Gene knockdown by ecdysone-based inducible RNAi in stable mammalian cell lines.
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.
Rangasamy
Danny
D
The John Curtin School of Medical Research, The Australian National University, PO Box 334, Canberra, Australian Capital Territory 2601, Australia. danny.rangasamy@anu.edu.au
Tremethick
David J
DJ
Greaves
Ian K
IK
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004440:Ecdysone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D014162:Transfection / Q000379:methods
D034741:RNA, Small Interfering; D004440:Ecdysone