Tetracycline-regulatable factors with distinct dimerization domains allow reversible growth inhibition by p16

Continuous regulation is required to maintain a given cell state(1,2) or to allow it to change in response to the environment(3,4). Studies of the mechanisms underlying such regulation have often been hindered by the inability to control gene expression at will. Among the inducible systems available for regulating gene expression in eukaryotes(5-8), the tetracycline (tet) regulatable system has distinct advantages(9-11). It is highly specific, non-toxic and non-eukaryotic, and consequently does not have pleiotropic effects on host cell genes. Previously this system also had drawbacks, as it did not extinguish gene expression completely, precluding the study of toxic or growth-inhibitory gene products. We report here the development of a facile reversible tetracycline-inducible retroviral system (designated RetroTet-ART) in which activators and repressors together are expressed in cells. Gene expression can now be actively repressed in the absence of tet and induced in the presence of tet, as we have engineered distinct dimerization domains that allow co-expression of homodimeric tet-regulated transactivators and transrepressors in the same cells, without the formation of non-functional heterodimers. Using this system, we show that growth arrest by the cell cycle inhibitor p16 is reversible and dependent on its continuous expression.