Report of research results:Generation of genetically modified pigs by zygote electroporation
Generation of genetically modified pigs by zygote electroporation
- Tatsuya Takemoto (Laboratory for embryology, Institute of Advanced Medical Science)
- Fuminori Tanihara (Faculty of Bioscience and Bioindustry)
- Takeshige Otoi (Faculty of Bioscience and Bioindustry)
Pigs are considered as one of the best animals for generating models of human diseases and for providing organs for xenotransplantation to human. Recently, the CRISPR/Cas9 gene editing system has been developed and suggested to have great promise for the generation of gene-targeting pigs. In previous studies, gene-targeted pigs have been generated either by cytoplasmic injection of Cas9 mRNA and sgRNA into zygotes or via somatic cell nuclear transfer (SCNT) using targeted fibroblasts (Fig. 1). However, both of these methods have disadvantages because they require special techniques to use micromanipulator system and are too time-consuming to produce mutant pigs on a large scale (e.g., by injecting hundreds of zygotes or producing hundreds of cloned embryos).
In this study, we dissolved this problem by a combination of in-vitro fertilization and electroporation techniques using CRISPR/Cas9 system (Fig. 1). We confirmed successful genome editing in in-vitro fertilized embryos without loss of survival and succeeded in the generation of Myostatin (MSTN) mutant pigs (Fig. 2). In vitro fertilization techniques in pigs has been well established. Electroporation does not require special skills and can be used to treat 40–50 embryos simultaneously, indicating that this method produces mutant pigs much more easily and quickly than previously reported. We believe that this method dramatically accelerates genome modifications in pigs in terms of the cost and techniques, and it will impact on the broad field of experimental animal research, biology, and medical science.
Fuminori Tanihara, Tatsuya Takemoto, Eri Kitagawa, Shengbin Rao, Lanh Thi Kim Do, Akira Onishi, Yukiko Yamashita, Chisato Kosugi, Hitomi Suzuki, Shoichiro Sembon, Shunichi Suzuki, Michiko Nakai, Masakazu Hashimoto, Akihiro Yasue, Munehide Matsuhisa, Sumihare Noji, Tatsuya Fujimura, Dai-ichiro Fuchimoto, Takeshige Otoi
Somatic cell reprogramming–free generation of genetically modified pigs
Science Advances 2 (9): e1600803 (2016). doi: 10.1126/sciadv.1600803