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

Danny Rangasamy; David J. Tremethick; Ian K. Greaves

doi:10.1038/nprot.2007.456

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

Danny Rangasamy^*, David J. Tremethick, Ian K. Greaves

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

21 Citations (Scopus)

Abstract

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.

Original language	English
Pages (from-to)	79-88
Number of pages	10
Journal	Nature Protocols
Volume	3
Issue number	1
DOIs	https://doi.org/10.1038/nprot.2007.456
Publication status	Published - Jan 2008

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

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