Figure 3

Confirmation of shRNA potency after lentiviral delivery. (a) shRNA potency evaluation after transduction with shRNA-encoding lentiviral vectors. HEK293 cells were transduced at an MOI of 10 (black bars) or << 1 (white bars) followed by puromycin selection for ten days to ensure that cells harbored only a single lentiviral insertion. In the two cases, cells were transfected with the psiCHECK-IL12B vector one day and ten days post-transduction, respectively, and luciferase activities were measured forty-eight hours post-transfection. Renilla luciferase activity was normalized to firefly luciferase activity and depicted relative to transduction with lentiviral vectors not encoding an shRNA (vehicle). (b) An IL-12B expressing HeLa cell line was transduced with shRNA-encoding lentiviral vectors at an MOI of 2. IL-12B mRNA levels were evaluated by qRT-PCR two days post-transduction. All assay were performed at least in triplicates and data are depicted as mean + SEM.