Controlled gene expression in mammalian cells via a regulatory cascade involving the tetracycline transactivator and...

Regulatory cascades or regulons control pathways at multiple points or multiple genes by one initial signal. In this paper, we describe the construction of an artificial regulatory cascade in CHO cells, which responded to various concentrations of tetracycline (Tc) and/or IPTG. The system consists of the constitutively produced transactivator (TTA) of the Tc operon (tet), which induced the expression of a lacI gene controlled by tet operator (tetO) and upstream CMV promoter (p*CMV) sequences. LacI repressed the activity of a cat gene by binding to lacO sites in its upstream RSV promoter (pRSV) region. However, this repression could be alleviated by exposure to Tc or IPTG, which inhibited the binding activities of TTA and LacI, respectively. Hence, treatment with either Tc or IPTG led to a tenfold increase in CAT activity. After the withdrawal of the inducer, cat expression reverted to basal levels. Regulation by Tc showed a phenotypic lag, and full induction was reached after 192 h, whereas IPTG addition led to full induction within 24 h. When cells were treated with both Tc and IPTG, full induction of cat was reached in 24 h and maintained thereafter while in the presence of Tc alone. This suggests that regulation by Tc is fast and that the phenotypic lag may be due to slow turnover of the LacI repressor. This TTA/lacI regulatory system may serve as an example in which cat expression was used as a reporter. The data indicate that regulatory cascades regulated at multiple points can be constructed with any cloned gene in mammalian cells.