Too much interference: injection of double-stranded RNA has nonspecific effects in the zebrafish embryo.
We have investigated the ability of double-stranded RNA (dsRNA) to inhibit gene expression in a vertebrate, the zebrafish, Danio rerio. Injection of dsRNA corresponding to the T-box gene tbx16/spadetail (spt) into early wild-type embryos caused a rapid and dramatic loss of tbx16/spt mRNA in the blastula. mRNAs from the papc, tbx6, and gata1 genes, which depend on tbx16/spt function for their expression, were reduced, apparently mimicking the spt mutant phenotype. However, mRNAs from a number of genes that are unaffected by the spt mutation, such as beta catenin, stat3, and no tail, were also lost, indicating that the "interference" effect of tbx16/spt dsRNA was not restricted to the endogenous tbx16/spt mRNA. We compared the effects of injecting dsRNA from the zebrafish tbx16/spadetail, nieuwkoid/bozozok, and Brachyury/no tail genes with dsRNA from the bacterial lacZ gene. In each case the embryos displayed a variable syndrome of abnormalities at 12 and 24 h postfertilization. In blind studies, we could not distinguish between the effects of the various dsRNAs. Consistent with a common effect of dsRNA, regardless of sequence, injection of dsRNA from the lacZ gene was likewise effective in strongly reducing tbx16/spt and beta catenin mRNA in the blastula. These findings indicate that, despite published reports, the current methodology of double-stranded RNA interference is not a practical technique for investigating zygotic gene function during early zebrafish development.
Oates
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Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
Bruce
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Ho
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D000818:Animals; D004625:Embryo, Nonmammalian; D020868:Gene Silencing; D017403:In Situ Hybridization; D008845:Microinjections; D010641:Phenotype; D012330:RNA, Double-Stranded / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D029961:Zebrafish Proteins
D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D020825:T-Box Domain Proteins; D029961:Zebrafish Proteins; C112539:tbx16 protein, zebrafish