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Background
Gene function can be identified through the loss, gain, or change of activity caused by
spontaneous or induced mutations. In many cases, however, the temporal regulation of its
activity in specific cells or in the intact animal by external mediators would give more
precise clues to its function. Control of gene activity is, in fact, imperative when the
product is cytotoxic or lethal during embryonic development. Today, the tetracycline
controllable transactivator system is best understood and widely used in mammalian cell
cultures (1-7) and more recently also in transgenic plants (8), and transgenic mice
(9-12). The system consists of two plasmids, one encoding the tetracycline controllable
transactivator protein (tTA) under control of a viral cytomegalovirus (CMV) promoter, and
the second being the tet operator (tetO) minimal promoter driving the gene of interest.
Using luciferase as a reporter gene, we have shown that the control elements integrated in cis on a single plasmid allow efficient and tight control of reporter gene expression in vitro and in vivo (13). In transgenic mice, the reporter gene was induced in various tissues to levels up to 800 fold more than the two-plasmid system. Highest transactivation was consistently found in striated muscle. We calculated that in the induced state, luciferase constitutes about 0.01 - 0.03% of the total protein in this tissue, based on the specific activity of the purified enzyme. To a lower extent, transactivation was also detected in brain, heart, kidney tongue and lungs. This tissue distribution is likely to reflect the activity pattern of the CMV promoter driving the tTA.
To study the repression efficiency of tetracycline, we supplied tetracycline either in drinking water or by tetracycline-pellets implanted s.c. A dose of 1 mg/ml in water could only partially suppress transactivation to 0.2 to 4 % of the induced values. Pellets containing 0.7 mg tetracycline, however, repressed the luciferase activity virtually to zero. As expected, subsequent withdrawal of the pellets results in complete re-expression of the reporter gene in the same tissues to pre-treatment levels within one week. In additional experiments, tetracycline was implanted in pregnant combi-tTA transgenic mice and offspring were analysed at day 12 post partum. Luciferase activity was completely repressed to baseline levels in all tissues analysed demonstrating that the tetracycline transmitted by the milk is apparently sufficient to suppress luciferase activity in 2-week old sucklings.
We conclude that the single plasmid version of the tetracycline system is functional in transgenic mice and moreover more efficacious than the two plasmid system. In addition, crossing and analysis of animals transgenic for the individual components of the system is unnecessary and genetic segregation of the control elements during breeding prevented. Experiments to replace the viral promoter by various eukaryotic promoters to obtain controllable gene expression in specific tissues in transgenic mice is currently in progress.
References
1. Gossen, M. and Bujard, H. 1992. Tight control of gene expression in mammalian cells by
tetracycline-responsive promoters. Proc Natl Acad Sci U S A 89 :5547- 5551.
2. Shan, B., Lee, W.H. 1994. Deregulated expression of E2F-1 induces S phase entry and leads to apoptosis. Mol Cell Biol 14 :8166-8173.
3. Resnitzky, D., Gossen, M., Bujard, H., and Reed, S.I. 1994. Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol Cell Biol 14:1669-1679.
4. Damke, H., Baba, T., Warnock, D.E., and Schmid, S.L. 1994. Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J Cell Biol 127:915-934.
5. Fruh, K., Gossen, M., Wang, K., Bujard, H., Peterson, P.A., and Yang, Y. 1994 Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: a newly discovered mechanism for modulating the multicatalytic proteinase complex. Embo J 13 :3236-3244.
6. Howe, J.R., Skryabin, B.V., Belcher, S.M., Zerillo, C.A., and Schmauss, C. 1995. The responsiveness of a tetracycline-sensitive expression system differs in different cell lines. J Biol Chem 270:14168-14174.
7. Baron U, Freundlieb S, Gossen M, Bujard H. 1995. Co-regulation of two gene activities by tetracycline via a bidirectional promoter. Nucleic Acids Res 23:3605- 3606
8. Weinmann, P., Gossen, M., Hillen, W., Bujard, H., Gatz, C. 1994. A chimeric transactivator allows tetracycline-responsive gene expression in whole plants. Plant J 5:559-569.
9. Furth, P.A., L. St Onge, H. Boger, P. Gruss, M. Gossen, A. Kistner, H. Bujard, and L. Hennighausen.1994. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc Natl Acad Sci U S A 91 :9302- 9306.
10. Passman, R.S. and Fishman, G.I. 1994. Regulated expression of foreign genes in vivo after germline transfer. J Clin Invest 94:2421-2425. 11. Efrat, S., Fuscodemane, D., Lemberg, H., Alemran, O. and Wang, X.R. 1995. Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene. Proc Natl Acad Sci USA 92:3576-3580.
12. Shockett P, Difilippantonio M, Hellman N, Schatz D.G. 1995. A modified tetracycline-regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice. Proc Natl Acad Sci USA 92:6522-6526.
13. Schultze N, Burki Y, Lang Y, Certa U, and Bluethmann H. 1996. Efficient control of gene expression by single step integration of the tetracycline system in transgenic mice. Nature Biotechnology 14:499-503, 1996
Keywords
inducible gene extression, tetracycline
Submitted by:Horst Bluethmann
Pharmaceutical Research Gene Technology
F. Hoffmann-La Roche Ltd.
CH-4070 Base
Switzerland
e-mail: horst.bluethmann@roche.com
last update
: June 1998