[
Methods Cell Biol,
1995]
DNA transformation assays in a whole organism provide experimental links between molecular structure and phenotype. Experiments with transgenic Caenorhabditis elegans start in general with the injection of DNA into the adult gonad. Effects on phenotype or gene expression patterns can be analyzed either in F1 progeny derived from the injected animals or in derived transgenic lines. Microinjection of C. elegans was first carried out by Kimble et al. (1982). Stinchcomb et al. (1985) then showed that injected DNA could be maintained for several generations in transgenic lines. The first selective methods for producing and maintaining transgenic lines were reported in 1986 (Fire, 1986). These methods have been considerably improved since then (Mello et al., 1991) , so that assays involving DNA transformation are now a standard part of the experimental repertoire for C. elegans.
[
2006]
RNA interference (RNAi) describes a conserved biological response to double-stranded RNA (dsRNA) resulting in the degradation of homologous messenger RNA. In the last few years, this process of sequence-specific, post-transcriptional gene silencing has become a key technique for rapidly assessing gene function in species ranging from plants to mammals. Fire et al. provided the first insight into the RNAi mechanism by identifying dsRNA as the trigger of RNAi in Caenorhabditis elegans in 1998 [1]. However, a similar gene-silencing phenomenon was reported in earlier studies in both plants and Neurospora [2,3]. The basic RNAi response starts with long dsRNA being processed into small interfering RNAs (siRNAs) by a ribonuclease (RNase) III enzyme, Dicer. Next, the siRNA is incorporated into the RNA-induced silencing complex (RISC). For target RNA recognition to occur, the siRNA duplex must be unwound, allowing binding of one siRNA strand to the target mRNA. This is followed by RISC cleavage of the homologous mRNA. Recent work has shown that the RNAi machinery is also involved in antiviral responses, transposon silencing, development and heterochromatin formation [4].
[
2005]
RNA interference (RNAi) is a recently discovered phenomenon in which doublestranded RNA (dsRNA) silences endogenous gene expression in a sequencespecific manner (Fire et al., 1998). Since its discovery, the use of RNAi has become widely employed in many organisms to specifically knock down gene function. RNAi shares a remarkable degree of similarity with silencing phenomena in other organisms (Cogoni et al., 1999a; Sharp, 1999). For instance, RNAi, posttranscriptional gene silencing in plants and cosuppression in fungi can all be activated by the presence of aberrant RNAs (Maine, 2000; Tijsterman et al., 2002a). Additionally, plant, worm, and fly cells or extracts undergoing RNA-mediated interference all contain small dsRNAs, around 25 nucleotides in length, identical to the sequences present in the silenced gene (Baulcombe, 1996; Hammond et al., 2000; Zamore et al., 2000; Catalanotto et al., 2000). The high degree of similarity between these RNA-mediated silencing phenomena supports the notion that they were derived from an ancient and conserved pathway used to regulate gene expression, presumably to eliminate defective RNAs and to defend against viral infections and transposons. (Zamore, 2002). Components of RNAi have also been implicated in developmental processes, suggesting that RNAi may play a broader role in regulating gene expression (Smardon et al., 2000; Knight et al., 2001; et al., Ketting et al., 2001). Although we have learned much about the general mechanisms underlying RNAi, a detailed understanding of how RNAi works remains to be elucidated. In this chapter we will discuss first the biology of RNAi, then the genes required for its function, and we will end with a discussion on recent findings that have implicated chromatin silencing in the mechanism of RNAi.