Efficient control of tetracycline-responsive gene expression from an autoregulated bi-directional expression vector

The tetracycline-responsive expression system is based on the ability of th e chimeric tTA and rtTA transactivators to stimulate specifically transcrip tion from a companion synthetic CMV* or TK* promoter element, and can provi de tightly regulated gene expression that can be induced up to five orders of magnitude in cultured cells and transgenic mice. A major problem with th e system is that high level expression of the tTA or rtTA transactivators c auses cellular toxicity. Under conditions of prolonged expression this resu lts in selective pressure against the stable incorporation of vectors expre ssing the tTA or rtTA transactivators, and makes the generation of stable c ell lines and transgenic mice problematic. In this report we describe the d evelopment of a set of autoregulated bi-directional expression vectors in w hich the weaker TK* promoter is used to direct expression of the rtTA or tT A transactivator and the stronger CMV* element is used to direct cDNA expre ssion. In this format the transactivator and response elements are encoded on the same vector, which simplifies the system and ensures that gene expre ssion is effectively skewed in favor of the cDNA while maintaining a contin uously low level of transactivator expression. We find that such an autoreg ulated system works equally well for both the tTA and rtTA transactivators, provided that they contain a nuclear localization signal. Similar to other versions of the tetracycline-responsive expression system, gene expression is tightly regulated and can be efficiently switched between the off and o n expression states by doxycycline. In contrast with other tetracycline-res ponsive systems, however, expression of the rtTA and tTA transactivators fr om the autoregulated TK* promoter is low enough such that there is no cellu lar toxicity associated with either expression state. By incorporating a se lectable marker into these vectors, all of the components required for usin g the system are now contained on a single plasmid construct, and we find t hat this format provides a more reliable and greatly simplified method for the generation of stable cell lines. (C) 1999 Elsevier Science B.V. All rig hts reserved.