**10.1. Gene knockouts**

One of the earliest genome engineering approaches applied to pigs was introduction of gene knockouts (KO). For any given gene, mutations which remove or disrupt the coding sequence can eliminate the expression of the gene and, provided that the KO is not lethal, create an organism which is entirely missing the gene product. The introduction of gene KO technology has been a key factor in the rapid advancement of the field of xenotransplantation [59].

As discussed above, there are several glycan molecules present in pigs which are absent in humans. These glycans are recognized by antibodies present in human serum which leads to rapid and extensive antibody-mediated damage to the porcine cells. Therefore, the elimination of the specific carbohydrate structures should help prevent human antibody recognition of the pig tissues. Unlike protein antigens which are directly coded by the DNA, glycosylation is due to the action of enzymes which create post-translational modifications of a variety of proteins produced by the cell. Therefore, glycosylation pathways must be examined to identify the key enzyme that creates the immunogenic glycan while otherwise leaving cellular metabolism intact.

The GGTA1 gene is responsible for creating the Gal alpha (1,3) Gal epitope in pigs. Although the specific reasons for this are unclear, human patients can express high levels of antibody specific for the Gal alpha (1,3) Gal epitope, presenting a major challenge to xenotransplantation [60]. The KO of the GGTA1 gene is one of the earliest genetic modifications of pigs for application in xenotransplantation, and results in greatly reduced human antibody recognition of porcine cells [61]. However, elimination of the GGTA1 gene alone has been shown to be insufficient due to a variety of other xenoantigens present in pig cells which are recognized by antibodies present in human serum. Generation of KO of CMAH [62], B4GALNT2 [28] and other xenoantigen genes have further decreased the reactivity of porcine cells to human serum. However, it is important to keep in mind that the greater the number of gene KO, especially when made in combination, may lead to detrimental effects on pig health.
