Post-transcriptional gene silencing (PTGS), which was initially considered a bizarre phenomenon limited to petunias and a few other plant species, is now one of the hottest topics in molecular biology (1). In the last few years, it has become clear that PTGS occurs in both plants and animals and has roles in viral defense and transposon silencing mechanisms. Perhaps most exciting, however, is the emerging use of PTGS and, in particular, RNA interference (RNAi) — PTGS initiated by the introduction of double-stranded RNA (dsRNA) — as a tool to knock out expression of specific genes in a variety of organisms (reviewed in 1-3).

How was RNAi discovered? How does it work? Perhaps more importantly, how can it be harnessed for functional genomics experiments? This article will briefly answer these questions and provide you with resources to find in depth information on PTGS and RNAi research.

More than a decade ago, a surprising observation was made in petunias. While trying to deepen the purple color of these flowers, Rich Jorgensen and colleagues introduced a pigment-producing gene under the control of a powerful promoter. Instead of the expected deep purple color, many of the flowers appeared variegated or even white. Jorgensen named the observed phenomenon "cosuppression", since the expression of both the introduced gene and the homologous endogenous gene was suppressed (1-5).

First thought to be a quirk of petunias, cosuppression has since been found to occur in many species of plants. It has also been observed in fungi, and has been particularly well characterized in Neurospora crassa, where it is known as "quelling" (1-3).

But what causes this gene silencing effect? Although transgene-induced silencing in some plants appears to involve gene-specific methylation (transcriptional gene silencing, or TGS), in others silencing occurs at the post-transcriptional level (post-transcriptional gene silencing, or PTGS). Nuclear run-on experiments in the latter case show that the homologous transcript is made, but that it is rapidly degraded in the cytoplasm and does not accumulate (1, 3, 6).

Introduction of transgenes can trigger PTGS, however silencing can also be induced by the introduction of certain viruses (2, 3). Once triggered, PTGS is mediated by a diffusible, trans-acting molecule. This was first demonstrated in Neurospora, when Cogoni and colleagues showed that gene silencing could be transferred between nuclei in heterokaryotic strains (1, 7). It was later confirmed in plants when Palauqui and colleagues induced PTGS in a host plant by grafting a silenced, transgene-containing source plant to an unsilenced host (8). From work done in nematodes and flies, we now know that the trans-acting factor responsible for PTGS in plants is dsRNA (1-3).

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).

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