title: Production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology.
abstract: Recent developments in genome editing technology using meganucleases demonstrate an efficient method of producing gene edited pigs. In this study, we examined the effectiveness of the transcription activator-like effector nuclease (TALEN) system in generating specific mutations on the pig genome. Specific TALEN was designed to induce a double-strand break on exon 9 of the porcine α1,3-galactosyltransferase (GGTA1) gene as it is the main cause of hyperacute rejection after xenotransplantation. Human decay-accelerating factor (hDAF) gene, which can produce a complement inhibitor to protect cells from complement attack after xenotransplantation, was also integrated into the genome simultaneously. Plasmids coding for the TALEN pair and hDAF gene were transfected into porcine cells by electroporation to disrupt the porcine GGTA1 gene and express hDAF. The transfected cells were then sorted using a biotin-labeled IB4 lectin attached to magnetic beads to obtain GGTA1 deficient cells. As a result, we established GGTA1 knockout (KO) cell lines with biallelic modification (35.0%) and GGTA1 KO cell lines expressing hDAF (13.0%). When these cells were used for somatic cell nuclear transfer, we successfully obtained live GGTA1 KO pigs expressing hDAF. Our results demonstrate that TALEN-mediated genome editing is efficient and can be successfully used to generate gene edited pigs.
authors.affiliation: MGENPLUS Biotechnology Research Institute, Seoul 08511, Korea.
authors.forename: Jung-Taek
authors.initials: JT
authors.lastname: Kang
authors.affiliation: MGENPLUS Biotechnology Research Institute, Seoul 08511, Korea.
authors.forename: Dae-Kee
authors.initials: DK
authors.lastname: Kwon
authors.affiliation: MGENPLUS Biotechnology Research Institute, Seoul 08511, Korea.
authors.forename: A-Rum
authors.initials: AR
authors.lastname: Park
authors.affiliation: MGENPLUS Biotechnology Research Institute, Seoul 08511, Korea.
authors.forename: Eun-Jin
authors.initials: EJ
authors.lastname: Lee
authors.affiliation: MGENPLUS Biotechnology Research Institute, Seoul 08511, Korea.
authors.forename: Yun-Jin
authors.initials: YJ
authors.lastname: Yun
authors.affiliation: MGENPLUS Biotechnology Research Institute, Seoul 08511, Korea.
authors.forename: Dal-Young
authors.initials: DY
authors.lastname: Ji
authors.affiliation: Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA 24061, USA.
authors.forename: Kiho
authors.initials: K
authors.lastname: Lee
authors.affiliation: MGENPLUS Biotechnology Research Institute, Seoul 08511, Korea.; Department of Animal Science & Technology, Sunchon National University, Suncheon 57922, Korea.
authors.forename: Kwang-Wook
authors.initials: KW
authors.lastname: Park
mesh_terms: D000818:Animals; D018958:CD55 Antigens / Q000235:genetics; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D005091:Exons / Q000235:genetics; D005700:Galactosyltransferases / Q000235:genetics*; D000072669:Gene Editing / Q000662:veterinary*; D055786:Gene Knockout Techniques; D006801:Humans; D053652:Nuclear Transfer Techniques; D013552:Swine; D000069896:Transcription Activator-Like Effector Nucleases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D018958:CD55 Antigens; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase; D000069896:Transcription Activator-Like Effector Nucleases
keywords: alpha-1,3-galactosyltransferase; cloned pig; human decay-accelerating factor; knockout; somatic cell nuclear transfer