Efficient shRNA-mediated inhibition of gene expression in zebrafish.
Despite the broad repertoire of loss of function (LOF) tools available for use in the zebrafish, there remains a need for a simple and rapid method that can inhibit expression of genes at later stages. RNAi would fulfill that role, and a previous report (Dong et al. 2009) provided encouraging data. The goal of this study was to further address the ability of expressed shRNAs to inhibit gene expression. This included quantifying RNA knockdown, testing specificity of shRNA effects, and determining whether tissue-specific LOF could be achieved. Using an F0 transgenic approach, this report demonstrates that for two genes, wnt5b and zDisc1, each with described mutant and morphant phenotypes, shRNAs efficiently decrease endogenous RNA levels. Phenotypes elicited by shRNA resemble those of mutants and morphants, and are reversed by expression of cognate RNA, further demonstrating specificity. Tissue-specific expression of zDisc1 shRNAs in F0 transgenics demonstrates that conditional LOF can be readily obtained. These results suggest that shRNA expression presents a viable approach for rapid inhibition of zebrafish gene expression.
Whitehead Institute for Biomedical Research, Cambridge, MA, USA.
Gianluca
G
De Rienzo
Jennifer H
JH
Gutzman
Hazel
H
Sive
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D004258:DNA Polymerase III / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D012313:RNA / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D051153:Wnt Proteins / Q000235:genetics* / Q000378:metabolism; D000071818:Wnt-5a Protein; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
C538728:DISC1 protein, zebrafish; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; C480282:Wnt5a protein, zebrafish; D029961:Zebrafish Proteins; D012313:RNA; D004258:DNA Polymerase III