Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells

Specific mRNA degradation mediated by double-stranded RNA (dsRNA) interfere nce (RNAi) is a powerful way of suppressing gene expression in plants, nema todes, and fungal, insect, and protozoan systems. However, only a few cases of RNAi have been reported in mammalian systems. Here, we investigated the feasibility of the RNAi strategy in several mammalian cells by using the e nhanced green fluorescent protein gene as a target, either by in situ produ ction of dsRNA from transient transfection of a plasmid harboring a 547-bp inverted repeat or by direct transfection of dsRNA made by in vitro transcr iption. Several mammalian cells including differentiated embryonic stem (ES ) cells did not exhibit specific RNAi in transient transfection. This long dsRNA, however, was capable of inducing a sequence-specific RNAi for the ep isomal and chromosomal target gene in undifferentiated ES cells. dsRNA at 8 .3 nM decreased the cognate gene expression up to 70%. However, RNAi activi ty was not permanent because it was more pronounced in early time points an d diminished 5 days after transfection. Thus, undifferentiated ES cells may lack the interferon response, similar to mouse embryos and oocytes. Regard less of their apparent RNAi activity, however, cytoplasmic extracts from ma mmalian cells produced a small RNA of 21 to 22 nucleotides from the long ds RNA. Our results suggest that mammalian cells may possess RNAi activity but nonspecific activation of the interferon response by longer dsRNA may mask the specific RNAi. The findings offer an opportunity to use dsRNA for inhi bition of gene expression in ES cells to study differentiation.