A., Greenleaf A. L. Molecular cloning of sequences from a Drosophila RNA polymerase II locus by P element transposon tagging. Cell. 1982 Dec;31(3 Pt 2):585–592. doi: 10.1016/0092-8674(82)90314-2. [DOI] [PubMed] [Google Scholar]
  19. Shinnick T. M., Lerner R. A., Sutcliffe J. G. Nucleotide sequence of Moloney murine leukaemia virus. Nature. 1981 Oct 15;293(5833):543–548. doi: 10.1038/293543a0. [DOI] [PubMed] [Google Scholar]
  20. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  21. Tada N., Kimura S., Hatzfeld A., Hämmerling U. Ly-m11: the H-3 region of mouse chromosome 2 controls a new surface alloantigen. Immunogenetics. 1980;11(5):441–449. doi: 10.1007/BF01567813. [DOI] [PubMed] [Google Scholar]
  22. Varmus H. E., Quintrell N., Ortiz S. Retroviruses as mutagens: insertion and excision of a nontransforming provirus alter expression of a resident transforming provirus. Cell. 1981 Jul;25(1):23–36. doi: 10.1016/0092-8674(81)90228-2. [DOI] [PubMed] [Google Scholar]
  23. Weinberg R. A. Integrated genomes of animal viruses. Annu Rev Biochem. 1980;49:197–226. doi: 10.1146/annurev.bi.49.070180.001213. [DOI] [PubMed] [Google Scholar]
  24. Wolf D., Rotter V. Inactivation of p53 gene expression by an insertion of Moloney murine leukemia virus-like DNA sequences. Mol Cell Biol. 1984 Jul;4(7):1402–1410. doi: 10.1128/mcb.4.7.1402. [DOI] [PMC free article] [PubMed] [Google Scholar]

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