Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells

Frequently, inhibition of gene expression has been caused not only by antisense mRNA but also, in some cases, by expression of sense mRNA which has been used as a control. Moreover, this gene silencing by sense mRNA was shown to be sequence specific for the homologous gene. These initially confusing observations have now been attributed to gene silencing by the production of a minute amount of double-stranded RNA (dsRNA) generated by transcription of the sense mRNA by its promoter and the antisense mRNA by a cryptic promoter within the construct (reviewed in references 3, 6, 11, 26, 27, and 33). The term RNA interference (RNAi) was defined after the discovery that injection of dsRNA into the nematode Caenorhabditis elegans led to specific silencing of the gene homologous to the delivered RNA (12). RNAi was also observed in fruit flies, zebra fish, and other animals, including mice (7, 17, 30, 34, 35). The posttranscriptional gene silencing of C. elegans (18, 19, 28) is closely linked to the mechanism of cosuppression in plants and quelling in fungi (14, 21–23, 29). Unlike other organisms, accumulation of very small amounts of dsRNA in mammalian cells results in the interferon response. This leads to an overall block of translation by inactivation of an elongation factor by protein kinase. In addition, dsRNA activates a latent 2′,5′-oligoadenylate synthase and increases synthesis of a 2′,5′-oligonucleotide, causing activation of RNase L and nonspecific mRNA degradation. These events result in the onset of apoptosis in mammalian cells (13, 16). The natural function of RNAi and cosuppression appears to be protection of the host genome against invasion by mobile genetic elements such as transposons and viruses, which produce dsRNA in host cells (14, 18, 20, 27). Such considerations have discouraged investigators from using RNAi in mammals. Recently, however, RNAi has been reported in several mammalian systems. Transfection of a plasmid carrying the full-length pro-α1(I) collagen gene into rodent fibroblasts decreased the endogenous pro-α1(I) collagen mRNA up to 90% (1). RNAi activity was also reported in CHO cells, although the amount of dsRNA required for interference was 2,500 times more than in Drosophila S2 cells (32). Sequence-specific RNAi has been demonstrated in the preimplantation mouse embryo and oocytes by direct injection of dsRNA (30, 35). When dsRNA corresponding to an active green fluorescent protein (GFP) gene was injected into mouse zygotes, dsRNAi was effective throughout the blastocyst stage and implantation until embryos reached 6.5 days of development, corresponding to a 40- to 50-fold increase in cell mass (35). With these findings, it becomes critical to determine whether RNAi can be applied in mammalian tissue culture for gene silencing. The hallmark of RNAi is its specificity. The dsRNA triggers a specific degradation of homologous mRNA only within the region of identity with the dsRNA (37). The ability of a few molecules of dsRNA to eliminate a much larger pool of endogenous mRNA suggests a catalytic or amplification component to the RNAi mechanism. Results from studies of RNAi in plants suggested a mechanism, in which an RNA-primed RNA polymerase can spread gene silencing by dsRNA (28). Another model involves a catalytic RNA degradation generated by the dsRNA molecule and as yet unknown protein components. Recently, dsRNAs were shown to be processed to small 21- to 22-bp sizes in Drosophila embryo extracts (10, 36, 37), cultured S2 cells (2, 15), and C. elegans (24), making it likely that such RNAs serve as the specificity determinants in the RNAi reaction. These results suggest that dsRNA molecules are initially activated by a process that does not require interactions with their cognate mRNA target. Activation would appear to be a limiting step in RNAi, as the reaction is saturated at relatively low levels of dsRNA in vivo (12), potentiated by preincubation with dsRNA (31), and inhibited by excess unrelated dsRNAs (36). Here, we investigated the feasibility of the RNAi strategy for gene silencing in several mammalian cell lines by using the enhanced GFP (EGFP) gene as a target. Our results show that undifferentiated mouse embryonic stem (ES) cells exhibit a sequence-specific RNAi at a dsRNA concentration similar to that needed in Drosophila S2 cells.We also compared the ability of different mammalian cell types to degrade dsRNA into small pieces of 21 to 22 bp, which is the initial step on RNAi activity.