title: GONAD: Genome-editing via Oviductal Nucleic Acids Delivery system: a novel microinjection independent genome engineering method in mice.
abstract: Microinjection is considered the gold standard technique for delivery of nucleic acids (NAs; transgenes or genome editing tools such as CRISPR/Cas9 systems) into embryos, for creating genetically modified organisms. It requires sophisticated equipment as well as well-trained and highly skilled personnel to perform the micro-injection technique. Here, we describe a novel and simple microinjection-independent technique, called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD). Using GONAD, we show that NAs (e.g., eGFP mRNA or Cas9 mRNA/sgRNAs) can be effectively delivered to pre-implantation embryos within the intact mouse oviduct by a simple electroporation method, and result in the desired genetic modification in the embryos. Thus GONAD can bypass many complex steps in transgenic technology such as isolation of zygotes, microinjection of NAs into them, and their subsequent transfer to pseudo-pregnant animals. Furthermore, this method can potentially be used for genome editing in species other than mice.
authors.affiliation: 1] Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan [2] Department of Bioproduction, Tokyo University of Agriculture, 196 Yasaka, Abashiri, Hokkaido, 099-2493, Japan.
authors.forename: Gou
authors.initials: G
authors.lastname: Takahashi
authors.affiliation: Mouse Genome Engineering Core Facility, Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, 68198, USA.
authors.forename: Channabasavaiah B
authors.initials: CB
authors.lastname: Gurumurthy
authors.affiliation: Department of Bioproduction, Tokyo University of Agriculture, 196 Yasaka, Abashiri, Hokkaido, 099-2493, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Wada
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Miura
authors.affiliation: Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima 890-8544, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sato
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Ohtsuka
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D054736:Elongation Factor 2 Kinase / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology; D005260:Female; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008845:Microinjections / Q000379:methods*; D010057:Oviducts / Q000166:cytology; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007041:Hypoxanthine Phosphoribosyltransferase; C517690:Eef2k protein, mouse; D054736:Elongation Factor 2 Kinase