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

Tatsuya Fujimura, Yoichi Takahagi, Tamotsu Shigehisa, Hiroshi Nagashima, Shuji Miyagawa, Ryota Shirakura, Hiroshi Murakami

Research output: Contribution to journalArticle

46 Citations (Scopus)

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 × 107 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.

Original languageEnglish
Pages (from-to)1372-1378
Number of pages7
JournalMolecular Reproduction and Development
Volume75
Issue number9
DOIs
Publication statusPublished - 1 Sep 2008

Keywords

  • Alpha 1,3-galactosyltransferase
  • Nuclear transfer
  • Transgenic knockout pig
  • Xenotransplantation

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