RNAi Is Discovered in Nematodes
The first evidence that dsRNA could lead to gene silencing came from work in the nematode Caenorhabditis elegans. Seven years ago, researchers Guo and Kemphues were attempting to use antisense RNA to shut down expression of the par-1 gene in order to assess its function. As expected, injection of the antisense RNA disrupted expression of par-1, but quizzically, injection of the sense-strand control did too (9).

This result was a puzzle until three years later. It was then that Fire and Mello first injected dsRNA — a mixture of both sense and antisense strands — into C. elegans (10). This injection resulted in much more efficient silencing than injection of either the sense or the antisense strands alone. Indeed, injection of just a few molecules of dsRNA per cell was sufficient to completely silence the homologous gene's expression. Furthermore, injection of dsRNA into the gut of the worm caused gene silencing not only throughout the worm, but also in its first generation offspring (10).

The potency of RNAi inspired Fire and Timmons to try feeding nematodes bacteria that had been engineered to express dsRNA homologous to the C. elegans unc-22 gene. Surprisingly, these worms developed an unc-22 null-like phenotype (11-13). Further work showed that soaking worms in dsRNA was also able to induce silencing (14). These strategies, whereby large numbers of nematodes are exposed to dsRNA, have enabled large-scale screens to select for RNAi-defective C. elegans mutants and have led to large numbers of gene knockout studies within this organism (15-18).

RNAi in Drosophila
RNAi has also been observed in Drosophila. Although a strategy in which yeast were engineered to produce dsRNA and then fed to fruit flies failed to work, microinjecting Drosophila embryos with dsRNA does effect silencing (2). Silencing can also be induced by "shooting" dsRNA into Drosophila embryos with a "gene gun" or by engineering flies to carry DNA containing an inverted repeat of the gene to be silenced. Over the last few years, these RNAi strategies have been used as reverse genetics tools in Drosophila organisms, embryo lysates, and cells to characterize various loss-of-function phenotypes (2, 19-23).

Glossary of Terms

Cosuppression - Silencing of an endogenous gene caused by the introduction of a transgene or infection by a virus. This term, which can refer to silencing at the post-transcriptional (PTGS) or transcriptional (TGS) level, has been primarily adopted by researchers working with plants.

Post-transcriptional Gene Silencing (PTGS) - Silencing of an endogenous gene caused by the introduction of a homologous dsRNA, transgene or virus. In PTGS, the transcript of the silenced gene is synthesized but does not accumulate because it is rapidly degraded. This is a more general term than RNAi, since it can be triggered by several different means.

Quelling - PTGS in Neurospora crassa induced by the introduction of a transgene.

RISC - RNA-induced silencing complex. A nuclease complex, composed of proteins and siRNA (see below), that targets and destroys endogenous mRNAs complementary to the siRNA within the complex.

RNA interference (RNAi) - Post-transcriptional gene silencing (PTGS) induced by the direct introduction of dsRNA. The term "RNA interference" was first used by researchers studying C. elegans.

siRNAs - Small interfering RNAs. Current models of PTGS indicate that these 21-23 nucleotide dsRNAs mediate PTGS. Introduction of siRNAs can induce PTGS in mammalian cells. siRNAs are apparently produced in vivo by cleavage of dsRNA introduced directly or via a transgene or virus. Amplification by an RNA-dependent RNA polymerase (RdRP) may occur in some organisms. siRNAs are incorporated into the RNA-induced silencing complex (RISC), guiding the complex to the homologous endogenous mRNA where the complex cleaves the transcript.