title: Pig cells that lack the gene for alpha1-3 galactosyltransferase express low levels of the gal antigen.
abstract: BACKGROUND
  The major antigen recognized on pig tissue by primate antibodies is a terminal galalpha1-3gal carbohydrate structure (gal antigen) present on glycolipids and glycoproteins. The production of animals from somatic cells allows for the inactivation of specific genes. It is anticipated that the complete inactivation of the gene encoding alpha1-3 galactosyltransferase, the enzyme that synthesizes the galalpha1-3gal linkage, will result in loss of that antigen from pig organs and tissue and will provide a survival benefit in pig-to-primate xenotransplants.
  
  
  METHODS
  Positive-negative selection was used to produce fetal-pig fibroblasts that were a heterozygous knockout (+/-) of the alpha1-3 galactosyltransferase gene. Nuclear transfer of these cells generated pig embryos and live born pigs with the appropriate genotype. Using a novel selection method with cells from (+/-) embryos, we produced homozygous (-/-) fetal-pig fibroblast cells.
  
  
  RESULTS
  Southern blot analysis of the alpha1-3 galactosyltransferase gene showed that we had produced (+/-) pig embryos, (+/-) live born pigs, and (-/-) pig-fetal fibroblast cells. Fluorescence-activated cell sorter (FACS) analysis with some, but not all, mouse anti-gal monoclonal antibodies and sensitized human serum showed that (-/-) cells still synthesized the gal antigen at 1 to 2% of the level of control heterozygous cells.
  
  
  CONCLUSIONS
  Fetal-pig fibroblasts homozygous for the knockout of the alpha1-3 galactosyltransferase gene appear to express low but detectable levels of the gal antigen.
authors.affiliation: Nextran Incorporated, Princeton, NJ 08540, USA.
authors.forename: Ajay
authors.initials: A
authors.lastname: Sharma
authors.forename: Bashoo
authors.initials: B
authors.lastname: Naziruddin
authors.forename: Cunqi
authors.initials: C
authors.lastname: Cui
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Martin
authors.forename: Hui
authors.initials: H
authors.lastname: Xu
authors.forename: Hua
authors.initials: H
authors.lastname: Wan
authors.forename: Ying
authors.initials: Y
authors.lastname: Lei
authors.forename: Caren
authors.initials: C
authors.lastname: Harrison
authors.forename: Jessie
authors.initials: J
authors.lastname: Yin
authors.forename: Jeannine
authors.initials: J
authors.lastname: Okabe
authors.forename: Christine
authors.initials: C
authors.lastname: Mathews
authors.forename: Aileen
authors.initials: A
authors.lastname: Stark
authors.forename: Connie S
authors.initials: CS
authors.lastname: Adams
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Houtz
authors.forename: Barry S
authors.initials: BS
authors.lastname: Wiseman
authors.forename: Guerard W
authors.initials: GW
authors.lastname: Byrne
authors.forename: John S
authors.initials: JS
authors.lastname: Logan
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002478:Cells, Cultured; D004187:Disaccharides / Q000276:immunology* / Q000378:metabolism*; D000939:Epitopes; D016332:Fetal Tissue Transplantation / Q000276:immunology*; D005347:Fibroblasts / Q000166:cytology / Q000276:immunology*; D005434:Flow Cytometry; D005700:Galactosyltransferases / Q000235:genetics*; D006579:Heterozygote; D013552:Swine
chemical_list: D000911:Antibodies, Monoclonal; D004187:Disaccharides; D000939:Epitopes; C055075:galactosyl-(1-3)galactose; D005700:Galactosyltransferases; C045097:UDP-galactose-lactosylceramide alpha 1-4-galactosyltransferase