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Anal Biochem. 1998 Aug 1;261(2):211-8.

Highly controlled heterologous gene expression through combined utilization of the tetracycline-repressible transactivator and the lac repressor.

Author information

1
Japanese Foundation for Cancer Research, Cancer Institute, 1-37-1 Kami-Ikebukuro, Tokyo, Toshima-ku, 170-8455, Japan.

Abstract

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 isopropyl thiogalactopyranoside (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 tetracycline-repressible transactivator and the lac repressor. Whereas the parental tetracycline-repressible promoter exhibited constitutive activities when stably introduced into the lymphoid cells, cDNA expression from the TcIP promoter was strongly inhibited by tetracycline and was potently induced by IPTG in stable transfectants. Hence, the TcIP promoter made it possible to achieve highly controlled cDNA expression in cells wherein the parental promoter did not function in an inducible manner. Potential application of this promoter was provided by expressing cyclin-dependent kinase inhibitor, p27(Kip1). Induced expression of p27(Kip1) by the TcIP promoter in the lymphoid cells strongly reduced cellular responsiveness to IL-2 or IL-3, consistent with the idea that p27(Kip1) is a critical target that must be inactivated by the interleukin-triggered mitogenic signals.

PMID:
9716424
DOI:
10.1006/abio.1998.2742
[Indexed for MEDLINE]

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