哺乳动物RNAi技术全面实验方法

WHENVIRUSESINFECTEUKARYOTICCELLS domly integrate into host genomes, double-stranded RNA (dsRNA) is frequently produced from the invading genes, either during viral replication or by aber

Wrant transcription from promoters located near the transgene insertion site. Eukaryotes such as plants, protists, and filamentous fungi and invertebrate and vertebrate animals have evolved a cellular defense system that responds to dsRNA and protects their genomes against these invading foreign elements. The dsRNA is rapidly processed by a cellular enzyme to small dsRNA fragments of distinct size and structure (Bernstein et al.2001), which then direct the sequence-specific degradation of the single-stranded mRNAs of the invading genes (Elbashir et al. 2001a). These short RNA duplexes were therefore named short interfering RNAs (siRNAs). The entire process of posttranscrip- tional dsRNA-dependent gene silencing is commonly referred to as RNA interference or RNAi (for recent reviews, see Hammond et al. 2001a; Matzke et al. 2001a; Sharp 2001;Tuschl 2001; Waterhouse et al. 2001; Hutvágner and Zamore 2002). In some instances, posttranscriptional gene silencing is also linked to transcriptional silencing (for reviews, see Wassenegger 2000; Bender 2001; Matzke et al. 2001b; Pal-Bhadra et al. 2002). Experimental introduction of dsRNA into cells has been used to disrupt the activity of cellular genes homologous in sequence to the introduced dsRNA (Fire et al. 1998). RNAi-based reverse genetic analysis now provides a rapid link between sequence data and biological function. RNAi is particularly useful for the analysis of gene function in Caenorhabditis elegans (for reviews, see Hope 2001; Kim 2001), but it is also widely used in other invertebrate animals (Kennerdell and Carthew 1998; Ngo et al. 1998; Brown et al. 1999). dsRNA of several hundred base pairs in length is typically required for effective gene silencing (Parrish et al. 2000; Elbashir et al. 2001b). Its application in vertebrate animals, including mammals, has proven to be more difficult because of the presence of additional dsRNA-triggered pathways that mediate nonspecific suppression of gene expression (Caplen et al. 2000; Nakano et al. 2000; Oates et al. 2000; Zhao et al. 2001). Fortunately, these nonspecific responses to dsRNA in vertebrates are not triggered by the siRNAs (Bitko and Barik 2001; Caplen et al. 2001; Elbashir et al. 2001c; Zhou et al. 2002). siRNAs can target genes as effectively as long dsRNAs (Elbashir et al. 2001b) and are widely used today for assessing gene function in cultured mammalian cells or early developing vertebrate embryos (Harborth et al. 2001; Elbashir et al. 2002; Zhou et al. 2002). siRNAs are also promising reagents for developing gene-specific therapeutics (Tuschl and Borkhardt 2002). This chapter concentrates on RNAi as it relates to mammalian systems and on the application of siRNAs for targeting genes expressed in somatic mammalian cell lines.