Efficient shRNA-Mediated Inhibition of Gene Expression in Zebrafish

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.

Stable Gene Silencing in Zebrafish with Spatiotemporally Targetable RNA Interference

“…Compared to previous reports (Dong et al 2009;De Rienzo et al 2012), our bipartite Gal4/UASbased design offers greater versatility and synergizes with the ongoing development of Gal4 enhancer trap lines in zebrafish.…”

“…This natural configuration is reported 12 times more efficient than simple hairpin designs. MiR-shRNAs are recently shown to be effective in vivo in mice (Dickins et al 2005;Premsrirut et al 2011;Zuber et al 2011) and zebrafish (Dong et al 2009;De Rienzo et al 2012). However, the capability of germ-line transmission is only reported by one study in zebrafish (Dong et al 2009), where functional efficacy and specificity of silencing were not quantitatively documented.…”

Exploring host–microbiota interactions in animal models and humans

“…The rapid development of organ primordia (within 5 d post-fertilization [dpf]) and onset of adult metamorphosis (within 14 dpf), together with small size, make zebrafish an invaluable resource for forward genetic and chemical screening. Methods for genetic screening include ENU mutagenesis, retrovirus-based insertional mutagenesis, zinc finger nucleases (Amacher 2008), morpholino-based gene knockdown (Nasevicius and Ekker 2000), conditional loss of function by RNAi (De Rienzo et al 2012), and, most recently, genome editing with the use of the TALEN system (Bedell et al 2012). There is a high degree of homology between zebrafish and mammals not only in the adaptive immune system, but also in the digestive system.…”

Zebrafish cancer: the state of the art and the path forward

“…First, through the use of TALlike effector nucleases (TALENs) and the CRISPR–Cas systems as extremely efficient and predictable methods of engineering genetic knockouts 86,87 . Second, through advances in RNA interference in the zebrafish 88 using miR-30-based approaches, which are now commonly used in mouse shRNA studies 89 . This will allow tissue-specific and inducible knockdowns of almost any candidate gene.…”