Establishment of reverse genetics in medaka

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To develop the system to study gene function at tissue level in medaka, we have established two methods in the present study. 1) Reverse genetics : TILLING(Targeting Induced Local Lesions IN Genome) is a reverse genetic strategy that combines random chemical mutagenesis with high-throughput discovery of induced mutations in the target genes. The method has been applied to a variety of plant and animal species. Screening of induced mutation is the most important step in TILLING. We developed the High Resolution Melting(HRM) assay, evaluated its efficacy for screening ENU-induced mutations in the medaka TILLING library. The HRM assay is fast, cost-effective, and robust in detecting variations in DNA. By HRM assay many nonsense mutations were identified and the phenotypes of these nonsense mutants confirmed their loss-of-function nature. 2) Regulation of gene expression in tissue : Infrared laser. evoked gene operator(IR-LEGO) is a microscope system optimized for heating cells at single cell level. Heat stress induces transcription of genes that are under the control of a heat shock promoter in individual targeted cells. We applied this technique to medaka fish carrying a heat shock promoter driven transgene and have succeeded the control of ectopic gene expression.