Regulated expression of sFRP-1 protein by the GeneSwitch system

The GeneSwitch system is a mifepristone-inducible expression system that provides exceptionally low uninduced and high-induced protein expression in mammalian cells. We have developed an adenovirus recombinant containing GeneSwitch protein driven by the GAL4-tk promoter, as well as recombinants containing sFRP-1 and luciferase reporter under the control of the GAL4-E1b promoter. Luciferase activity in A549 cells infected with the GeneSwitch and Luciferase viruses is very low in ethanol-treated cells, while the level of luciferase activity increases 200-fold in cells treated with mifepristone. Conditional expression of functional sFRP-1 is demonstrated in A549, human osteoblast, and CHO cell lines by either the co-infection of cells with sFRP-1 and GeneSwitch viruses or the infection of GeneSwitch expressing cell lines with sFRP-1 virus and subsequent treatment with mifepristone. The expression of sFRP-1 is seen as early as 4h post-mifepristone treatment, reaching the highest levels at 20h. The sFRP-1 protein is present in conditioned media, and the protein is functional based upon its ability to inhibit the Wnt-mediated activation of TCF-Luciferase reporter activity. The regulated expression of sFRP-1 utilizing adenovirus vectors provides an opportunity to address the contribution of sFRP-1 in the regulation of stem cell differentiation, maturation, and their function by modulating the Writ signaling. (C) 2004 Elsevier Inc. All rights reserved.