U6 promoter-driven siRNA injection has nonspecific effects in zebrafish

RNA interference (RNAi) is a posttranscriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA), which causes degradation of homologous mRNAs. RNAi has been observed in a wide range of eukaryotes, including fungi, plants and animals. In vertebrates, long dsRNA activates the interferon response and yields nonspecific degradation of mRNA. In contrast, small interference RNA (siRNA) duplexes with a length of 21-23 nucleotides trigger specific gene silencing and thus are widely used in gene function studies. The use of siRNA for gene silencing in zebrafish has rarely been reported. In this report, we studied mammalian U6 promoter-driven siRNA-mediated RNA interference in zebrafish. The well characterized genes Myf5, Dlg3 and Nacre were selected as targets. Two to four target siRNAs were synthesized with incorporation of the U6 promoter. Constructs were introduced into early zebrafish embryos through microinjection, followed by in situ hybridization and embryonic development was monitored to determine whether U6 promoter-driven siRNAs could efficiently suppress specific gene expression. We showed that these siRNAs could partially suppress endogenous gene expression and that the siRNA efficiency varied at different targeted positions. However, the U6 promoter-driven siRNAs may also have induced nonspecific gene suppression (off-target effects). It appears that, despite the findings of previous reports, the current methodology of siRNA interference is not practical for studying gene function during early zebrafish development.