Improving the efficiency of RNA interference in mammals

RNA interference (RNAi) is the process in which dsRNA leads to gene silencing, by either inducing the sequence-specific degradation of complementary mRNA or inhibiting translation. RNAi has been successfully applied as a powerful gene-silencing approach to various organisms, including Caenorhabditis elegans, plants, Drosophila melanogaster and mouse oocytes. The use of long dsRNAs in mammalian systems has been limited primarily because the introduction of dsRNA that is longer than 30 nucleotides (nt) induces a nonspecific interferon response. However, short 21–22-nt dsRNA molecules — known as small interfering RNAs (siRNAs) — could be used to target mammalian genes by RNAi while evading the interferon response. RNAi in mammals is triggered by various types of molecule, including synthetic siRNAs, plasmid-based short hairpin RNAs (shRNAs) or endogenous hairpin micro RNAs (miRNAs). It is now possible to carry out gene-silencing experiments in various cell types and cell lines, as well as in living animals. This is largely due to the development of DNA-vector-based siRNA-expression systems that allow the stable and prolonged silencing of target genes, together with a wider choice of delivery methods. Germline transmission of cells that contain an shRNA transgene — such as embryonic stem cells in mice and fertilized eggs in rats — has also been accomplished. The specificity and efficiency of RNAi in mammals has improved greatly thanks to advances in rational design, the ability to screen for the most effective siRNAs and tools that allow the inducible suppression of endogenous genes. Genome-wide functional RNAi screens, which were previously carried out exclusively in worms and flies, have now begun to revolutionize large-scale loss-of-function studies in mammals. The application of RNAi in mammals has the potential to allow the systematic analysis of gene expression and holds the promise of therapeutic gene silencing. RNA interference (RNAi) is the process in which dsRNA leads to gene silencing, by either inducing the sequence-specific degradation of complementary mRNA or inhibiting translation. RNAi has been successfully applied as a powerful gene-silencing approach to various organisms, including Caenorhabditis elegans, plants, Drosophila melanogaster and mouse oocytes. The use of long dsRNAs in mammalian systems has been limited primarily because the introduction of dsRNA that is longer than 30 nucleotides (nt) induces a nonspecific interferon response. However, short 21–22-nt dsRNA molecules — known as small interfering RNAs (siRNAs) — could be used to target mammalian genes by RNAi while evading the interferon response. RNAi in mammals is triggered by various types of molecule, including synthetic siRNAs, plasmid-based short hairpin RNAs (shRNAs) or endogenous hairpin micro RNAs (miRNAs). It is now possible to carry out gene-silencing experiments in various cell types and cell lines, as well as in living animals. This is largely due to the development of DNA-vector-based siRNA-expression systems that allow the stable and prolonged silencing of target genes, together with a wider choice of delivery methods. Germline transmission of cells that contain an shRNA transgene — such as embryonic stem cells in mice and fertilized eggs in rats — has also been accomplished. The specificity and efficiency of RNAi in mammals has improved greatly thanks to advances in rational design, the ability to screen for the most effective siRNAs and tools that allow the inducible suppression of endogenous genes. Genome-wide functional RNAi screens, which were previously carried out exclusively in worms and flies, have now begun to revolutionize large-scale loss-of-function studies in mammals. The application of RNAi in mammals has the potential to allow the systematic analysis of gene expression and holds the promise of therapeutic gene silencing.