Scientists successfully produced four genetically modified pigs lacking the α1,3-galactosyltransferase (α1,3 GT) gene, which is responsible for producing α1,3-galactose (α1,3 Gal) epitopes. These epitopes are major xenoantigens that cause hyperacute rejection in pig-to-human xenotransplantation. The study used a bacterial toxin, toxin A from Clostridium difficile, to select for cells with the second allele of the α1,3 GT gene knocked out. This method allowed the researchers to identify a T-to-G single point mutation in the second allele of the α1,3 GT gene, which inactivated the α1,3 GT protein. The pigs produced were α1,3 GT double knockout (DKO) and did not carry antibiotic-resistance genes, making them safer for human use. The study also demonstrated that the DKO pigs did not produce α1,3 Gal epitopes, as confirmed by immunological tests. The pigs were healthy and showed no signs of cataracts or other abnormalities at 7 weeks of age. The results indicate that the removal of α1,3 Gal epitopes from pig cells does not prevent normal development, even though pig cells express up to 500 times more α1,3 Gal epitopes than mouse cells. The study highlights the potential of these pigs for xenotransplantation due to their lack of α1,3 Gal epitopes and the absence of antibiotic-resistance genes. The findings also suggest that the point mutation in the α1,3 GT gene is stable and will be maintained in the genome of these pigs and their offspring. This research represents a significant step toward the development of safe xenotransplantation therapies.Scientists successfully produced four genetically modified pigs lacking the α1,3-galactosyltransferase (α1,3 GT) gene, which is responsible for producing α1,3-galactose (α1,3 Gal) epitopes. These epitopes are major xenoantigens that cause hyperacute rejection in pig-to-human xenotransplantation. The study used a bacterial toxin, toxin A from Clostridium difficile, to select for cells with the second allele of the α1,3 GT gene knocked out. This method allowed the researchers to identify a T-to-G single point mutation in the second allele of the α1,3 GT gene, which inactivated the α1,3 GT protein. The pigs produced were α1,3 GT double knockout (DKO) and did not carry antibiotic-resistance genes, making them safer for human use. The study also demonstrated that the DKO pigs did not produce α1,3 Gal epitopes, as confirmed by immunological tests. The pigs were healthy and showed no signs of cataracts or other abnormalities at 7 weeks of age. The results indicate that the removal of α1,3 Gal epitopes from pig cells does not prevent normal development, even though pig cells express up to 500 times more α1,3 Gal epitopes than mouse cells. The study highlights the potential of these pigs for xenotransplantation due to their lack of α1,3 Gal epitopes and the absence of antibiotic-resistance genes. The findings also suggest that the point mutation in the α1,3 GT gene is stable and will be maintained in the genome of these pigs and their offspring. This research represents a significant step toward the development of safe xenotransplantation therapies.