A tetracycline-responsive β-galactosidase mammalian expression vector

Abstract

▼Often, it is of interest to have a reporter in which transcription is controlled. Although many eukaryotic promoters have been identified and cloned, they are often leaky and/or lack responsiveness in mammalian cell lines. Moreover, the inducers are generally deleterious to the host cell or have significant drawbacks. Many chimeric transcription factors have been developed to ameliorate these problems. Early attempts at producing chimeric transcription factors used the glucocorticoid receptor (Ref. 1). More recently, the bacterial lac and tet operons have been exploited. The tetbased systems are not as ‘leaky’ as their lac-based counterparts. Two tetracycline (Tc)-responsive binary systems have been described (Ref. 2, 3). Both utilize a regulatory sequence composed of a heptameric repeat of the tetO 19 bp inverted repeat fused to the immediate early sequences of the cytomegaloviral enhancer (tetO7CMV). The systems differ in kinetics related to the addition of Tc based on the modified chimeric transcription factor utilizing the Tc resistence protein (tetR). The chimera presently referred to as tTA is composed of the tetR protein, fused to the so-called ‘transactivating’ C-terminus of the co-adaptor protein, VP-16, from HSV-1. Whereas the tetRKRAB chimera consists of the highly conserved Kruppel-associated box (KRAB) domain of the Kox1 zincfinger protein family fused to the tetracycline repressor protein. Accordingly, the motifs differ in their kinetics and mechanism of associations. The tTA system is induced by the absence of Tc. In the uninduced state, basal levels of expression tend to be relatively high, owing to the CMV sequences. The tetRKRAB system makes use of the transcriptional silencing influence of the KRAB domain. This system maintains much lower levels of basal activity in the