Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

New Genetic Methods for Mammalian Cells

Abstract

The power of genetics for the elucidation of gene function has been clearly established. In mammalian systems, the limited scope of available methods for the genetic manipulation of cultured cells has discouraged the widespread application of genetic approaches for the study of many current issues in molecular biology. Several recent technological developments hold out the promise of facilitating the manipulation of genes in mammalian cells. Three particular areas are emphasized in this review: 1) mutational analysis using nonsense suppression; 2) gene regulation by rapid, controlled amplification; and 3) gene targeting by homologous recombination.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Horowitz, N.H. and Leupold, U. .1951. Some recent studies bearing on the one gene-one enzyme hypothesis. Cold Spring Harbor Symp. Quant. Biol. 16:65–72.

    Article  CAS  Google Scholar 

  2. Campbell, A. .1961. Sensitive mutants of bacteriophage lambda. Virology 14:22–32.

    Article  CAS  Google Scholar 

  3. Basilico, C. .1977. Temperature-sensitive mutations in animal Cells. Adv. Cancer Res. 24:223–266.

    Article  CAS  Google Scholar 

  4. Marcus, M., Fainsod, A., and Diamond, G. 1985. The genetic analysis of mammalian Cell-cycle mutants. Ann. Rev. Genet. 19:389–421.

    Article  CAS  Google Scholar 

  5. Dermody, J.J., Wojcik, B.E., Du, H., and Ozer, H.L. .1986. Identification of temperature-sensitive DNA mutants of Chinese hamster Cells affected in Cellular and viral DNA synthesis. Mol. Cell. Biol. 6:4594–4601.

    Article  CAS  Google Scholar 

  6. Benzer, S. and Champe, S.P. 1962. A change from nonsense to sense in the genetic code. Proc. Natl. Acad. Sci. USA 48:1114–1121.

    Article  CAS  Google Scholar 

  7. Epstein, R.H., Bolle, A., Steinberg, C.M., Kellenberger, E. Boy de la Tour, E., Chevalley, R. Edgar, R.S., Slisman, M., Denhardt, G.H., and Lielausis, A. 1963. Physiological studies of conditional-lethal mutants of phage T4D. Cold Spring Harbor Symp. Quant. Biol. 28:375–392.

    Article  CAS  Google Scholar 

  8. Celis, J.E. and Smith, J.D. 1979. Nonsense Mutations and tRNA Suppressors. Academic Press, London.

  9. Miller, J.H., Coulondre, C., Hofer, M., Schmeissner, U., Sommer, H., and Schmitz, A. 1979. Genetic studies of the lac represser. IX. The generation of altered proteins by the suppression of nonsense mutations. J. Mol. Biol. 131:191–222.

    Article  CAS  Google Scholar 

  10. Sedivy, J.M., Capone, J.P., RajBhandary, U.L., and Sharp, P.A. 1987. An inducible mammalian amber suppressor: propagation of a poliovirus mutant. Cell 50:379–389.

    Article  CAS  Google Scholar 

  11. Laski, F.A., Belagaje, R., RajBhandary, U.L., and Sharp, P.A. 1982. An amber suppressor tRNA gene derived by site specific mutagenesis: cloning and expression in mammalian Cells. Proc. Natl. Acad. Sci. USA 79:5813–5817.

    Article  CAS  Google Scholar 

  12. Temple, G.F., Dozy, A.M., Roy, K.L., and Kan, Y.W. 1982. Construction of a functional human suppressor tRNA gene: an approach to gene therapy of thalassemia. Nature 296:537–540.

    Article  CAS  Google Scholar 

  13. Laski, F.A., Belagaje, R., Hudziak, R.M., Capecchi, M.R., Palese, P., RajBhandary, U.L., and Sharp, P.A. 1984. Synthesis of an ochre suppressor transfer RNA gene and expression in mammalian Cells. EMBO J. 3:2445–2452.

    Article  CAS  Google Scholar 

  14. Capone, J.P., Sharp, P.A., and RajBhandary, U.L. 1985. Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene. EMBO J. 4:213–221.

    Article  CAS  Google Scholar 

  15. Hudziak, R.M., Laski, F.A., RajBhandary, U.L., Sharp, P.A., and Capecchi, M.R. 1982. Establishment of mammalian Cell lines containing multiple nonsense mutations and functional suppressor transfer RNA genes. Cell 31:137–146.

    Article  CAS  Google Scholar 

  16. Young, J.F., Capecchi, M.R., Laski, F.A., RajBhandary, U.L., Sharp, P.A., and Palese, P. 1983. Measurement of suppressor transfer RNA activity. Science 221:873–875.

    Article  CAS  Google Scholar 

  17. Capone, J.P., Sedivy, J.M., Sharp, P.A., and RajBhandary, U.L. 1986. Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian Cells. Mol. Cell. Biol. 6:3059–3067.

    Article  CAS  Google Scholar 

  18. DePamphilis, M.L. and Bradley, M.K. 1986. Replication of SV40 and polyoma virus chromosomes. In: The Papovaviridae, Volume 1. N. P. Salzman, (Ed.). Plenum Press, NY. p.99–246

    Chapter  Google Scholar 

  19. Brockman, W.W. and Nathans, D. 1974. The isolation of simian virus 40 variants with specifically altered genomes. Proc. Natl. Acad. Sci. USA 71:942–946.

