A novel tool that enables straightforward, qualitative assessment of promoters that actively drive expression in your cells of interest.
Identifying the optimal promoter for your cells of interest using the SMARTchoice promoter selection plate allows for increased functionality and performance of lentiviral shRNA and shMIMIC microRNA - resulting in successful gene down-regulation in any cell of interest, including transformed, primary, embryonic stem and neuronal cells.
Identify active promoters in adherent cells using the SMARTchoice Promoter Selection Plate
In a single matrixed experiment, the SMARTchoice Promoter Selection Plate enables the evaluation of seven different constitutive promoters at a range of multiplicities of infection (MOIs) in your cells of interest.
After transduction of cells, wells can be quickly and visually evaluated for TurboGFP intensity using fluorescence microscopy, high-content imaging or detection using a microplate reader. The most active promoter (with best TurboGFP expression) will provide the highest expression of your SMARTvector reagent in your cells. Then simply order SMARTvector reagent with your choice of promoter and reporter!
Figure 1: The 96-well SMARTchoice Promoter Selection Plate Layout
The SMARTchoice Promoter Selection Plate enables straightforward qualitative assessment of promoters that actively drive expression
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.
Select the optimal promoter for maximum shRNA and shMIMIC expression
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.