RNAi rescue by ectopic expression - (Mar/26/2008 )
A gene was knockdown by vector based shRNA. Loss of the gene resulted in change in cell morphology. To comfirm that this change is due to the specific effect of loss of the target gene, I plan to reintroduce the gene using lentivirus. Because the ectopic gene is driven by CMV promoter, its high expression level should overcome RNAi. But will the shRNA already exist in the cells interfere with the infection? The shRNA target site is in the coding region.
Wouldn't it be better to have an siRNA-refractory protein (i.e. engineer silent mutations in the siRNA target site so that it is no longer targeted by the siRNA)? That way you won't have to worry about the expression by CMV
Thank you miRNA man,
Yes. I had done what you said.