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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
Author(s) -
Fujimura Tatsuya,
Takahagi Yoichi,
Shigehisa Tamotsu,
Nagashima Hiroshi,
Miyagawa Shuji,
Shirakura Ryota,
Murakami Hiroshi
Publication year - 2008
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.20890
Subject(s) - biology , somatic cell , galactosyltransferase , alpha (finance) , somatic cell nuclear transfer , gene , antigen , genetics , microbiology and biotechnology , selection (genetic algorithm) , biochemistry , embryogenesis , medicine , construct validity , nursing , blastocyst , patient satisfaction , artificial intelligence , computer science , enzyme
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 × 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 αGal antigens were not present in the cells of the cloned DKO pigs. Mol. Reprod. Dev. 75: 1372–1378, 2008. © 2008 Wiley‐Liss, Inc.