Figure 6From: New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesisEstablishment for the fugu TILLING method. Mutagenesis was carried out with injection of ENU (conc., 70Â mg/kg body weight) into founder fugu 3 times at weekly intervals. Half of the founder fish died around 6Â weeks after the last ENU treatment. Sperm was not obtained from the founder until 5Â weeks after the last ENU treatment. To collect the sperm from the founder, gonadotrophin (HCG/SPH) was injected once 3Â weeks after the last ENU treatment. The sperm was collected 3Â weeks after the hormone treatment and then cryopreserved for a long-term TILLING resource. For screening strategy, the hatched fry were obtained in vitro fertilization with the collected sperm and eggs from wild-type female. Genomic DNA extracted from the fry was used to screen the induced mutations in Mstn gene with HRM analysis. The screened mutations were confirmed by direct sequencing and CEL I assay. These approaches demonstrate that the TILLING method was established in fugu.Back to article page