ABSTRACT
The key approaches to elucidate the function of a gene in an organism are either to look for the dominant mutant of the gene that ablates the activity of that gene or to experimentally ablate the genetic message in vivo. Several loss-of-function (LOF) approaches, such as gene knockout by homologous recombination, antisense approach, and ribozyme technology, have been applied in the past. Of these arsenals, gene targeting by homologous recombination is the most commonly used technology to determine gene function; however, it is expensive and time consuming and many organisms are not amenable to it. RNA interference (RNAi) is a new tool of reverse genetics that has coalesced during the last decade. RNA silencing is a posttranscriptional regulatory
mechanism induced by double-stranded RNA (dsRNA), which inhibit the gene expression through specific degradation of homologous mRNA.1 Even though RNAi was first discovered in plants, its first elegant demonstration was first shown in an animal, the nematode worm Caenorhabiditis elegans. Fire, Mello, and colleagues, while attempting to use antisense RNA as an approach to inhibit the gene expression, found that the mixture of sense and antisense RNA (dsRNA) was at least tenfold or more potent than either sense or antisense RNAs alone. They also showed that the inhibition by dsRNA treatment closely resembled the loss-of-function or gene knockout phenotypes.2 RNAi was shown to be similar to PTGS triggered by a transgenic DNA, called co-suppression in plants,3 and to a related phenomenon termed quelling, observed in filamentous fungus Neurospora crassa.4 In the beginning, utility of RNAi was limited to mammalian cell lines due to the innate immune and interferon-related responses triggered by dsRNA.5 However, the discovery by Tuschl and colleagues that chemically synthesized short interfering RNAs (siRNAs) of 21 to 25 nt can bring about effective silencing of gene expression without inducing the interferon response opened the gate for large-scale application of RNAi in a wide range of organisms.6 RNA interference by double-stranded RNA has now been demonstrated in diverse groups of organisms such as protozoa, animals (invertebrates and vertebrates), plants, and fungi.7
