chapter  7
Plasmid-Mediated Intracellular Expression of siRNAs
WithRenu Wadhwa, Masayuki Sano, Makoto Miyagishi, Kazunari Taira
Pages 10

RNA interference (RNAi) refers to obstruction of the flow of genetic information at the RNA level, resulting in the silencing of gene expression. It is triggered by the presence of double-stranded RNA. Synthetic short interfering RNAs (siRNAs) of ~21 nt were recently shown to be effective for specific gene silencing in mammalian cells without evoking the interferon response.1 Following this discovery, many reports describing successful expression and effect of vector-based siRNA in mammalian cells allowing delivery of siRNAs into mammalian cells by a DNA vector (low-cost alternative to the chemically synthesized siRNAs) have appeared. This has initiated a new way of studying gene function in higher eukaryotes. The effect of RNAi strongly depends on the target sequence and vector systems. Various groups, including our own, have developed plasmid based expression systems for siRNA.2-8 Viral vectors including retero-, adeno-and lentivirus are useful in facilitating the entry of siRNA in cells and tissues that are difficult to transfect. Retroviral vectors have been favored for

their ability to integrate exogenous genes into the genome of host cells. Replicationdefective lentiviral vectors can be used to introduce exogenous sequences into nonproliferating ones such as haematopoietic stem cells. Adenoviral vectors that are able to transduce exogenous sequences into quiescent cells and sustain their long-term expression can also be used.9-11 Such rapidly developing virus-based siRNA vectors may become potentially useful tools for therapy of dominant human diseases. This chapter describes the strategies and construction of plasmid-based expression systems for siRNA in mammalian cells.