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Cointegration of DNA Molecules Introduced into Mammalian Cells by Electroporation

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Somatic Cell and Molecular Genetics

Abstract

Electroporation was used to introduce a mixture of two plasmid-cloned genes into Chinese hamster ovary (CHO) cells, and the location of the two genes was subsequently determined by fluorescence in situ hybridization (FISH). The 25 kb Chinese hamster gene for dihydrofolate reductase (dhfr) in the form of a cosmid-derived 40 kb BglI fragment and the SV40 promoter-driven E. coli gene for guanine phosphoribosyltransferase (gpt) were co-electroporated and gpt+transfectants selected. Clones that had also integrated a single copy of the dhfr gene were studied by 2-color fluorescence in situ hybridization (FISH) to localize the integration site(s) of the exogenous DNA in metaphase chromosomes. All 9 clones examined showed co-localization of the two transgenes. The chromosomal site of integration was different in each clone. Co-integration was confirmed by co-amplification experiments. We conclude that, even when provided at low concentrations, separate soluble DNA molecules become linked upon gene transfer by electroporation, either by intracellular ligation prior to integration, or by co-integration at a common site in a given recipient cell.

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LITERATURE CITED

  1. Graham, F.L., and van der Eb, A.J. (1973). Virology 52:456–467.

    PubMed  Google Scholar 

  2. Perucho, M., Hanahan, D., and Wigler, M. (1980). Cell 22:309–317.

    PubMed  Google Scholar 

  3. Robins, D.M., Ripley, S., Henderson, A.S., and Axel, R. (1981). Cell 23:29–39.

    PubMed  Google Scholar 

  4. Wigler, M., Sweet, R., Sim, G.K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, S., and Axel, R. (1979). Cell 16:777–786.

    PubMed  Google Scholar 

  5. Wigler, M., Perucho, M., Kurtz, D., Dana, S., Pellicer, A., Axel, R., and Silverstein, S. (1980). Proc. Natl. Acad. Sci. U.S.A. 77:3567–3570.

    PubMed  Google Scholar 

  6. Shigekawa, K., and Dower, W.J. (1988). Biotechniques 6:742–751.

    PubMed  Google Scholar 

  7. Potter, H. (1993). Meth. Enzymol. 217:461–478.

    PubMed  Google Scholar 

  8. Chaney, W.G., Howard, D.R., Pollard, J.W., Sallustio, S., and Stanley, P. (1986). Som. Cell Molec. Genet. 12:237–244.

    Google Scholar 

  9. Boggs, S.S., Gregg, R.G., Borenstein, N., and Smithies, O. (1986). Exper. Hematol. 14:988–994.

    Google Scholar 

  10. Kuo, C.L., and Hood, L. (1987). Proc. Natl. Acad. Sci. U.S.A. 84:7614–7618.

    PubMed  Google Scholar 

  11. Lamon-Fava, S., Ordovas, J.M., Mandel, G., Forte, T.M., Goodman, R.H., and Schaefer, E.J. (1987). J. Biol. Chem. 262:8944–8947.

    PubMed  Google Scholar 

  12. Weeks, R.S., and Sibley, C.H. (1988). J. Immunol. 15:1312–1320.

    Google Scholar 

  13. Urlaub, G., and Chasin, L.A. (1980). Proc. Natl. Acad. Sci. U.S.A. 77:4216–4220.

    PubMed  Google Scholar 

  14. Urlaub, G., Mitchell, P.J., Kas, E., Chasin, L.A., Funanage, V.L., Myoda, T.T., and Hamlin, J.L. (1986). Somat. Cell and Molec. Genet. 12:555–566.

    Google Scholar 

  15. Mulligan, R.C., and Berg, P. (1980). Science 209:1422–1427.

    PubMed  Google Scholar 

  16. Urlaub, G., Carothers, A.M., and Chasin, L.A. (1985). Proc. Natl. Acad. Sci. U.S.A. 82:1189–1193.

    PubMed  Google Scholar 

  17. Nunberg, J.H., Kaufman, R.J., Schimke, R.T., Urlaub, G., and Chasin, L.A. (1978). Proc. Nat. Acad. Sci. U.S.A. 75:5553–5556.

    Google Scholar 

  18. Carothers, A.M., Urlaub, G., Ellis, N., and Chasin, L.A. (1983). Nuclei. Acids Res. 11:1997–2012.

    Google Scholar 

  19. Lowy, I., Pellicer, A., Jackson, J.F., Sim, G-K., Silverstein, S., and Axel, R. (1980). Cell 22:817–823.

    PubMed  Google Scholar 

  20. Chen, I-T., and Chasin, L.A. (1993). Mol. Cell. Biol. 13:289–300.

    PubMed  Google Scholar 

  21. Trask, B.J. (1991). Methods Cell Biol. 35:3–35.

    PubMed  Google Scholar 

  22. Carothers, A.M., Urlaub, G., Steigerwalt, R.W., Chasin, L.A., and Grunberger, D. (1986). Proc. Natl. Acad. Sci. U.S.A. 83:6519–6523.

    PubMed  Google Scholar 

  23. Gallois, P., Lindsey, K., Malone, R., Kreis, M., and Jones, M.G.K. (1992). Nucleic Acids Res. 20:3977–3982.

    PubMed  Google Scholar 

  24. Toneguzzo, F., Keating, A., Glynn, S., and McDonald, K. (1988). Nucleic Acids Res. 16:5515–5532.

    PubMed  Google Scholar 

  25. Barsoum, J. (1990). DNA Cell Biol. 9:293–300.

    PubMed  Google Scholar 

  26. Reid, L.H., Shesely, E.G., Kim, H.S., and Smithies, O. (1991). Mol. Cell. Biol. 11:2769–2777.

    PubMed  Google Scholar 

  27. van Duin, M., Westerveld, A., and Hoeijmakers, J.H. (1985). Mol. Cell. Biol. 5:734–741.

    PubMed  Google Scholar 

  28. Rubin, J.S. (1988). Somat. Cell Molec. Genet. 14:613–621.

    PubMed  Google Scholar 

  29. Yorifuji, T., and Mikawa, H. (1990). Mutation Res. 243:121–126.

    PubMed  Google Scholar 

  30. Fujimaki, K., Aratani, Y., Fujisawa, S., Motomura, S., Okubo, T., and Koyama, H. (1996). Somat. Cell Molec. Genet. 22:279–290.

    PubMed  Google Scholar 

  31. Merrihew, R.V., Marburger, K., Pennington, S.L., Roth, D.B., and Wilson, J.H. (1996). Mol. Cell. Biol. 16:10–18.

    PubMed  Google Scholar 

  32. Rouet, P., Smih, F., and Jasin, M. (1994). Proc. Natl. Acad. Sci. U.S.A. 91:6064–6068.

    PubMed  Google Scholar 

  33. Reff, M.E., and Pfarr, D.S. (1993). In: Gene Amplification in Mammalian Cells, R. Kellems, editor, Marcel Dekker, N.Y. pp. 355–367.

    Google Scholar 

  34. Sauer, B. (1998). Methods. 14:381–392.

    PubMed  Google Scholar 

  35. Ma, C., Martin, S., Trask, B., and Hamlin, J.L. (1993). Genes & Dev. 7:605–620.

    Google Scholar 

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Authors and Affiliations

  1. Department of Biological Sciences, Columbia University, New York, New York, 10027. Present address: The Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California 94141

    Chao Chen & Lawrence A. Chasin

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  1. Chao Chen
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  2. Lawrence A. Chasin
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Chen, C., Chasin, L.A. Cointegration of DNA Molecules Introduced into Mammalian Cells by Electroporation. Somat Cell Mol Genet 24, 249–256 (1998). https://doi.org/10.1023/B:SCAM.0000007127.80657.10

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  • DOI: https://doi.org/10.1023/B:SCAM.0000007127.80657.10

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