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Design and cloning of lentiviral vectors expressing small interfering RNAs

Abstract

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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Figure 1: Cloning scheme for Option A.
Figure 2: Cloning scheme for Option B.
Figure 3: An example of an efficient lentiviral silencing vector.

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Acknowledgements

The authors wish to thank Kenneth Fringpong and Knut Madden of Invitrogen Corporation for their assistance in development of the L-pDest-cmv-gfp vector.

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Correspondence to Inder M Verma.

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Tiscornia, G., Singer, O. & Verma, I. Design and cloning of lentiviral vectors expressing small interfering RNAs. Nat Protoc 1, 234–240 (2006). https://doi.org/10.1038/nprot.2006.36

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