Elbashir et al. showed that 21–23 nt dsRNA fragments successfully trigger RNAi in an in vitro system using drosophila lysate. In addition, they demonstrated that chemically synthesized 21 nt siRNA duplexes specifically suppress the expression of endogenous and heterologeous genes in different mammalian cell lines, including human 293 and HeLa cells (8). A key discovery from these studies was that no non-specific gene-silencing effects were seen in mammalian cells by transfection of short dsRNA sequences (<30 nt). These results showed that 21 nt siRNA duplexes can be used as a new tool for studying gene function in mammalian cells, and may eventually find a use as gene-specific therapeutics.

Work by Caplen et al., (9) confirmed and extended the reports of siRNA-mediated RNAi in mammalian cell extracts. They demonstrated that identically sized synthetic siRNAs can induce gene-specific inhibition of expression in C. elegans, human, and mouse cells. Consistent with this hypothesis, numerous studies have since shown that dsRNA-induced gene silencing occurs in a number of different eukaryotic species (7, 11–21). The finding that the size of functional dsRNA fragments is conserved in plants and animals suggests a highly conserved mechanism in nature (10).

How Does RNAi Lead to Gene Silencing?

The basic mechanism of RNAi is thought to be a multi-step process:

In cultured mammalian cells, RNAi is mediated by 21nt RNA duplexes with symmetric 2-nt 3' overhangs. These siRNAs are introduced into a cell by transfection and lead to degradation of mRNA having the same sequence, thereby silencing gene expression. The specific pathways and mechanism of RNAi in mammalian cells are currently under intense investigation.