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Hyperacute rejection (HAR) depends on the response of xenoreactive antibodies principally against porcine α-Gal epitope. Methods eliminating HAR include GGTA1 inactivation, regulation of the complement system and modification of the oligosaccharide structure of surface proteins in donor's cells. Transgenic animals designed for the purpose of xenotransplantation with single modification do not display full reduction of the α-Gal epitope level, which means that a accumulation of several modifications in one transgenic individual is needed. The aim of the study was to create a molecular and cytogenetic profile of a double transgenic animal with α1,2-fucosyltransferase and α-galactosidase expression. As a result of interbreeding of an individual with α1,2-fucosyltransferase expression with an individual with α-galactosidase expression 12 living piglets were obtained. PCR revealed the pCMVFUT gene construct was present in four individuals and pGAL-GFPBsd in three, including one with a confirmed integration of both the gene constructs. Fluorescence in situ hybridization confirmed the site of transgene integration, which corresponded to the mapping site of the transgenes which occurred in the parental generations. Karyotype analysis did not show any changes in the structure or the number of chromosomes (2n = 38, XX). As for the results pertaining to the single transgenic individuals, expression analysis demonstrated a high extent of α-Gal epitope level reduction on the surface of cells, whereas human serum cytotoxicity tests revealed the smallest decrease in longevity of cells in the obtained double transgenic individual (4.35 %). The tests suggest that the co-expression of both the transgenes leads to a considerable reduction of the α-Gal antigen level on the surface of cells and a decrease of xenotransplant immunogenicity.

Double Transgenic Pigs with Combined Expression of Human α1,2-Fucosyltransferase and α-Galactosidase Designed to Avoid Hyperacute Xenograft Rejection