Reverse Genetic Approaches in Zebrafish

Zebrafish (Danio rerio) is a well-established vertebrate animal model. A comprehensive collection of reverse genetics tools has been developed for studying gene function in this useful organism. Morpholino is the most widely used reagent to knock down target gene expression post-transcriptionally. For a long time, targeted genome modification has been heavily relied on large-scale traditional forward genetic screens, such as ENU (N-ethyl-N-nitrosourea) mutagenesis derived TILLING (Targeting Induced Local Lesions IN Genomes) strategy and pseudo-typed retrovirus mediated insertional mutagenesis. Recently, engineered endonucleases, including ZFNs (zinc finger nucleases) and TALENs (transcription activator-like effector nucleases), provide new and efficient strategies to directly generate site-specific indel mutations by inducing double strand breaks in target genes. Here we summarize the major reverse genetic approaches for loss-of-function studies used and emerging in zebrafish, including strategies based on genome-wide mutagenesis and methods for site-specific gene targeting. Future directions and expectations will also be discussed.