Organ xenografts in discordant combinations such as pig-to-man undergo hyperacute rejection due to the presence of naturally occurring human anti-pig xenoantibodies. The galactose alpha(1,3)-galactose epitope on glycolipids and glycoproteins is the major porcine xenoantigen recognized by these xenoantibodies. This epitope is formed by alpha(1,3)-galactosyltransferase, which is present in all mammals except man, apes, and Old World monkeys. We have generated mice lacking this major xenoantigen by inactivating the alpha(1,3)-galactosyltransferase gene. These mice are viable and have normal organs but develop cataracts. Substantially less xenoantibody from human serum binds to cells and tissues of these mice compared with normal mice. Similarly, there is less activation of human complement on cells from mice lacking the galactose alpha(1,3)-galactose epitope. These mice confirm the importance of the galactose alpha(1,3)-galactose epitope in human xenoreactivity and the logic of continuing efforts to generate pigs that lack this epitope as a source of donor organs.

Transgenic expression of the human complement regulatory molecule CD59 in mice and genetic deletion of the major xenoantigen galactose α 1,3 galactose (Gal KO) each resulted in partial protection of spleen cells from lysis by human serum. These protective effects were additive when the two genetic modifications were combined. However, when the effects of these genetic modifications were examined in an ex vivo model in which mouse hearts were perfused with human plasma, it was Gal KO which was the modification which determined protection. CD59 expression alone was not protective and CD59 expression in combination with Gal knockout did not result in a significant additional increase in protection over and above that provided by Gal knockout alone. The likely explanation for this discrepancy between the in vitro and ex vivo data is that the H2‐Kb promoter used to drive CD59 expression results I in substantially less expression on endothelium than on spleen cells.

The galactose a 1‐3 galactose terminal disaccharide (Gal epitope) has been identified as the major porcine xenoantigen recognised by xenoantibody in human plasma. Elimination or suppression of the epitope or antibody will be a major factor in overcoming hyperacute rejection. Inhibition of the antibody by depletion or elimination of the epitope by gene knockout may reveal the importance of other xenoantibodies, and in addition elimination of the epitope may unmask or produce other xenoantibody combinations. This study aims to determine the relative importance of anti‐Gal antibody and Gal epitope elimination in a functional model of xenotransplantation, ex vivo perfusion of mouse hearts with human plasma on a Langendorff apparatus. Perfusion of mouse hearts with human plasma depleted of anti‐Gal antibody demonstrates a protective effect compared to hearts perfused with undepleted plasma with prolongation of survival time from 24.1 to 44.5 min. Similarly, elimination of the epitope is also protective. Hearts from Gal knockout mice, which were generated by gene targeting of the al,3 galactosyltransferase gene, and hearts from appropriate control mice were perfused with human plasma. Gal knockout mice hearts demonstrated an increase in survival time from 10.2 to 33.8 min compared to control hearts. This was accompanied by a decrease in C3c and IgM, but little change in IgG deposition. The protective effect is incomplete, probably due to the effect of antibodies against non‐Gal xenoantigens. There was no functional evidence for generation of neo‐antigens in the Gal KO mice that were I recognised by naturally occurring human xenoantibodies.

Methods to improve the production of transgenic animals are being developed. Conventional transgenesis, involving microinjection of DNA into fertilized eggs, has a number of limitations. These result from the inability to control both the site of transgene insertion and the number of gene copies inserted. The approach described seeks to overcome these problems and to allow single copy insertion of transgenes into a defined site in animal genomes. The method involves the use of embryonic stem cells, gene targeting and the FLP recombinase system.