Gene down-regulation with short hairpin RNAs and validation of specificity by inducible rescue in mammalian cells.
The principal problem with RNA interference (RNAi) experiments is off-target effects. The most vigorous demonstration of the specificity is the rescue of the RNAi effects with an RNAi-resistant target gene. By combining the expression of short hairpin RNA (shRNA) and rescue cDNA in the same vector, both the validation of shRNA specificity and the generation of shRNA-expressing cell lines can easily be accomplished. If the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied, by conditionally turning off the rescue protein. The use of model systems is detailed in these protocols.