We have demonstrated efficient targeting of two endogenous zebrafish genes using shRNAs, at mid- to late embryonic stages. The genes targeted are of interest in our laboratory, with described mutant and morphant phenotypes. The findings demonstrate that RNAi methodology works efficiently and specifically in the zebrafish, and add new shRNA reagents for functional analyses.
The results show for the first time in zebrafish, that shRNA expression can decrease RNA levels significantly, at least by 70% in mosaic F0 transgenic embryos. In mammalian cells, a 70% knockdown of endogenous RNA levels by transfected shRNAs is considered reasonable25
(J. Doench and D. Root, Broad Institute, personal communication), and this LOF is clearly achievable in zebrafish. The siRNAs produced from the shRNAs most likely led to RNA cleavage, since the perfect match between siRNA guide strand and target used predicts RNA cleavage.19
Consistently, endogenous RNA levels decreased significantly. Additionally, qPCR using primers near the 5′ end of the zDisc1
gene or flanking the siRNA target site gave similar results, indicating that the entire RNA had been degraded (not shown). The F0 transgenic approach used to express the shRNAs is rapid and effective, but does give some mosaicism that may result in less than maximal LOF. In order to obtain uniform shRNA expression, inducible lines can be prepared. These may be necessary to allow viability after extensive LOF, and could use the Gal4/UAS strategy, or the various inducible Cre/lox approaches available.6,8
It seems very likely that inducible expression of shRNA can be achieved, but this remains to be tested.
The ability to prevent a phenotype by injection of cognate mRNA that does not bind the targeting siRNA, provided, for the first time, a stringent demonstration of specificity. The data further indicated that off-target effects of the shRNAs tested are small. In mammalian systems, rescue assays are generally not performed for shRNA-mediated LOF but are considered the “gold standard”.37
The zebrafish community established this stringent criterion to confirm MO specificity and we chose to require it of the shRNAs tested. Adopting this criterion for shRNAs would set a very high standard by the zebrafish community. For both wnt5b
, phenotypes observed with the shRNA were not as severe as those seen in the morphant, which required p53 to suppress necrosis, but more like the mutant phenotype.31
This suggests that toxic effects of shRNAs may be generally lower than those observed with MOs.
shRNAs that were able to target the genes tested mapped to both the 3′UTR, which shRNAs traditionally target,19
and to the open reading frame (ORF). This indicates that in zebrafish, shRNAs can be designed to either part of the gene. While four hairpin sequences per gene tested yielded 2 per gene that elicited a phenotype, testing more (perhaps 6) per gene may give more efficient knockdown or may be necessary for some genes.
Many tissue-specific promoters have been identified in zebrafish and our demonstration of tissue-restricted effects of zDisc1
shRNAs suggests that assaying shRNA effects in this way will be most useful. In these initial assays, we did not examine effects of shRNA past 24
hpf, into later stages of organogenesis and function, and persistence of shRNA effects in larvae, juveniles, and adults, remains to be addressed.
What else has not been done in this analysis? We have not looked for phenotypes at blastula stages, because our test genes do not show a phenotype before the end of gastrulation. It is not clear whether shRNA processing activity (particularly Ago2) is limiting in the early zebrafish, as it appears to be in Xenopus,26
and this will need to be tested. However, since MOs are effective in the early embryo, this activity is less critical than effectiveness of shRNAs in older embryos.
We do not have a large-scale measure of how frequently genes can be efficiently targeted by shRNAs. However, in our lab, we have successfully targeted 5 genes (of 6 tested) by shRNA expression (wnt5b, zDisc1, aldoaa, kif22,
although the extensive assays presented in this article have not yet been performed on some of these (HLS, unpublished, constructs available on request). In the Dong et al
, 2009 study,27
gene was targeted by shRNA injection and the gata-1
gene was targeted by shRNA expression from the CMV promoter, but RNA quantification and rescue assays were not performed. Together, these data suggest that shRNAs may be able to target many genes in the zebrafish. However, a more extensive assessment of targeting efficiency and off-target effects will need to come from a future study.
In sum, we have presented results that support usefulness of shRNAs for zebrafish LOF analysis, suggesting that this approach could be used to develop an RNAi resource that would benefit the large group of researchers in the community.