Time course of induction in vitro

The time course of transgene induction was assessed to determine if there was a noticeable delay in the onset of transgene induction by the auto-inducible GeneSwitch® system, compared to the constitutive CMV-GeneSwitch® system. COS-1 cells were transfected with a 1:1 mixture of pGS1158 (CMV-GeneSwitch v3.1)/pAP1205 (6xGAL4/TATA-SEAP) or pGS1382 (4xGAL4/tk-GeneSwitch v3.1)/pAP1205 and cell culture supernatant samples were collected 1, 2, 3, 4, 5, 7, 14 and 24 hours after addition of MFP. An increase in SEAP expression was observed within one hour of MFP addition for both GeneSwitch® systems. Therefore, no obvious lag in the induction of SEAP activity was detected for pGS1382 (auto-inducible GeneSwitch v3.1). The induction level reached at the 24-hour time point was 236-fold. A gradual, time-dependent increase in basal transgene expression was observed in cells transfected with pGS1382/pAP1205, in the absence of MFP. This finding indicates that the 4xGAL4/tk promoter has detectable activity of its own.

Figure Legend

The time course of transgene induction was generated by transfecting COS-1 cells with a 1:1 mixture of pGS1158 (CMV-GeneSwitch v3.1)/pAP1205 (6xGAL4/TATA-SEAP) (squares) or pGS1382 (4xGAL4/tk-GeneSwitch v3.1)/pAP1205 (circles).  Open and closed symbols indicate the absence or presence of MFP at 10^-8 M, respectively. Cell culture supernatant samples were taken 1, 2, 3, 4, 5, 7, 14 and 24 hours after media change and assayed for SEAP activity. The fold induction due to MFP administration is indicated at 24 hours. Data are mean values ± SD of six independent transfections.