title: Production of alpha 1,3-galactosyltransferase gene-deficient pigs by somatic cell nuclear transfer: a novel selection method for gal alpha 1,3-Gal antigen-deficient cells.
abstract: The objective of the present study was to isolate alpha 1,3-galactosyltransferase (GalGT)-gene double knockout (DKO) cells using a novel simple method of cell selection method. To obtain GalGT-DKO cells, GalGT-gene single knockout (SKO) fetal fibroblast cells were cultured for three to nine passages and GalGT-null cells were separated using a biotin-labeled IB4 lectin attached to streptavidin-coated magnetic beads. After 15-17 days of additional cultivation, seven GalGT-DKO cell colonies were obtained from a total of 2.5 x 10(7) GalGT-SKO cells. A total of 926 somatic nuclear transferred embryos reconstructed with the DKO cells were transferred into eight recipient pigs, producing four farrowed, three liveborns, and six stillborns. Absence of GalGT gene in the cloned pigs was confirmed by PCR and Southern blotting. Flow cytometric analysis revealed that alphaGal antigens were not present in the cells of the cloned DKO pigs.
authors.affiliation: The Animal Engineering Research Institute, Midorigahara, Tsukuba, Ibaraki, Japan. t.fujimura@nipponham.co.jp
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Fujimura
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Takahagi
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Shigehisa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nagashima
authors.forename: Shuji
authors.initials: S
authors.lastname: Miyagawa
authors.forename: Ryota
authors.initials: R
authors.lastname: Shirakura
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Murakami
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000941:Antigens / Q000235:genetics / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods; D004187:Disaccharides / Q000172:deficiency* / Q000235:genetics / Q000276:immunology; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005700:Galactosyltransferases / Q000235:genetics*; D006801:Humans; D008280:Magnetics; D008297:Male; D053652:Nuclear Transfer Techniques; D011247:Pregnancy; D013552:Swine / Q000235:genetics / Q000276:immunology; D014183:Transplantation, Heterologous
chemical_list: D000941:Antigens; D004187:Disaccharides; C055075:galactosyl-(1-3)galactose; D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine