Abstract
Expression plasmids containing the human α1-antitrypsin (α1 AT) promoter fused to either adenine phosphoribosyltransferase (aprt) or xanthine-guanine phosphoribosyltransferase (gpt) coding sequences were sequentially introduced into APRT− HPRT− rat hepatoma cells. Stable transfectants expressing both transgenes were isolated and characterized. Nonexpressing variants were subsequently obtained by selecting against expression of one or both transgenes. Variants isolated by selecting against expression of either transgene alone generally displayed deficiency phenotypes incis, as only three of 20 clones tested were affected for expression of α1AT mRNA. In contrast, double selection yielded predominantlytrans effects: 12 of 14 lines tested showed impaired ability to express their chromosomal α1AT genes. Furthermore, expression of several other liver genes, including the gene encoding the HNF-1trans-activator, was repressed in many of the variant lines. Thus, double selection using chimeric transgenes is a useful approach for generating variant cell lines deficient in expression of specific mammalian genes.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Literature cited
Johnson, P.F., and McKnight, S.L. (1989).Annu. Rev. Biochem. 58799–839.
Ashman, C.R., and Davidson, R.L. (1987).Proc. Natl. Acad. Sci. U.S.A. 843354–3358.
Pellegrini, S., John, J., Shearer, M., Kerr, I.M., and Stark, G.R. (1989).Mol. Cell. Biol. 94605–4612.
Thacker, J., Debenham, P.G., Stretch, A., and Webb, M.B.T. (1983).Mutat. Res. 1119–23.
Tindall, K.R., Stankowski, L.F., Jr., Machanoff, R., and Hsie, A.W. (1984).Mol. Cell. Biol. 41411–1415.
Gebara, M.M., Drevon, C., Harcourt, S.A., Steingrimsdottir, H., James, M.R., Burke, J.F., Arlett, C.F., and Lehmann, A.R. (1987).Mol. Cell. Biol. 71459–1464.
DeSimone, V., and Cortese, R. (1988). InOxford Surveys on Eukaryotic Genes, Vol. 5 (ed.) McLean, N. (Oxford University Press, Oxford), pp. 51–96.
DeSimone, V., Ciliberto, G., Hardon, E., Paonessa, G., Palla, F., Lundber, L., and Cortese, R. (1987).EMBO J. 62759–2766.
Li, Y., Shen, R.F., Tsai, S.Y., and Woo, S.L.C. (1988).Mol. Cell. Biol. 84362–4369.
Monaci, P., Nicosia, A., and Cortese, R. (1988).EMBO J. 72075–2087.
Bulla, G.A., DeSimone, V., Cortese, R., and Fournier, R.E.K. (1992).Genes Dev. 6316–327.
Pitot, H.C., Peraino, C., Morse, P.A., and Potter, V.R. (1964).Natl. Cancer Inst. Monogr. 13229–242.
Chen, T.R. (1977).Exp. Cell Res. 104255–262.
Dush, M.K., Sikela, J.M., Khan, S.A., Tischfield, J.A., and Sambrook, R.J. (1985).Proc. Natl. Acad. Sci. U.S.A. 822731–2735.
Mulligan, R.C., and Berg, P. (1981).Mol. Cell. Biol. 1449–459.
Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982).Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
Chu, G., Hayakawa, H., and Berg, P. (1987).Nucleic Acids Res. 151311–1326.
Favaloro, J., Freisman, R., and Kamen, R. (1980).Methods Enzymol. 65718–749.
Cowan, N.J., Dobner, P.R., Fuchs, E.V., and Cleveland, D.W. (1983).Mol. Cell. Biol. 31738–1745.
Sargent, T.D., Wu, J.R., Sala-Trepat, J.M., Wallace, R., Reyes, A.A., and Bonner, J. (1979).Proc. Natl. Acad. Sci. U.S.A. 763256–3260.
Simon, M.P., Besmond, C., Cottreau, D., Weber, A., Chaumet-Riffaud, P., Dreyfus, J.C., Trepat, J.S., Marie, J., and Kahn, A. (1983).J. Biol. Chem. 25814576–14584.
Surh, L.C., Morris, S.M., O'Brien, W.E., and Beaudet, A.L. (1988).Nucleic Acids Res. 169352.
Yoo-Warren, H., Monahan, J.E., Short, J., Short, H., Bruzel, A., Wynshaw-Boris, A., Meisner, H.M., Samols, D., and Hanson, R.W. (1983).Proc. Natl. Acad. Sci. U.S.A. 803656–3660.
Frain, M., Swart, G., Monaci, P., Nicosia, A., Stämpfli, S., Frank, R., and Cortese, R. (1989).Cell 59145–157.
Feinberg, A.P., and Vogelstein, B. (1983).Anal. Biochem. 1326–13.
Bell, G.I., Karam, J.H., and Rutter, W.J. (1981).Proc. Natl. Acad. Sci. U.S.A. 785759–5763.
Mendel, D.B., and Crabtree, G.R. (1991).J. Biol. Chem. 266677–680.
Sladek, F.M., Zhong, W., Lai, E., and Darnell Jr., J.E. (1990).Genes Dev. 42353–2365.
Tripodi, M., Abbott, C., Vivian, N., Cortese, R., and Lovell-Badge, R. (1991).EMBO J. 103177–3182.
Tian, J.-M., and Schibler, U. (1991).Genes Dev. 52225–2234.
Kuo, C.J., Conley, P.B., Chen, L., Sladek, F.M., Darnell, J.E., and Crabtree, G.R. (1992).Nature 355457–461.
Deschatrette, J., Moore, E.E., Dubois, M., and Weiss, M.C. (1980).Cell 191043–1051.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Bulla, G.A., Fournier, R.E.K. Direct selection of hepatoma cell variants deficient in α1-antitrypsin gene expression. Somat Cell Mol Genet 18, 361–370 (1992). https://doi.org/10.1007/BF01235759
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF01235759