Targeted disruption of the α1,3-galactosyltransferase gene in cloned pigs

Abstract

Galactose-alpha1,3-galactose (alpha1,3Gal) is the major xenoantigen causing hyperacute rejection in pig-to-human xenotransplantation. Disruption of the gene encoding pig alpha1,3-galactosyltransferase (alpha1,3GT) by homologous recombination is a means to completely remove the alpha1,3Gal epitopes from xenografts. Here we report the disruption of one allele of the pig alpha1,3GT gene in both male and female porcine primary fetal fibroblasts. Targeting was confirmed in 17 colonies by Southern blot analysis, and 7 of them were used for nuclear transfer. Using cells from one colony, we produced six cloned female piglets, of which five were of normal weight and apparently healthy. Southern blot analysis confirmed that these five piglets contain one disrupted pig alpha1,3GT allele.

Targeting efficiency of a-1,3-galactosyl transferase gene in pig fetal fibroblast cells

“…Recent studies that disrupted genes in somatic cells for nuclear transfer have used positive selection only using Neo r gene (McCreath et al, 2000;Denning et al, 2001;Lai et al, 2002;Ramsoondar et al, 2003). Large variations in the number of targeted colonies versus the number of random-integrated colonies (1/20 to 1/500) for gene targeting in somatic cells have been reported (Denning et al, 2001;Dai et al, 2002;Ramsoondar et al, 2003). In our experiments, the efficiency of single selection with G418 was 1 in 354 that was similar to that of reports.…”

“…Nuclear transfer of somatic cells in pigs is the most promising procedure to achieve knockout of α-1,3-galactosyl transferase (13-GT) gene for pigto-human xenotransplatation (Betthanser et al, 2000;Onishi, et al, 2000;Polejaeva, et al, 2000;Harrison et al, 2002). Production of viable pig and sheep with targeted insertion at several gene loci following nuclear transfer with gene-targeted cells has been reported (McCreath et al, 2000;Denning et al, 2001;Dai et al, 2002;Lai et al, 2002;Phelps et al, 2003;Ramsoondar et al, 2003).…”

The contribution of farm animals to human health

“…These structures are known as 1,3-a-gal-epitopes and are primarily produced by activity of 1,3-a-galactosyltransferase (a-gal). Piglets in which one allele of a-gal locus had been knocked out by homologous recombination in primary donor cells that were employed in nuclear transfer were recently generated [34,35]. The birth of four healthy piglets with disruption of both allelic loci for a-gal has meanwhile also been published.…”

Relations between animal transgenesis and reproduction

“…Interestingly, the galactosyl transferase gene in the pig has been knocked out in this manner with no major difficulty [21,22]. For an unknown reason, pig cells are less sensitive than ruminant cells to factors which reduce cloning efficiency.…”