Gene knockdown by ecdysone-based inducible RNAi in stable mammalian cell lines

Nat Protoc. 2008;3(1):79-88. doi: 10.1038/nprot.2007.456.


RNA interference (RNAi) is a powerful tool for the functional analysis of essential genes in the mammalian genome. Here, we present a simple ecdysone-based inducible RNAi approach that allows high induction and adjustable control of short hairpin RNA (shRNA) expression for silencing gene expression in a wide range of mammalian cells. This protocol describes the following: the design and cloning of inducible shRNA; testing and validation of gene knockdown; and methodology for establishing stable cell lines. This step-by-step protocol offers a quick and cost-effective approach for addressing the function of genes essential for cell cycle regulation and development and can be completed in less than 6 weeks.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cloning, Molecular
  • Ecdysone / pharmacology*
  • Gene Expression Regulation / drug effects
  • Genetic Techniques
  • Genetic Vectors
  • Humans
  • Mice
  • RNA Interference*
  • RNA, Small Interfering / metabolism
  • Transfection / methods


  • RNA, Small Interfering
  • Ecdysone