Production of α1,3-Galactosyltransferase-Deficient Pigs

TLDR

α1,3‑galactosyltransferase generates α1,3‑gal epitopes that trigger hyperacute rejection in pig‑to‑human xenotransplantation, making their removal essential for transplant success. We disrupted both alleles of the α1,3GT gene in cloned pigs by first targeting one allele and then selecting for a toxin‑resistant second‑allele knockout. Sequencing revealed a T→G point mutation in exon 9 that inactivates α1,3GT, and three rounds of cloning produced four healthy double‑knockout piglets that lack antibiotic‑resistance genes and offer a safer source of α1,3‑gal‑deficient organs.

Abstract

The enzyme α1,3-galactosyltransferase (α1,3GT or GGTA1) synthesizes α1,3-galactose (α1,3Gal) epitopes (Galα1,3Galβ1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of α1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the α1,3GT gene in cloned pigs. A selection procedure based on a bacterial toxin was used to select for cells in which the second allele of the gene was knocked out. Sequencing analysis demonstrated that knockout of the second allele of the α1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the α1,3GT protein. Four healthy α1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the α1,3GT gene hold significant value, as they would allow production of α1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use.