Too Much Interference: Injection of Double-Stranded RNA Has Nonspecific Effects in the Zebrafish Embryo

Abstract

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.

A zebrafish vasa morphant abolishes Vasa protein but does not affect the establishment of the germline

“…The vasa dsRNA-injected embryos had neither significant decrease in the number of vasa RNA-expressing cells nor of Vasa protein-expressing cells compared with the controls (data not shown). Our RNAi results are in general agreement with Oates et al (2000), who tested the effect of dsRNA injection in zebrafish and did not observe a specific phenotype related to the targeted mRNA.…”

In vivo and in vitro knockdown of FREP2 gene expression in the snail Biomphalaria glabrata using RNA interference

“…It has been reported that off-target effects can be a potential problem for RNAi [47][48][49]. We first demonstrated that limited or no cross reactivity occurs between FREP2 and myoglobin, and then showed that with FREP subfamilies non-specific knockdown of expression was not observed except for FREP6.…”

Efficient Target-Selected Mutagenesis in Zebrafish

“…A few years ago, RNA interference seemed to be a promising new approach for specifically inactivating genes in zebrafish (Wargelius et al 1999;Li et al 2000), but success rates in zebrafish were low and varying, with reports of major nonspecific effects on embryonic development reported as well (Oates et al 2000;Zhao et al 2001). The most widely used reverse genetics method in zebrafish is undoubtedly the use of morpholinos that are targeted to the translation initiation site of a specific transcript, thereby inhibiting its translation (Nasevicius and Ekker 2000).…”