Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1992 May;12(5):2391–2395. doi: 10.1128/mcb.12.5.2391

Production of homozygous mutant ES cells with a single targeting construct.

R M Mortensen 1, D A Conner 1, S Chao 1, A A Geisterfer-Lowrance 1, J G Seidman 1
PMCID: PMC364411  PMID: 1569957

Abstract

We have developed a simple method for producing embryonic stem (ES) cell lines whereby both alleles have been inactivated by homologous recombination and which requires a single targeting construct. Four different ES cell lines were created that were heterozygous for genes encoding two guanine nucleotide-binding protein subunits, alpha i2 and alpha i3, T-cell receptor alpha, and beta-cardiac myosin heavy chain. When these heterozygous cells were grown in high concentrations of G418, many of the surviving cells were homozygous for the targeted allele and contained two copies of the G418 resistance gene. This scheme provides an easy method for obtaining homozygous mutationally altered cells, i.e., double knockouts, and should be generally applicable to other genes and to cell lines other than ES cells. This method should also enable the production of cell lines in which more than one gene have had both alleles disrupted. These mutant cells should provide useful tools for defining the role of particular genes in cell culture.

Full text

PDF
2391

Images in this article

2393Image
on p.2393
2393Image
on p.2393

Selected References

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

  1. Campbell C. E., Worton R. G. Segregation of recessive phenotypes in somatic cell hybrids: role of mitotic recombination, gene inactivation, and chromosome nondisjunction. Mol Cell Biol. 1981 Apr;1(4):336–346. doi: 10.1128/mcb.1.4.336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cruz A., Coburn C. M., Beverley S. M. Double targeted gene replacement for creating null mutants. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7170–7174. doi: 10.1073/pnas.88.16.7170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Doetschman T., Gregg R. G., Maeda N., Hooper M. L., Melton D. W., Thompson S., Smithies O. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature. 1987 Dec 10;330(6148):576–578. doi: 10.1038/330576a0. [DOI] [PubMed] [Google Scholar]
  4. Groden J., Nakamura Y., German J. Molecular evidence that homologous recombination occurs in proliferating human somatic cells. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4315–4319. doi: 10.1073/pnas.87.11.4315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hayday A. C., Diamond D. J., Tanigawa G., Heilig J. S., Folsom V., Saito H., Tonegawa S. Unusual organization and diversity of T-cell receptor alpha-chain genes. 1985 Aug 29-Sep 4Nature. 316(6031):828–832. doi: 10.1038/316828a0. [DOI] [PubMed] [Google Scholar]
  6. Joyner A. L., Skarnes W. C., Rossant J. Production of a mutation in mouse En-2 gene by homologous recombination in embryonic stem cells. Nature. 1989 Mar 9;338(6211):153–156. doi: 10.1038/338153a0. [DOI] [PubMed] [Google Scholar]
  7. Mansour S. L., Thomas K. R., Capecchi M. R. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature. 1988 Nov 24;336(6197):348–352. doi: 10.1038/336348a0. [DOI] [PubMed] [Google Scholar]
  8. Mortensen R. M., Zubiaur M., Neer E. J., Seidman J. G. Embryonic stem cells lacking a functional inhibitory G-protein subunit (alpha i2) produced by gene targeting of both alleles. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7036–7040. doi: 10.1073/pnas.88.16.7036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Nelson F. K., Frankel W., Rajan T. V. Mitotic recombination is responsible for the loss of heterozygosity in cultured murine cell lines. Mol Cell Biol. 1989 Mar;9(3):1284–1288. doi: 10.1128/mcb.9.3.1284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Paludan K., Duch M., Jørgensen P., Kjeldgaard N. O., Pedersen F. S. Graduated resistance to G418 leads to differential selection of cultured mammalian cells expressing the neo gene. Gene. 1989 Dec 28;85(2):421–426. doi: 10.1016/0378-1119(89)90435-6. [DOI] [PubMed] [Google Scholar]
  11. Potter T. A., Zeff R. A., Frankel W., Rajan T. V. Mitotic recombination between homologous chromosomes generates H-2 somatic cell variants in vitro. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1634–1637. doi: 10.1073/pnas.84.6.1634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Rajan T. V., Moffat L. F., Frankel W. N. Rate and mechanism of generation of beta 2-microglobulin mutants from a heterozygous murine cell line. J Immunol. 1990 Sep 1;145(5):1598–1602. [PubMed] [Google Scholar]
  13. Schwartzberg P. L., Goff S. P., Robertson E. J. Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells. Science. 1989 Nov 10;246(4931):799–803. doi: 10.1126/science.2554496. [DOI] [PubMed] [Google Scholar]
  14. Thomas K. R., Capecchi M. R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987 Nov 6;51(3):503–512. doi: 10.1016/0092-8674(87)90646-5. [DOI] [PubMed] [Google Scholar]
  15. Wasmuth J. J., Vock Hall L. Genetic demonstration of mitotic recombination in cultured Chinese hamster cell hybrids. Cell. 1984 Mar;36(3):697–707. doi: 10.1016/0092-8674(84)90350-7. [DOI] [PubMed] [Google Scholar]
  16. Zijlstra M., Li E., Sajjadi F., Subramani S., Jaenisch R. Germ-line transmission of a disrupted beta 2-microglobulin gene produced by homologous recombination in embryonic stem cells. Nature. 1989 Nov 23;342(6248):435–438. doi: 10.1038/342435a0. [DOI] [PubMed] [Google Scholar]
  17. Zimmer A., Gruss P. Production of chimaeric mice containing embryonic stem (ES) cells carrying a homoeobox Hox 1.1 allele mutated by homologous recombination. Nature. 1989 Mar 9;338(6211):150–153. doi: 10.1038/338150a0. [DOI] [PubMed] [Google Scholar]
  18. te Riele H., Maandag E. R., Clarke A., Hooper M., Berns A. Consecutive inactivation of both alleles of the pim-1 proto-oncogene by homologous recombination in embryonic stem cells. Nature. 1990 Dec 13;348(6302):649–651. doi: 10.1038/348649a0. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES