Getting Specific with RNAi-Transgenic Mice

Abstract

RNA interference, mediated by 21- to 22-nucleotide double-stranded (ds)-RNA (called small interfering RNA, or siRNA), has proven effective in determining gene function in cultured mammalian cells and in mice by targeting a specific mRNA and reducing or abolishing its translation into protein. Shinagawa and Ishii have now modified this method to generate the equivalent of a tissue-specific knockout mouse model. Longer (about 500 nucleotides) ds-RNA was expressed from a vector using an RNA polymerase II-dependent promoter that is active in only a subset of mouse tissues. In addition, the long ds-RNA generated lacked the 7-methylguanosine cap structure at its 5′ end and poly(A) tail from its 3′ end, thereby blocking its export from the nucleus to the cytosol. By remaining in the nucleus, the long ds-RNA presumably does not trigger an antiviral interferon response that mammalian cells can elicit to this type of RNA, thus causing a general block in translation. Restricted localization to the nucleus would allow ribonucleases, such as dicer, to cleave the long ds-RNA into siRNA, which can then move into the cytosol to degrade target mRNA. An expression vector for long ds-RNA that targets mRNA encoding Ski, a protein that associates with histone deacetylases and operates in transcriptional repression, was injected into fertilized mouse oocytes. siRNA of 21 to 22 nucleotides derived from the long ds-RNA was detected in specific mouse embryonic tissue and corresponded to decreased levels of endogenous Ski mRNA. Developing embryos exhibited many of the phenotypes observed in Ski -deficient mice generated by conventional transgenic techniques. The use of expression vectors with various tissue-specific promoters could allow for more efficient generation of tissue-specific knockout mice. T. Shinagawa, S. Ishii, Generation of Ski -knockdown mice by expressing a long double-stranded RNA from an RNA polymerase II promoter. Genes Dev . 17 , 1340-1345 (2003). [Abstract] [Full Text]