Retroviral vectors for establishing tetracycline-regulated gene expression in an otherwise recalcitrant cell line

Here we report the successful incorporation of tetracycline-mediated gene expression in a mouse mammary epithelial cell line, HC11, in which conventional approaches failed. We generated retroviruses in which tTA expression was controlled by one of three promoters: a synthetic tetracycline responsive promoter (TRE), the elongation factor 1-alpha promoter (EF1α) or the phosphoglycerate kinase-1 promoter (PGK), and compared the resulting cell lines to one generated using a cytomegalovirus immediate early gene promoter (CMV). In contrast to cells produced using the CMV and PGK promoters, those produced using the EF1α and TRE promoters expressed high levels of β-galactosidase in a tetracycline-dependent manner.These novel retroviral vectors performed better than the commercially available system and may have a more general utility in similarly recalcitrant cell lines.The ability to control the spatial and temporal expression of a transgene, either in cell culture or in transgenic animals, is a valuable tool in gene function studies. Constitutive overexpression of a transgene over long periods in culture may have several undesired effects that are inimical to its functional characterisation. This is particularly true in the case of gene products with growth inhibitory or toxic effects. Temporal and spatial control of transgene expression is conferred by the choice of heterologous promoter. Many methods have been described to achieve this aim in mammalian cells. Some of these methods involve the use of endogenous mammalian proteins. These include the use of heat shock proteins [1-3], metallothionein [4], the glucocorticoid receptor [5], the estrogen receptor [6], the progesterone receptor [7] and the aryl hydrocarbon receptor [8]. The ecdysone receptor of Drosophila melanogaster has also been used to confer ligand-dependent activation on heterologous transgenes in vivo and in vitro[9]. Other approaches have harnessed components from two well-understood paradigms of prokary