title: Generation of interleukin-2 receptor gamma gene knockout pigs from somatic cells genetically modified by zinc finger nuclease-encoding mRNA.
abstract: Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration.
authors.affiliation: Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan ; Meiji University International Institute for Bio-Resource Research (MUIIBR), Kawasaki, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Watanabe
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Nakano
authors.forename: Hitomi
authors.initials: H
authors.lastname: Matsunari
authors.forename: Taisuke
authors.initials: T
authors.lastname: Matsuda
authors.forename: Miki
authors.initials: M
authors.lastname: Maehara
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kanai
authors.forename: Mirina
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Yukina
authors.initials: Y
authors.lastname: Matsumura
authors.forename: Rieko
authors.initials: R
authors.lastname: Sakai
authors.forename: Momoko
authors.initials: M
authors.lastname: Kuramoto
authors.forename: Gota
authors.initials: G
authors.lastname: Hayashida
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Asano
authors.forename: Shuko
authors.initials: S
authors.lastname: Takayanagi
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Arai
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Umeyama
authors.forename: Masaki
authors.initials: M
authors.lastname: Nagaya
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Hanazono
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nagashima
mesh_terms: D000818:Animals; D001483:Base Sequence; D002469:Cell Separation; D002478:Cells, Cultured; D002999:Clone Cells; D003851:Deoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D055786:Gene Knockout Techniques / Q000379:methods*; D006801:Humans; D053631:Interleukin Receptor Common gamma Subunit / Q000172:deficiency* / Q000235:genetics*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D013552:Swine; D016335:Zinc Fingers
chemical_list: D053631:Interleukin Receptor Common gamma Subunit; D012333:RNA, Messenger; D003851:Deoxyribonucleases