Control of siRNA expression using the Cre-loxP recombination system.
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.
Kasim
Vivi
V
Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Hongo, Tokyo 113-8656, Japan.
Miyagishi
Makoto
M
Taira
Kazunari
K
D000595:Amino Acid Sequence; D000818:Animals; D001287:Attachment Sites, Microbiological / Q000235:genetics*; D001483:Base Sequence; D002457:Cell Extracts; D002460:Cell Line; D001517:Coleoptera; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D019426:Integrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D011401:Promoter Regions, Genetic / Q000235:genetics; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002457:Cell Extracts; D019913:Nuclear Localization Signals; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA; D014764:Viral Proteins; D008156:Luciferases; C045073:Cre recombinase; D019426:Integrases