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.
AUTOR(ES)
Hu, M C
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=362889Documentos Relacionados
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