Abstract
Continuous regulation is required to maintain a given cell state1,2 or to allow it to change in response to the environment3,4. Studies of the mechanisms underlying such regulation have often been hindered by the inability to control gene expression at will. Among the inducible systems available for regulating gene expression in eukaryotes5,7,8, the tetracycline (tet) regulatable system has distinct advantages9,10,11. It is highly specific, non-toxic and non-eukaryotic, and consequently does not have pleiotropic effects on host cell genes. Previously this system also had drawbacks, as it did not extinguish gene expression completely, precluding the study of toxic or growth-inhibitory gene products. We report here the development of a facile reversible tetracycline-inducible retroviral system (designated RetroTet-ART) in which activators and repressors together are expressed in cells. Gene expression can now be actively repressed in the absence of tet and induced in the presence of tet, as we have engineered distinct dimerization domains that allow co-expression of homodimeric tet-regulated transactivators and transrepressors in the same cells, without the formation of non-functional heterodimers. Using this system, we show that growth arrest by the cell cycle inhibitor p16 is reversible and dependent on its continuous expression.
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References
Blau, H.M. & Baltimore, D. Differentiation requires continuous regulation. J. Cell. Biol. 112, 781– 783 (1991).
Blau, H.M. Differentiation requires continuous active control. Annu. Rev. Biochem. 61, 1213–1230 ( 1992).
McKay, R. Stem cells in the central nervous system. Science 276 , 66–71 (1997).
Dupin, E., Ziller, C. & Le Douarin, N.M. The avian embryo as a model in developmental studies: chimeras and in vitro clonal analysis. Curr. Top. Dev. Biol. 36, 1–35 (1998 ).
No, D., Yao, T.P. & Evans, R.M. Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc. Natl Acad. Sci. USA 93, 3346–3351 (1996).
Rivera, V.M. et al. A humanized system for pharmacologic control of gene expression. Nature Med. 2, 1028–1032 (1996).
Wang, Y., O'Malley, B.W. Jr, Tsai, S.Y. & O'Malley, B.W. A regulatory system for use in gene transfer. Proc. Natl Acad. Sci. USA 91, 8180– 8184 (1994).
Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl Acad. Sci. USA 89, 5547–5551 (1992).
Kringstein, A.M., Rossi, F.M.V., Hofmann, A. & Blau, H.M. Graded transcriptional response to different concentrations of a single transactivator. Proc. Natl Acad. Sci. USA 95, 13670– 13675 (1998).
Gossen, M. et al. Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766– 1769 (1995).
Bohl, D., Naffakh, N. & Heard, J.M. Long-term control of erythropoietin secretion by doxycycline in mice transplanted with engineered primary myoblasts. Nature Med. 3, 299–305 ( 1997).
Moosmann, P., Georgiev, O., Le Douarin, B., Bourquin, J.P. & Schaffner, W. Transcriptional repression by RING finger protein TIF1 β that interacts with the KRAB repressor domain of KOX1. Nucleic Acids Res. 24, 4859– 4867 (1996).
Deuschle, U., Meyer, W.K. & Thiesen, H.J. Tetracycline-reversible silencing of eukaryotic promoters. Mol. Cell. Biol. 15, 1907– 1914 (1995).
Hinrichs, W. et al. Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance. Science 264, 418 –420 (1994).
Kisker, C., Hinrichs, W., Tovar, K., Hillen, W. & Saenger, W. The complex formed between Tet repressor and tetracycline-Mg2+ reveals mechanism of antibiotic resistance. J. Mol. Biol. 247, 260–280 ( 1995).
Zhao, J. & Aoki, T. Nucleotide sequence analysis of the class G tetracycline resistance determinant from Vibrio anguillarum. Microbiol. Immunol. 36, 1051–1060 (1992).
Hillen, W. & Berens, C. Mechanisms underlying expression of Tn10 encoded tetracycline resistance. Annu. Rev. Microbiol. 48, 345–369 ( 1994).
Nakauchi, H. et al. Molecular cloning of Lyt-2, a membrane glycoprotein marking a subset of mouse T lymphocytes: molecular homology to its human counterpart, Leu- 2/T8, and to immunoglobulin variable regions. Proc. Natl Acad. Sci. USA 82, 5126–5130 ( 1985).
Sherr, C.J. Cancer cell cycles. Science 274, 1672– 1677 (1996).
Poulos, N.E., Farmer, A.A., Chan, K.W. & Stanbridge, E.J. Design of a novel bicistronic expression vector with demonstration of a p16INK4-induced G(1)-S block(1). Cancer Res. 56, 1719– 1723 (1996).
Elledge, S.J. Cell cycle checkpoints: preventing an identity crisis. Science 274, 1664–1672 ( 1996).
Brockes, J.P. Amphibian limb regeneration: rebuilding a complex structure. Science 276, 81–87 ( 1997).
Sah, D.W., Ray, J. & Gage, F.H. Bipotent progenitor cell lines from the human CNS. Nature Biotechnol . 15, 574–580 ( 1997).
Morgenstern, J.P. & Land, H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18, 3587–3596 (1990).
Galbraith, D.W., Anderson, M.T. & Herzenberg, L.A. Flowcytometric analysis and sorting of cells based on GFP accumulation. Meth. Cell. Biol. (in press).
Hofmann, A., Nolan, G.P. & Blau, H.M. Rapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette. Proc. Natl Acad. Sci. USA 93, 5185–5190 ( 1996).
Riviere, I., Brose, K. & Mulligan, R.C. Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells. Proc. Natl Acad. Sci. USA 92, 6733–6737 (1995).
Springer, M.L. & Blau, H.M. High-efficiency retroviral infection of primary myoblasts. Somat. Cell. Mol. Genet. 23, 203–209 ( 1997).
Pear, W.S., Nolan, G.P., Scott, M.L. & Baltimore, D. Production of high-titer helper-free retroviruses by transient transfection. Proc. Natl Acad. Sci. USA 90, 8392– 8396 (1993).
Spicher, A., Etter, A., Bernard, V., Tobler, H. & Muller, F. Extremely stable transcripts may compensate for the elimination of the gene fert-1 from all Ascaris lumbricoides somatic cells. Dev. Biol. 164, 72– 86 (1994).
Yu, S.F. et al. Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc. Natl Acad. Sci. USA 83, 3194–3198 (1986).
Acknowledgements
We thank G. Nolan, K. Cimprich, P. Jackson and M. Springer for helpful critique. We are grateful to T. Aoki, C.J. Sherr, G. Nolan, M. Anderson and U. Deuschle for providing cDNAs. We thank A. Aslanian and E. Sanjines for technical assistance. This work was supported by postdoctoral fellowships from the Human Frontiers in Science Program (LT 623/96) to F.M.V.R., from the Swiss National Science Foundation (823A-46704) to A.S., by a summer undergraduate research fellowship from the Howard Hughes Medical Institute to A.M.K. and grants from the NIH (AG09521, CA59717 and HD18179) to H.M.B.
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Rossi, F., Guicherit, O., Spicher, A. et al. Tetracycline-regulatable factors with distinct dimerization domains allow reversible growth inhibition by p16. Nat Genet 20, 389–393 (1998). https://doi.org/10.1038/3871
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DOI: https://doi.org/10.1038/3871
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