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Sujuan Jia, Yong-Dong Wang, Gang Wu, Hannah Henson, Michael R Taylor; Rapid Identification of Causative Mutations from Blind Zebrafish by Whole Exome Sequencing. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6398.
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Zebrafish is a useful model for ocular diseases. One of the most significant advantages of zebrafish is the ability to perform large-scale forward genetic screens for the unbiased identification of genetic pathways involved in visual disorders. However, the high cost of time and labor for the positional cloning of mutations has limited this benefit. Here we explored exome sequening to identify the causative mutations in blind zebrafish from a large-scale visual behavior screen.
Blind zebrafish larvae (Mu) at 5 days postfertilization were identified and separated from their wild type (WT) and heterozygous (Het) siblings based on the optokinetic response (OKR). Genomic DNA was isolated from each pool of 20 Mu or 20 WT/Het larvae. Exome-enriched libraries were prepared with the Agilent SureSelect XT zebrafish kit and subjected to 108-bp pair-end Illumina sequencing. cDNA from both Mu and Het/WT larvae were sequenced to evaluate the identified candidate genes. The expression patterns of the mutated genes were investigated by whole-mount in situ hybridization. The retinal morphology of the mutants was analyzed by immunostaining using cell-specific markers.
Using the zebrafish reference genome sequence (Zv9), we defined highly conserved 4 to 5 Mb DNA regions in 2 Mu lines when compared to the WT/Het pools. Within 2 months, we identified and validated the splice-site mutations responsible for both zebrafish mutants. The defective genes, syntaxin binding protein 1b (stxbp1b) and n-ethylmaleimide-sensitive factor a (nsfa), both play important functions in synaptic transmission.
Exome sequencing significantly accelerated the identification of mutations in blind zebrafish from a large-scale genetic screen. The cloned blind zebrafish lines provide useful tools for understanding the role of defective neurotransmitter release in inherited retinal diseases.
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