RNA interference (RNAi) is a phenomenon whereby double-stranded RNA (dsRNA) induces the sequence-dependent degradation of a cognate mRNA in animal or plant cells (1
). The mechanism responsible for dsRNA-induced gene silencing, which proceeds via a two-step mechanism, appears to have been strongly conserved during evolution (5
). In the first step, long dsRNAs are recognized by a nuclease in the RNase III family known as Dicer, which cleaves the dsRNA into small interfering RNAs (siRNAs) (7
) of 21–23 nt. These siRNAs are incorporated into a multicomponent nuclease complex, known as RISC, that is then responsible for the destruction of cognate mRNAs (9
Since dsRNAs act as inactivating agents of specific genes, they have been utilized as tools for the functional analysis of genes in a nematode, the fruit fly and plants (12
). It has been reported that, in mammalian cells, long dsRNAs induce the sequence-specific silencing of genes in mouse embryonal carcinoma cells and embryonic stem cells (15
). However, long dsRNAs (of >30 nt in length) activate a dsRNA-dependent protein kinase (PKR) and 2′,5′-oligoadenylate synthetase (17
) in mammalian somatic cells and the activities of these enzymes lead to a non-specific reduction in levels of mRNAs.
It was reported recently that synthetic 21 nt siRNAs specifically suppressed the expression of endogenous genes in several lines of mammalian cells (18
). Use of these 21 nt siRNA duplexes circumvented the activation of PKR and 2′,5′-oligoadenylate synthetase and suggested that siRNAs might be useful as gene-inactivating agents in mammals. However, the efficacy of siRNAs is dependent on identification of a specific target site within a target mRNA (19
). To obtain effective siRNAs, it is necessary, although both costly and time-consuming, to design and synthesize many different siRNAs.
In this report, we demonstrate that siRNAs generated in vitro
by recombinant human Dicer (re-hDicer) significantly suppressed not only the exogenous expression of a puromycin-resistance gene but also the endogenous expression of H-ras
. As reported recently, it is possible to produce short dsRNAs using RNase III from Escherichia coli
). However, since dsRNAs of 12–15 nt in length, on average, are generated by this RNase III (21
), it is better to use re-hDicer, which generates a more uniform population of 21–23 nt siRNAs. Use of re-hDicer to generate siRNAs might provide a powerful tool for studies of the mechanism of RNAi and the functions of various genes in mammalian cells, with potential utility in a clinical setting.