222 PRODUCTION OF KNOCKOUT RATS BY USING ZINC FINGER NUCLEASES AND TAL EFFECTOR NUCLEASES

Abstract

The rat has been used as an important animal for understanding human diseases. Genetically engineered rat strains are used as a human disease model in various research fields. Genetically engineered rat strains are now being routinely produced, not only as transgenic animals but also using gene knockout techniques. Recently, zinc finger nucleases (ZFN) and TAL effector nucleases (TALEN) have enabled editing targeted genes without using embryonic stem cells. These techniques have been applied for production of the knockout and knockin animals. We here studied that the effects of gene targeting by ZFN and TALEN introduced into rat embryos for efficient production of knockout rats. We custom-designed ZFN and TALEN plasmids targeted rat interleukin 2 receptor gamma (Il2rg) gene. Each mRNA was transcribed in vitro from these plasmids. Final concentration of mRNA was adjusted at 10 ng μL^–1 in sterilized water for microinjection. Messenger RNA was injected into rat pronuclear stage embryos. The embryos were then cultured in vitro to the 2-cell stage, and were transferred into oviducts of pseudopregnant females. The rate of development of offspring of embryos and effects of editing targeted genes were examined. Of 41 two-cell embryos introduced ZFN after embryo transfer, 9 embryos (22%) developed to offspring. Three offspring (33%) had an edited targeted gene locus. In the embryos introduced TALEN, 30% (6 offspring) of embryos developed to offspring after embryo transfer and all offspring had an edited targeted gene locus. This study demonstrated that the ZFN and TALEN mRNA was active after introduction into rat embryos. Knockout rats could be produced by introduction of ZFN and TALEN into rat embryos. ZFN and TALEN will provide a powerful new approach for targeted gene editing not only in rats but also in other animal species.