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 4 h post-mifepristone treatment, reaching the highest levels at 20 h. 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 Wnt signaling.
Bhat
Ramesh A
RA
Women's Health Research Institute, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA. bhatr@wyeth.com
Stauffer
Barbara
B
Komm
Barry S
BS
Bodine
Peter V N
PV
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000494:pharmacology; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D051821:Lymphoid Enhancer-Binding Factor 1; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics*; D015735:Mifepristone / Q000494:pharmacology*; D008957:Models, Genetic; D010006:Osteoblasts / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D051153:Wnt Proteins; D000094742:Secreted Frizzled-Related Proteins
D017077:Culture Media, Conditioned; D004268:DNA-Binding Proteins; D000431:Ethanol; D006727:Hormone Antagonists; D036341:Intercellular Signaling Peptides and Proteins; D008156:Luciferases; D051821:Lymphoid Enhancer-Binding Factor 1; D008565:Membrane Proteins; D015735:Mifepristone; D012313:RNA; D011994:Recombinant Proteins; D014157:Transcription Factors; D051153:Wnt Proteins; C117636:SFRP1 protein, human; D000094742:Secreted Frizzled-Related Proteins