Transgene is tightly regulated by Geneswitch system in 293 cells

Abstract

Introduction: Inducible transgene expression is a novel strategy in vascular gene therapy. Since the inducible nitric oxide synthase (iNOS) gene has many potent or possibly detrimental effects if mis-regulated, we insert the human iNOS gene into an inducible system, Geneswitch™ (Invitrogen) and tested transgene induction in HEK293 cells. Methods: The DNA plasmid pSwitch, pGenev5His-iNOS or pGenev5His-lacZ was introduced into HEK293 cells for 1hr by TransIT Express™ (Mirus). Then culture media were added and the cells were incubated for 24 hrs. Subsequently, the cells were induced by a ligand, mifepristone (10−12~10−7M) for 24~48 hrs. Finally, nitrite levels in culture media were measured by Greiss assay and iNOS expression was assessed by western blot. LacZ expression was detected by X-gal staining. Results: Without mifepristone, the basal expression of iNOS was undetectable. At doses of mifepristone of 10−10~10−7 M, functional iNOS protein was induced as determined by western blot and Greiss assay. At doses ≤ 10−11M, iNOS expression was detected only at baseline levels. LacZ expression was similarly induced and showed a mifepristone dose-dependent expression pattern. Conclusions: Geneswitch™ provided very low basal expression of iNOS and lacZ. Both iNOS and lacZ expression were tightly regulated by the ligand. Geneswitch™ may hold promise for cellular therapy or for generation of transgenic animals.