    Article  CAS  Google Scholar 

  20. Tooze, J. 1980. The Molecular Biology of Tumor Viruses. DNA Tumor Viruses, 2nd ed. Cold Spring Harbor Laboratory, NY.

    Google Scholar 

  21. Rio, D.C., Clark, S.G., and Tjian, R. 1986. A mammalian host-vector system that regulates expression and amplification of transfected genes by temperature induction. Science 227:23–28.

    Article  Google Scholar 

  22. Botchan, M., Topp, W., and Sambrook, J. 1979. Studies on simian virus 40 excision from Cellular chromosomes. Cold Spring Harbor Symp. Quant. Biol. 43:709–719.

    Article  CAS  Google Scholar 

  23. Kern, F.G. and Basilico, C. 1986. An inducible eukaryotic host-vector expression system: amplification of genes under the control of the polyoma late promoter in a Cell line producing a thermolabile large T antigen. Gene 43:237–245.

    Article  CAS  Google Scholar 

  24. Portela, A., Melero, J.A., de la Luna, S., and Ortin, J. 1986. Construction of Cell lines that regulate by temperature the amplification and expression of influenza virus non-structural protein genes. EMBO J. 5:2387–2392.

    Article  CAS  Google Scholar 

  25. Kaufman, R.D. and Sharp, P.A. 1982. Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene. J. Mol. Biol. 159:601–621.

    Article  CAS  Google Scholar 

  26. Davie, E.W. and Fujikawa, K. 1975. Basic mechanisms in blood coagulation. Ann. Rev. Biochem. 44:799–829.

    Article  CAS  Google Scholar 

  27. Barbacid, M. 1987. Ras genes. Ann. Rev. Biochem. 56:779–828.

    Article  CAS  Google Scholar 

  28. Orr-Weaver, T.L., Szostak, J.W., and Rothstein, R.J. 1981. Yeast transformation: a model system for study of recombination. Proc. Natl. Acad. Sci. USA 78:6354–6358.

    Article  CAS  Google Scholar 

  29. Rothstein, R. 1983. One-step gene disruption in yeast. Meth. Enzymol. 101:202–211.

    Article  CAS  Google Scholar 

  30. Mammalian homologous recombination. 1984. In: Cold Spring Harbor Symp. Quant. Biol. 49:123–198.

  31. Subramani, S. and Berg, P. 1983. Homologous and nonhomologous recombination in monkey Cells. Mol. Cell. Biol. 3:1040–1052.

    Article  CAS  Google Scholar 

  32. Lin, F.-L., Sperle, K., and Sternberg, N. 1985. Recombination in mouse L Cells between DNA introduced into Cells and homologous chromosomal sequences. Proc. Natl. Acad. Sci. USA 82:1391–1395.

    Article  CAS  Google Scholar 

  33. Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A., and Kucherlapati, R.S. 1985. Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination. Nature 317:230–234.

    Article  CAS  Google Scholar 

  34. Thomas, K.R., and Capecchi, M.R. (1987). Site-directed mutagenesis by gene targeting in mouse embryo-derived stem Cells. Cell 51:503–512.

    Article  CAS  Google Scholar 

  35. Smithies, O., Koralewski, M.A., Song, K.-Y., and Kucherlapati, R.S. 1984. Homologous recombination with DNA introduced into mammalian Cells. Cold Spring Harbor Symp. Quant. Biol. 48:161–170.

    Article  Google Scholar 

  36. Thomas, K.R., Folger, K.R., and Capecchi, M.R. 1986. High frequency targeting of genes to specific sites in the mammalian genome. Cell 44:419–428.

    Article  CAS  Google Scholar 

  37. Song, K.-Y., Schwartz, F., Maeda, N., Smithies, O., and Kucherlapati, R. 1987. Accurate modification of a chromosomal plasmid by homologous recombination in human Cells. Proc. Natl. Acad. Sci. USA 84:6820–6824.

    Article  CAS  Google Scholar 

  38. Doetschman, T., Gregg, R.G., Maeda, N., Hooper, M.L., Melton, D.W., Thompson, S., and Smithies, O. 1987. Targeted correction of a mutant HPRT gene in mouse embryonic stem Cells. Nature 330:576–578.

    Article  CAS  Google Scholar 

  39. Lin, F.-L., Sperle, K., and Sternberg, N. 1984. Model for homologous recombination during transfer of DNA into mouse L Cells: role for DNA ends in the recombination process. Mol. Cell. Biol. 4:1020–1034.

    Article  CAS  Google Scholar 

  40. Kucherlapati, R.S., Eves, E.M., Song, K.-Y., Morse, B.S., and Smithies, O. 1984. Homologous recombination between plasmids in mammalian Cells can be enhanced by treatment of input DNA. Proc. Natl. Acad. Sci.USA 81:3153–3157.

    Article  CAS  Google Scholar 

  41. Wake, C.T., Vernaleone, F., and Wilson, J.H. 1985. Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells. Mol. Cell. Biol. 5:2080–2089.

    Article  CAS  Google Scholar 

  42. Chang, X.-B. and Wilson, J.H. 1987. Modification of DNA ends can decrease end joining relative to homologous recombination in mammalian cells. Proc. Natl. Acad. Sci. USA 84:4959–4963.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sedivy, J. New Genetic Methods for Mammalian Cells. Nat Biotechnol 6, 1192–1196 (1988). https://doi.org/10.1038/nbt1088-1192

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1088-1192

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing