title: Somatic cell reprogramming-free generation of genetically modified pigs.
abstract: Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.
authors.affiliation: Laboratory of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, 2272-1 Ishii, Myozai-gun, Tokushima 779-3233, Japan.; Diabetes Therapeutics and Research Center, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan.
authors.forename: Fuminori
authors.initials: F
authors.lastname: Tanihara
authors.affiliation: Division of Embryology, Fujii Memorial Institute of Medical Sciences, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Takemoto
authors.affiliation: Research and Development Center, NH Foods Ltd., 3-3 Midorigahara, Tsukuba, Ibaraki 300-2646, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Kitagawa
authors.affiliation: Research and Development Center, NH Foods Ltd., 3-3 Midorigahara, Tsukuba, Ibaraki 300-2646, Japan.
authors.forename: Shengbin
authors.initials: S
authors.lastname: Rao
authors.affiliation: Laboratory of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, 2272-1 Ishii, Myozai-gun, Tokushima 779-3233, Japan.
authors.forename: Lanh Thi Kim
authors.initials: LT
authors.lastname: Do
authors.affiliation: Laboratory of Animal Reproduction, Department of Animal Science and Resources, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa, 252-0880, Japan.; Transgenic Pig Research Unit, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki, 305-0901, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Onishi
authors.affiliation: Division of Embryology, Fujii Memorial Institute of Medical Sciences, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Yamashita
authors.affiliation: Division of Embryology, Fujii Memorial Institute of Medical Sciences, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan.
authors.forename: Chisato
authors.initials: C
authors.lastname: Kosugi
authors.affiliation: Division of Embryology, Fujii Memorial Institute of Medical Sciences, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Transgenic Pig Research Unit, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki, 305-0901, Japan.
authors.forename: Shoichiro
authors.initials: S
authors.lastname: Sembon
authors.affiliation: Transgenic Pig Research Unit, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki, 305-0901, Japan.
authors.forename: Shunichi
authors.initials: S
authors.lastname: Suzuki
authors.affiliation: Transgenic Pig Research Unit, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki, 305-0901, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Nakai
authors.affiliation: Laboratory for Embryogenesis, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Hashimoto
authors.affiliation: Department of Orthodontics and Dentofacial Orthopedics, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Yasue
authors.affiliation: Diabetes Therapeutics and Research Center, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan.
authors.forename: Munehide
authors.initials: M
authors.lastname: Matsuhisa
authors.affiliation: Department of Life System, Institute of Technology and Science, Tokushima University, 2-1 Minami-Jyosanjima-cho, Tokushima 770-8506, Japan.
authors.forename: Sumihare
authors.initials: S
authors.lastname: Noji
authors.affiliation: Research and Development Center, NH Foods Ltd., 3-3 Midorigahara, Tsukuba, Ibaraki 300-2646, Japan.
authors.forename: Tatsuya
authors.identifier: 0000-0001-9677-733X
authors.initials: T
authors.lastname: Fujimura
authors.affiliation: Transgenic Pig Research Unit, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki, 305-0901, Japan.
authors.forename: Dai-Ichiro
authors.initials: D
authors.lastname: Fuchimoto
authors.affiliation: Laboratory of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, 2272-1 Ishii, Myozai-gun, Tokushima 779-3233, Japan.
authors.forename: Takeshige
authors.initials: T
authors.lastname: Otoi
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics* / Q000254:growth & development; D064113:CRISPR-Cas Systems; D065150:Cellular Reprogramming / Q000235:genetics*; D005307:Fertilization in Vitro; D009154:Mutation; D055435:Myostatin / Q000235:genetics*; D053652:Nuclear Transfer Techniques; D017393:RNA Editing / Q000235:genetics; D013552:Swine / Q000235:genetics; D015053:Zygote / Q000254:growth & development
chemical_list: D055435:Myostatin
keywords: Somatic cell; gmo; somatic cell nuclear transfer