Strength of promoter activity is different from one biological context to another and can affect experimental outcomes.
Figure 1. Promoter activity varies across several human and rodent cell lines. Cells were plated at a density of 50,000 cells per well in a 24-well plate and transduced at MOI = 15 with SMARTvector 2.0 Empty Vector Control Particles expressing TurboGFP. Promoter activity was assessed at 72 hours post-transduction by the fluorescence intensity of TurboGFP.
Promoter activity is correlated to functional gene silencing.
Figure 2. Promoters driving the expression of TurboGFP have differential activity from cell to cell. Multiple cell lines were transduced with SMARTvector Non-targeting shRNA Control Particles which delivered vectors expressing TurboGFP driven by seven different cellular and viral promoters. Control wells contained untreated cells. Fluorescent images were obtained 72 hours post-transduction. Visual assessment of TurboGFP expression in human PC-3, human OVCAR-8, human Jurkat and mouse NIH/3T3 cells shows a varying degree of promoter activity across cell lines.
SMARTchoice shRNA Promoter Selection Plate layout
Figure 1. The 96-well SMARTchoice shRNA Promoter Selection Plate layout.
| hCMV: |
human cytomegalovirus |
| mCMV: |
mouse cytomegalovirus |
| hEF1α: |
human elongation factor 1α |
| mEF1α: |
mouse elongation factor 1α |
| CAG: |
chicken β actin hybrid |
| PGK: |
mouse phosphoglycerate kinase |
| UBC: |
human ubiquitin C |
The SMARTchoice shRNA Promoter Selection Plate enables straightforward qualitative assessment of promoters that actively drive expression.
Figure 2. 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.
Relative knockdown in NIH/3T3 cells.
Figure 3. Selection of the mouse CMV promoter for mouse NIH/3T3 cells using the SMARTchoice Promoter Selection Plate results in greater gene silencing. Gene silencing experiments targeting mouse GAPDH and RHOA genes demonstrate that shRNAs expressed by the mCMV promoter are significantly more effective at silencing the target gene in NIH/3T3 cells when compared to the same shRNAs expressed by the hCMV promoter. Cells were transduced with lentiviral particles at MOI = 20, 10 and 5; gene expression levels were detected and normalized to controls 72 hours post-transduction using Thermo Scientific Solaris qPCR Gene Expression Assays and Master Mix.
Relative knockdown in A539 cells.
Figure 4. shRNAs expressed from the mouse CMV promoter result in significant gene silencing in human A549 cells. One shRNA targeting human GAPDH and three shRNAs targeting human RHOA were delivered individually to A549 cells with expression driven by either the mCMV or hCMV promoter. Results demonstrate that all four shRNAs expressed from the mCMV promoter significantly silenced GAPDH or RHOA compared to the same shRNAs driven by the hCMV promoter. Cells were transduced with lentiviral particles at MOI = 20, 10 and 5; gene silencing was measured using Solaris™ qPCR Gene Expression reagents 72 hours post-transduction.
SMARTvector 2.0 Lentiviral shRNA Control Particles Give Consistent Results Across Cell Lines
Figure 1.SMARTvector 2.0 Lentiviral shRNA Controls: Non-Targeting Control and GAPD. K562, HUVEC and SH-SY5Y cells were transduced at three MOIs and assessed for mRNA knockdown 72 hours post-transduction using a branched DNA assay (Panomics, Inc.)