Published online 23 April 2004
Nucleic Acids Research, 2004, Vol. 32, No. 7 e66
© 2004 Oxford University Press
Control of siRNA expression using the Cre–loxP recombination system
1 Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Hongo, Tokyo 113-8656, Japan and 2 Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan
*To whom correspondence should be addressed at Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Hongo, Tokyo 113-8656, Japan. Tel: +81 3 5841 8828; Fax: +81 3 5841 7340; Email: taira{at}chembio.t.u-tokyo.ac.jp
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Received February 24, 2004; Revised and Accepted March 24, 2004
Gene silencing mediated by RNA interference (RNAi) was first discovered in Caenorhabditis elegans, and was subsequently recognized in various other organisms. In mammalian cells, RNAi can be induced by small interfering RNAs (siRNAs). In earlier studies, our group developed a vector-based system for expression of siRNA under control of a polymerase III promoter, the U6 promoter, which can induce RNAi in living cells. We here describe a system for controlling the U6 promoter-driven expression of siRNA using the Cre–loxP recombination system. We constructed a ‘Cre-On’ siRNA expression vector which could be switched on upon excision catalyzed by Cre recombinase, which was delivered to cells directly from the medium as a fusion protein. An examination of the effectiveness of RNAi against a reporter gene revealed that addition of TAT-NLS-Cre (where NLS is a nuclear localization signal and TAT is a peptide of 11 amino acids derived from HIV) to the medium resulted in plasmid recombination, generation of siRNA and suppression of reporter activity. This system should allow us to induce RNAi in a spatially, temporally, cell type-specifically or tissue-specifically controlled manner and potentiate the improved application of RNAi in both an experimental and a therapeutic context.
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