Hu, Mickey C.-T. and Davidson, Norman (1990) A combination of derepression of the lac operator-repressor system with positive induction by glucocorticoid and metal ions provides a high-level-inducible gene expression system based on the human metallothionein-IIA promoter. Molecular and Cellular Biology, 10 (12). pp. 6141-6151. ISSN 0270-7306. http://resolver.caltech.edu/CaltechAUTHORS:HUMmcb90
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We and others have introduced the use of the lac operator-repressor system as a method for providing inducible gene expression for gene transfer experiments in animal cells (M. C.-T. Hu, and N. Davidson, Cell 48:555-566, 1987; J. Figge, C. Wright, C. J. Collins, T. M. Roberts, and D. M. Livingston, Cell 52:713-722, 1988). To improve the dynamic range of such an inducible system, we have investigated the effects of combining the relief by isopropyl-beta-D-thiogalactoside (IPTG) of negative control by the lac system with positive induction by the natural inducers glucocorticoids and cadmium ion for a system based on the human metallothionein-IIA gene promoter. We used the chloramphenicol acetyltransferase gene as a reporter gene and inserted a lacO sequence into the promoter between the GC box and metal-responsive element 1, between metal-responsive element 1 and the TATA box, or between the TATA box and the transcription start site. Surprisingly, all of these insertions had a significant inhibitory effect on promoter activity even in the absence of repressor. However, with these lacO-containing constructs, the levels of gene expression after induction by glucocorticoid, Cd2+, or both were considerably reduced in cells engineered to express the lac repressor. Derepression by IPTG, coupled with induction by both dexamethasone and Cd2+ ion, then provided a high level of induced expression, i.e., by a factor of approximately 100 over the basal level of expression. However, inserting the lacO sequence well upstream just before the glucocorticoid-responsive element had much smaller effects on expression levels in both repressor-negative and repressor-positive cells. This study describes a new, high-level-inducible promoter system for gene transfer experiments. The observed effects are discussed in terms of current models of the mechanisms by which transcription factors control gene expression.
|Additional Information:||Copyright © 1990 by the American Society for Microbiology. Received 11 June 1990/Accepted 24 September 1990 We are grateful to Michael Karin (University of California, San Diego) for generously providing a -286 5' deletion mutant of the hMT-IIA promoter linked to the HSV tk gene. We thank Marie Krempin for assistance with cell cultures. This work was supported by a grant from Amgen, Inc., and by a Public Health Service research grant from the National Institutes of Health.|
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|Deposited On:||10 Dec 2006|
|Last Modified:||26 Dec 2012 09:21|
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