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

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

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The John Curtin School of Medical Research, The Australian National University, PO Box 334, Canberra, Australian Capital Territory 2601, Australia.


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.

[Indexed for MEDLINE]

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