Unit

UNIT 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible Rescue in Mammalian Cells

  1. Hoi Tang Ma,
  2. Randy Y.C. Poon

Published Online: 1 DEC 2010

DOI: 10.1002/0471143030.cb2702s49

Current Protocols in Cell Biology

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.

Author Information

  1. Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong

Publication History

  1. Published Online: 1 DEC 2010
  2. Published Print: DEC 2010

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. Curr. Protoc. Cell Biol. 49:27.2.1-27.2.12. © 2010 by John Wiley & Sons, Inc.

Keywords:

  • inducible expression;
  • RNA interference;
  • short hairpin RNA