Dear Researchers,
I would like to see how a cell responds by switching p53 status, ie knocking down mutant p53 and expressing wt-p53 simultaneously. I am considering co-transfection of plasmids containing shRNA to mutant-p53 and wt-p53 (with altered 3' UTR). Does this approach work?
Thanks
The approach is likely to work. In the absence of any stress to cells, the transfected cells should express reduced levels of mut-p53 and wt-p53. Depending upon the levels of the wt-p53 and cell type, you may or may not see alterations in cell proliferation. Because most cell types keep the levels of wt-p53 in control, you may not be able to select overexpressor cells (because cells with increased levels of p53 will be growth arrested).
I never used it myself, but I heard it was feasible to take advent of 3' and 5'UTR to target one allele but not the other. If necessary you could also change codon usage of one allele near the 3'UTR to extend region that would be different in the two alleles. If codons are properly selected it should not change greatly relative expression level.
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