Efficient shRNA-Mediated Inhibition of Gene Expression in Zebrafish

1 Whitehead Institute for Biomedical Research , Cambridge Massachusetts.
Zebrafish (Impact Factor: 1.95). 07/2012; 9(3):97-107. DOI: 10.1089/zeb.2012.0770
Source: PubMed


Abstract 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.

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Available from: Jennifer H Gutzman, Jun 02, 2015
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    • "Here, artificially constructed miRNAs based on the endogenous miRNA backbone of mir30e are inserted within a transgene and transcribed from a pol II promoter. However , conflicting results in zebrafish indicate that the use of shRNAs may not be applicable for all target genes and its functionality needs to be thoroughly tested on a case-bycase basis (Dong et al. 2009; Kelly and Hurlstone 2011; De Rienzo et al. 2012). Nevertheless, the use of shRNAs seems a promising approach to down-regulate gene expression levels in a controlled and tissue-specific manner in fish model systems (Dong et al. 2009). "
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    • "Reports of successful knockdown via RNAi have been countered by reports of broad nonspecificity1213141516171819. Recently, shRNA expression vectors have been generated to produce siRNAs mimicking miRNA pathways2337 but it remains to be determined how useful or efficient these methods will be in generating stable knockdown lines. For mCherry knockdown, we observed stable, ongoing silencing. "
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