title: Production of genetically modified porcine blastocysts by somatic cell nuclear transfer: preliminary results toward production of xenograft-competent miniature pigs.
abstract: Galα1-3Gal (α-Gal epitope) is the major xenoantigenic epitope responsible for hyperacute rejection upon pig-to-human xenotransplantation. Endo-β-galactosidase C (EndoGalC) from Clostridium perfringens can digest the α-Gal epitope. In this study, gene-engineered primary cultured porcine embryonic fibroblasts (PEF) expressing EndoGalC were obtained and subjected to somatic cell nuclear transfer (SCNT) to test whether xenograft-competent pigs can be created. The EndoGalC-expressing PEF clones exhibited highly reduced expression of α-Gal epitope, as revealed by cytochemical staining with BS-I-B(4) isolectin, a lectin that specifically binds to α-Gal epitope, and FACS analysis. The pattern of low level of α-Gal epitope expression continued for at least 6 months (more than 10 generations) after isolation. SCNT of nuclei from these cells resulted in the generation of blastocysts that displayed nearly complete loss of α-Gal epitope from their cell surface. This is the first study to demonstrate that SCNT using EndoGalC-expressing PEFs as donors would be useful for production of genetically modified cloned pigs suitable for xenotransplantation.
authors.affiliation: Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Himaki
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watanabe
authors.forename: Haiei
authors.initials: H
authors.lastname: Chi
authors.forename: Mitsutoshi
authors.initials: M
authors.lastname: Yoshida
authors.forename: Kazuchika
authors.initials: K
authors.lastname: Miyoshi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sato
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001755:Blastocyst / Q000276:immunology* / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002999:Clone Cells / Q000276:immunology / Q000378:metabolism; D004622:Embryo, Mammalian; D005260:Female; D005347:Fibroblasts / Q000276:immunology / Q000378:metabolism; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D006026:Glycoside Hydrolases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006084:Graft Rejection / Q000235:genetics / Q000276:immunology; D006801:Humans; D016056:Immunodominant Epitopes / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D023421:Models, Animal; D053652:Nuclear Transfer Techniques; D037121:Plant Lectins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013552:Swine; D013556:Swine, Miniature / Q000196:embryology* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D014183:Transplantation, Heterologous / Q000276:immunology*
chemical_list: D000954:Antigens, Surface; C045475:Griffonia simplicifolia lectins; D016056:Immunodominant Epitopes; D037121:Plant Lectins; D012333:RNA, Messenger; D006026:Glycoside Hydrolases; C057030:endo-beta-D-galactosidase C