Tetracycline-regulatable expression vectors tightly regulate in vitro gene expression of secreted proteins

The regulation of gene expression by the tetracycline system has attracted a high level of interest in the recent past. However, expression of secreted proteins has not been evaluated precisely. In this study, we constructed two versions of a one-plasmid system containing the elements necessary for the regulation of gene expression. The regulatable elements and the selectable marker (Neo^r) were set up in two different configurations, pTRIN31 and pTRIN76. With these two regulatable versions, the levels of protein expression after transfection into the NIH/3T3 cell line were measured by insertion of three different genes encoding the secreted proteins (hGH, ApoE3, hGM-CSF). The maximum levels of gene expression obtained with the pTRIN76-derived plasmids were 100 ng/24 h/10⁶ cells for hGH, 427 ng/24 h/10⁶ cells for ApoE3 and 108 ng/24 h/10⁶ cells for hGM-CSF. For the pTRIN31-derived plasmids the maximum levels were 2.7 ng/24 h/10⁶ cells for hGH and 47 ng/24 h/10⁶ for ApoE3. Both plasmids give rise to an expression of the transfected gene that can be tightly regulated by three different molecules: tetracycline, minocycline and doxycycline. The levels of the secreted proteins are below the detectable level when the reporter genes are repressed. This repression is reversible within 48 h after the regulator has been removed from the medium.