論文

Inbred strains of organisms are genetically highly uniform and thus useful for life science research. We have previously reported the ongoing generation of the zebrafish IM strain from the India (IND) strain through full sib-pair mating for 16 generations. However, the IM fish laid a small number of offspring and had a short lifespan, implying the need for discreet care in breeding. Here, we report the subsequent establishment of IM strain as well as the generation of a new inbred zebrafish strain, Mishima-AB (M-AB). M-AB was derived from the *AB strain by full sib-pair mating for over 20 generations, which fulfills the general criterion for the establishment of an inbred strain. In contrast to the IM case, maintenance of the M-AB strain by sib-pair mating required almost no special handling. Genome sequencing of IM individuals from the 47th generation and M-AB individuals from the 27th generation revealed that SNP-based genomic heterogeneity across whole-genome nucleotides was 0.008% and 0.011%, respectively. These percentages were much lower than those of the parental IND (0.197%) and *AB (0.086%) strains. These results indicate that the genomes of these inbred strains were highly homogenous. We also demonstrated the successful microinjection of antisense morpholinos, CRISPR/Cas9, and foreign genes into M-AB embryos at the 1-cell stage. Overall, we report the establishment of a zebrafish inbred strain, M-AB, which is capable of regular breeding and genetic manipulation. This strain will be useful for the analysis of genetically susceptible phenotypes such as behaviors, microbiome features and drug susceptibility.

The congenic strain, an inbred strain containing a small genomic region from another strain, is a powerful tool to assess the phenotypic effect of polymorphisms and/or mutations in the substituted genomic region. Recent substantial progress in the genetic studies of complex traits increases the necessity of congenic strains and, therefore, a quick breeding system for congenic strains has become increasingly important in model organisms such as mouse and medaka. Traditionally, more than ten generations are necessary to produce a congenic strain. In contrast, a quick method has been reported previously for the mouse, in which the use of genetic markers reduces the required number of backcross generations to about a half that of the traditional method, so that it would take around six generations to obtain a congenic strain. Here, we present an even quicker congenic production system, which takes only about four generations. The system can produce medaka congenic strains having part of the HNI-II (an inbred medaka strain derived from the northern Japanese population, Oryzias sakaizumii) genome in the HdrR-II1 (another inbred strain from the southern Japanese population, O. latipes) background. In this system, the availability of frozen sperm and genotype data of the BC1 male population makes it possible to start marker-assisted congenic production after obtaining the BC2 population. Our evaluation revealed that the system could work well to increase the percentage of recipient genome as expected, so that a congenic strain may be obtained in about one year.