Figure 1: The 96-well SMARTchoice Promoter Selection Plate Layout
Figure 2: The SMARTchoice Promoter Selection Plate enables straightforward qualitative assessment of promoters that actively drive expression
Legend: Human A549, HEK293T and Jurkat cells were transduced with concentrated lentiviral particles arrayed in the SMARTchoice Promoter Selection Plate. TurboGFP expression was assessed by fluorescence microscopy 72 hours post-transduction. Images clearly demonstrate that the most functional promoter in A549 cells is mCMV, whereas the hCMV promoter is most active in HEK293T, and the mEF1α promoter is most active in Jurkat cells. TU = transducing unit.
Figure 3. Promoters vary in activity in different cells which effects lentiviral microRNA expression and, consequently, down-regulation of target gene expression. Mouse NIH/3T3 cells were transduced with shMIMIC lentiviral microRNA particles expressing mmu-miR-122, which regulates ALDOA, and a non-targeting control (siNTC). Transduction Medium contained 3 µg/mL Polybrene with no serum; cells were incubated overnight with lentiviral particles at MOIs 30, 15 and 7.5. After 72 hours post-transduction, TurboGFP fluorescent intensity was observed by microscopy indicating promoter activity; the mouse CMV (mCMV) promoter is most active in mouse NIH/3T3 cells. Additionally, expression of ALDOA and GAPDH (Reference gene) was measure by RT-qPCR. Relative expression of ALDOA was normalized to GAPDH, and then to siNTC-treated cells using a ΔΔCq method. Expression of miR-122 by the mCMV promoter resulted in the greatest down-regulation of ALDOA.