LAC TET DUAL-INDUCIBLE SYSTEM FUNCTIONS IN MAMMALIAN-CELL LINES/

The Escherichia coli Lac repressor (Lac system) and tetracycline respo nsive promoter (Tet system) systems have been used individually to reg ulate gene expression at the cellular as well as at the organismal lev els. In this study, these two systems were combined (designated Lac/Te t dual-inducible system) to regulate two inducible genes simultaneousl y in a single cell. The isopropyl-beta-D-thiogalactopyranoside (IPTG) and tetracycline (used for the operation of the Lac and the Tet system s) were non-cytotoxic to the cells when added together into the cells at around the optimal concentrations (IPTG: less than or equal to 5 mM ; tetracycline: <1.5 mu g). The rate and efficiency of induction and r epression of two inducible genes regulated by the Lac/Tet dual-inducib le system were similar to the results obtained when one inducible gene is regulated by one inducible systems in a single cell. The Lac/Tet d ual-inducible system could function in many cell lines. which was demo nstrated by regulating the expression of beta-galactosidase and lucife rase reporter genes in five tumor cell lines by transient transfection analysis. The feasibility of introducing a second inducible system in to an already established inducible cell line was confirmed. Finally, we showed that the Lac/Tet dual-inducible system functions at translat ional and at functional levels in a stable cell line named 7-4-b, whic h contains the Ha-ras and bcl-2 inducible genes. In conclusion, this s tudy extends the application of prokaryotic inducible systems from the regulation of a single gene to two genes and helps clarify the relati onship between two genes and the effects of two genes on cells.