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Figure 10-18. Medaka TILLING diagram. Schematic illustration of our medaka TILLING library and a flow chart of how the mutants are obtained. Red frame indicates the library. G0 males are mutagenized using ENU and cross with wild-type females. Only males of F1 serve as the library that constructed the “genomic DNA archive” for screening PCR templates and “sperm archive” for recovering mutant progeny. Blue frame indicates ways for screening mutants. In this scheme, three mutation detection systems are described; “direct sequencing method,” “Cel1 method,” and “TGCE method.” Each strategy is mentioned in the text. A process after identification of mutation in a target gene is indicated in the green frame. F1 mutant fish that include a mutation in the target gene is recovered by artificial insemination using cryopreserved sperm archive. F2 progeny are heterozygote (heterozygote/wild type ratio is 1:1). To obtain homozygotic mutants, cross “hetero” with “hetero.” (Modified from Mitani et al. 2006, with permission of Karger AG, Basel.) |