A MODIFIED TETRACYCLINE-REGULATED SYSTEM PROVIDES AUTOREGULATORY, INDUCIBLE GENE-EXPRESSION IN CULTURED-CELLS AND TRANSGENIC MICE

A system for tetracycline-regulated inducible gene expression was desc ribed recently which relies on constitutive expression of a tetracycli ne-controlled transactivator (tTA) fusion protein combining the tetrac ycline repressor and the transcriptional activation domain of VP16 [Go ssen, hi, and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-55 51], This system yielded only low levels of transactivator protein, pr obably because tTA is toxic, To avoid this difficulty, we placed the t TA gene under the control of the inducible promoter to which tTA binds , making expression of tTA itself inducible and autoregulatory, When u sed to drive expression of the recombination activating genes 1 and 2 (RAG-1 and RAG-2), the autoregulatory system yielded both substantiall y higher levels of variable (diversity) joining [V(D)J] recombination activity (70-fold on average) and inducible expression in a much large r fraction of transfected cells (autoregulatory, 90%, vs, constitutive , 18%), In addition, this system allowed the creation of transgenic mi ce in which expression of a luciferase transgene was inducible tens to hundreds of times the basal levels in most tissues examined, Induced levels of expression were highest in thymus and lung and appear to be substantially higher than in previously reported inducible luciferase transgenic mice created with the constitutive system, With the modifie d system, inducible transactivator mRNA and protein were easily detect ed in cell lines by RNA and Western blotting, and transactivator mRNA was detected by RNA blotting in some tissues of transgenic mice, This autoregulatory system represents an improved strategy for tetracycline -regulated gene expression both in cultured cells and in transgenic an imals.