Background

Because the conventional genotyping of genome edited animals largely depends on PCR-based genotyping, it is necessary to extract genomic DNA. In medaka, tail fin is an accessible and regenerable tissue, so that it is frequently used for genotyping to keep fish alive. To cut tail fin, fish must be fed for a few weeks to grow big enough (ca. 1 cm in body length). Feeding and taking care of many fishes are time-consuming and laborious. Therefore, reducing the number of fish to be fed in the early stage of breeding, such as embryos and early larvae, is desired to reduce the tasks. However, it is hard to take tail fin from embryos and early larvae alive because they are too small and fragile. To overcome this issue, a simple and non-invasive method for genotyping is essential. Therefore, we developed a new method that can non-invasively determine the genotype of each individual at embryonic stage simply by observing the fluorescence. We demonstrated the benefit of this method in a gene knock-in experiment targeting the growth associated protein 43 (gap43) gene expressed in the central nervous system (CNS) at 4 days post fertilization (dpf) (Murakami et al., 2017). This gene is suitable for the gene knock-in experiment, because its spatio-temporal expression pattern has been already revealed by RT-PCR and in situ hybridization in a previous study (Fujimori et al., 2008). If the expression pattern of a targeted gene in wild-type is unknown, it needs to be analyzed by RT-PCR, or any other technique, in order to confirm the correspondence between it and that of reporter gene in gene knock-in strains.