shRNA rescue with site directed mutagenesis - oligo design - (Aug/31/2011 )
I would like to do a rescue from my shRNA knock down and analyze the RNA on gene expression microarray.
Of all shRNA sequences tested, only one of all, which is in the coding region, resulted in a proper knock down.
To exclude false positives, I now would like to re-induce the expression with a mutated cDNA. I already have the unmutated cDNA in my backbone and would use the Invitrogen site directed mutagenesis kit to insert point mutations.
Does anyone know, how many mutations I need to insert when designing my primers and what else I would have to consider? There was also some discussion in our lab, whether this rescue is still "up to date" or rather other approaches should be addressed (another shRNA targeting elsewhere in the gene - but I've already tried 5 different shRNA sequences, 3 different siRNAs resulted even in a "knock up").
Any help would be appreciated!!
Thanks a lot!
Is the purpose of mutating the cDNA to differentiate it from endogenous mRNA? If so, one mutation is enough and the mutation should be synonymous. Such rescue experiment is scientifically meaningful. Ideally you want to get at least another shRNA to work--knock down the target gene and causes similar phenotypic changes.
Thank you very much, yes I want to differentiate it from the endogenous RNA which is targeted by the shRNA. What do you mean exactly by synonymous ? Does the mutation have to be in any specific region of the shRNA?
Another question just came to my mind (I am quite new to all this). If the other shRNAs I tried only resulted in a weak (max. 50%) knock down on RNA level - could it help to just increase the MOI of my lentivirus delivering the shRNA?
Thank you very much again!!
Since you want to rescue RNAi knockdown by overexpressing the same gene. The overexpressed gene should be functional, which means a mutation you are going to introduce does not alter the protein sequence, or synonymous mutation.
I think you can increase MOI to achieve a similar knockdown efficiency as the other shRNA.
Thanks a lot for your help!! I will try both (rescue and increase MOI) and see what works best.