I have a gene that was knockdown by vector based shRNA. The loss of the gene resulted in change in cell morphology and affected other gene and protein expression of possible downstream targets of the gene of interest. To comfirm that this change is due to the specific effect of the loss of the target gene and no off target effect of the shRNA knockdown was introduced, I plan to reintroduce the gene using pCMV vector transfection. Because the ectopic gene is driven by pCMV promoter, wil its high expression level overcome the shRNA, or will the shRNA that is already in the cells interfere with the transfection? Note: the shRNA target site is in the coding region.
Hi Razan, I may miss something in your question... The usual approach for a "rescue" experiment under shRNA is to transfect the gene for the protein that is KOed but with some silent mutations in the sequence targeted by the shRNA (same final protein but mRNA is resistant to silencing). An easy shortcut is to take the homolog from a different species, which may already have plenty of silent mutations. I hope this helps
I agree with Thomas. Expressing a protein coding gene that is resistant to your shRNA would be best to rescue your phenotype. Other things to consider: You may want to verify your phenotype with other shRNA sequences targeting the same gene. If your effect is true, by knocking down the same gene using different shRNA targets should produce same phenotype. You may also want to consider a non-targeting shRNA as control. Many shRNA expression cassettes uses RNA pol III promoters which are very strong and can cause cell toxicity....Good luck.
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