title: Gene down-regulation with short hairpin RNAs and validation of specificity by inducible rescue in mammalian cells.
abstract: 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.
authors.affiliation: Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.
authors.forename: Hoi Tang
authors.initials: HT
authors.lastname: Ma
authors.forename: Randy Y C
authors.initials: RY
authors.lastname: Poon
mesh_terms: D000818:Animals; D002460:Cell Line; D015536:Down-Regulation; D055785:Gene Knockdown Techniques / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D055029:Inverted Repeat Sequences; D008322:Mammals / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D013045:Species Specificity
chemical_list: D034741:RNA, Small Interfering