UNIT 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible Rescue in Mammalian Cells
Published Online: 1 DEC 2010
Copyright © 2010 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Cell Biology
How to Cite
Ma, H. T. and Poon, R. Y. 2010. Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible Rescue in Mammalian Cells. Current Protocols in Cell Biology. 49:27.2:27.2.1–27.2.12.
- Published Online: 1 DEC 2010
- Published Print: DEC 2010
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. Curr. Protoc. Cell Biol. 49:27.2.1-27.2.12. © 2010 by John Wiley & Sons, Inc.
- inducible expression;
- RNA interference;
- short hairpin RNA