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Nat Protoc. 2006;1(1):234-40.

Design and cloning of lentiviral vectors expressing small interfering RNAs.

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The Salk Institute for Biological Studies, Laboratory of Genetics, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.


RNA interference (RNAi) has emerged as a powerful technique to downregulate gene expression. The use of polIII promoters to express small hairpin RNAs (shRNAs), combined with the versatility and robustness of lentiviral vector-mediated gene delivery to a wide range of cell types offers the possibility of long-term downregulation of specific target genes both in vitro and in vivo. The use of silencing lentivectors allows for a rapid and convenient way of establishing cell lines (or transgenic mice) that stably express shRNAs for analysis of phenotypes produced by knockdown of a gene product. Here we present two possible protocols describing the design and cloning of silencing lentiviral vectors. These protocols can be completed in less than 3 weeks.

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