Ambion's newest line of plasmid siRNA expression vectors, pSilencer 4.1-CMV, feature polymerase (pol) II promoters that drive transcription of both an siRNA template and an antibiotic resistance gene. The CMV promoter is used to drive siRNA expression. This strong promoter is active in a broad range of cell types and performs better than most pol III promoters under long term selection.
One use of plasmid-based siRNA expression vectors is for long term gene silencing studies in mammalian cells. Typically these vectors encode a hairpin siRNA under the control of a polymerase III promoter and an antibiotic resistance gene driven by a polymerase II promoter. While it is possible to use such vectors to produce stable cell lines expressing siRNAs that silence their targets, there are, to date, very few published accounts of doing so (Xia 2002). One explanation for the difficulty in producing such clones is possible interference between the pol II and III promoters. Although the exact mechanism is not understood, it is hypothesized that when pol II and III promoters are in close proximity, interference of transcription between the two promoters can occur. This suggests that clones derived from a transfection/selection experiment will either express the siRNA or the antibiotic resistance gene but rarely express both. To overcome this potential problem, we have constructed three pSilencer 4.1-CMV siRNA Expression Vectors that express a hairpin siRNA as well as an antibiotic resistance gene from pol II promoters. The CMV promoter expresses the siRNA, and an SV40 promoter expresses the antibiotic resistance gene. To demonstrate the utility of these CMV promoter vectors, the vectors were engineered to express an siRNA targeting GAPDH. The vectors were then transfected into HeLa cells, which were in turn put under long term antibiotic selection. This strategy produced cell lines that have had reduced levels of GAPDH mRNA and protein for over 8 months.
As shown in Figure 1, several pSilencer 4.1-CMV transfected clones had reduced GAPDH protein and mRNA expression compared to non-transfected HeLa cells. Panel B shows immunofluorescence data demonstrating the relative fluorescence differences between a clone with reduced levels of GAPDH versus a clone that did not show significant GAPDH reduction.
Figure 1. Long Term Stable Reduction in GAPDH Levels. Both mRNA expression levels and protein levels were analyzed in these same clones using either real-time PCR or immunofluorescence analysis. (A) Clones that showed low levels of GAPDH protein expression also showed low levels of GAPDH mRNA expression. (B) Representative immunostaining data showing the relative fluorescence differences between a clone having GAPDH reduced and another clone that did not have a significant amount of GAPDH knocked down. Immunofluorescence was performed using gene specific primary antibodies and a fluorescein conjugated secondary antibody and the resulting reduction in protein expression was examined by fluorescence microscopy. The cells were mounted with DAPI to stain nuclei (Blue). Images were digitally captured and quantified using Metamorph software.
The pSilencer 4.1-CMV siRNA Expression Vectors are provided with 1) linearized and purified vector ready for ligation; 2) a DNA insert encoding a GAPDH-specific siRNA; 3) a circular, negative control pSilencer vector that expresses a scrambled control siRNA; and 4) 1X DNA Annealing Solution.
Cat# Product Name Size 5775 pSilencer 4.1-CMV puro 20 rxns 5777 pSilencer 4.1-CMV hygro 20 rxns 5779 pSilencer 4.1-CMV neo 20 rxns 5790 pSilencer adeno 1.0-CMV System 5 virus preparations