HIGHLY CONTROLLED HETEROLOGOUS GENE-EXPRESSION THROUGH COMBINED UTILIZATION OF THE TETRACYCLINE-REPRESSIBLE TRANSACTIVATOR AND THE LAC REPRESSOR

To achieve strictly on-off switching of a desired cDNA expression in a broader range of mammalian cell types, we introduced the Escherichia coli lac operator sequences into the tetracycline-repressible promoter and created a chimeric promoter (designated here as the TcIP promoter ) whose activity was reciprocally controlled by tetracycline and isopr opyl thio-galactopyranoside (IPTG). cDNAs were connected downstream of the TcIP promoter and stably transfected into interleukin (IL-2 or IL -3)-dependent lymphoid cells that ectopically coexpress the tetracycli ne-repressible transactivator and the lac repressor. Whereas the paren tal tetracycline-repressible promoter exhibited constitutive activitie s when stably introduced into the lymphoid cells, cDNA expression from the TcIP promoter was strongly inhibited by tetracycline and was pote ntly induced by IPTG in stable transfectants. Hence, the TcIP promoter made it possible to achieve highly controlled cDNA expression in cell s wherein the parental promoter did not function in an inducible manne r. Potential application of this promoter was provided by expressing c yclin-dependent kinase inhibitor, p27(Kip1). Induced expression of p27 (Kip1) by the TcIP promoter in the lymphoid cells strongly reduced cel lular responsiveness to IL-2 or IL-3, consistent with the idea that p2 7(Kip1) is a critical target that must be inactivated by the interleuk in-triggered mitogenic signals. (C) 1998 Academic Press.