Establishment of Reverse Genetics in Medaka : Screening for Induced Point Mutations in Medaka with TILLING

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One of the most important tools in biological research is mutational analysis. Our understanding of the basic mechanism of disease has been transformed by the systematic application of mutational analysis. One of the most widely used approaches is forward genetics, which is driven by the identification of mutant phenotypes. Another approach is reverse genetics. The most widely used reverse genetics method in zebrafish and Medaka is undoubtedly the use of morpholinos, but this method is not a substitute for mutations because it is a transient method and only suited for early developmental stages. Recently a general reverse genetics method was reported, which can identify mutations in genes that are known only by their sequence. The method, called TILLING (Targeting Induced Local Lesions IN Genome), includes random mutagenesis, followed by screening for induced mutations in target genes at the genomic DNA level. We have applied this method for establishment of reverse genetics in Medaka. Adult Cab male Medaka were mutagenized with ENU and then outcrossed with Cab female to generate F1 progeny for the library. We established 5771 ENU-mutagenized F1 male fishes. To construct a library, genomic DNA and testis samples were isolated and cryopreserved from each F1 fish. Screening for mutations was done using TGCE (Temperature Gradient Capillary electrophoresis). A pilot screening revealed that the average per-base mutation frequency was 1 in 350kbp. Rev1 protein is an essential player in the production of both spontaneous and DNA damage-induced mutations. We have identified 16 mutations in Rev1 gene, which include 2 nonsense and 14 missense mutations.