The study describes the production of α1,3-galactosyltransferase (α1,3GT)–deficient pigs, which are crucial for xenotransplantation due to the presence of α1,3Gal epitopes that cause hyperacute rejection. The researchers used a bacterial toxin, Clostridium difficile toxin A, to select for cells with a knocked-out second allele of the α1,3GT gene. Four healthy female piglets were produced through three rounds of cloning, each carrying a point mutation in the α1,3GT gene, which inactivates the enzyme. This method avoids the need for antibiotic-resistance genes, making the pigs safer for human use. The piglets were successfully born and showed no signs of α1,3Gal epitopes on their cell surfaces, indicating the successful knockout of the second α1,3GT allele. The study also demonstrates that the mutation is stable and does not affect the overall development or health of the piglets.The study describes the production of α1,3-galactosyltransferase (α1,3GT)–deficient pigs, which are crucial for xenotransplantation due to the presence of α1,3Gal epitopes that cause hyperacute rejection. The researchers used a bacterial toxin, Clostridium difficile toxin A, to select for cells with a knocked-out second allele of the α1,3GT gene. Four healthy female piglets were produced through three rounds of cloning, each carrying a point mutation in the α1,3GT gene, which inactivates the enzyme. This method avoids the need for antibiotic-resistance genes, making the pigs safer for human use. The piglets were successfully born and showed no signs of α1,3Gal epitopes on their cell surfaces, indicating the successful knockout of the second α1,3GT allele. The study also demonstrates that the mutation is stable and does not affect the overall development or health of the piglets.