A Collection of Transgenic Medaka Strains for Efficient Site-Directed Transgenesis Mediated by phiC31 Integrase

Genetic analysis is facilitated by the efficient production of transgenic strains expressing a DNA of interest as a single copy at a designated chromosomal location. However, technical progress toward this goal in medaka fish (Oryzias latipes), a vertebrate model organism, has been slow. It is well known that phiC31 integrase enables efficient site-directed transgenesis by catalyzing the recombination of an attP DNA motif in a host genome with an attB motif in a targeting vector. This system was pioneered in medaka using the Sleeping Beauty transposon system, and the attP site was established at three chromosomal locations. However, this number appeared insufficient with regard to genetic linkage between the attP-landing site and a genetically modified locus of interest. Here, to establish a collection of transgenic strains of medaka, we introduced an attP motif into the medaka genome using the Ac/Ds maize transposon system and established 12 independent transgenic strains harboring a single copy of the attP motif in at least 11 of the 24 medaka chromosomes. We designed an attB-targeting vector that was integrated efficiently and precisely into the attP-landing site, and with which the DNA of interest was efficiently transmitted to germline cells. Extraneous sequences in the integrants derived from the bacterial backbone of the attB-targeting vector as well as a transgenic fluorescence marker present in the attP-landing site were removable through flippase-mediated recombination. Further, an advanced targeting vector with a heart-specific recombination marker served as a useful tool for easily screening phiC31 integrase-mediated recombinant G₀ embryos, leading to the efficient establishment of transgenic strains. Thus, our resources advance genetic research in medaka.