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Drive Long-Term Silencing with CMV Vectors

href="http://www.ambion.com/catalog/CatNum.php?5775">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
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