Profiling Human Transgene Performance Assist in Selecting Best Suited Specimens and Tissues for Swine Organ Xenotransplantation.

Xenotransplantation of pig organs receives substantial attention for being comparable to human's. However, compatibility constraints involving hyper-acute rejection (HAR) still block clinical applications. Transgenesis of human complement regulatory proteins has been proposed to overcome xenorejection. Pigs expressing human- have been widely tested in experimental surgery. Still, no standardized method has been developed to determine tissue expression of human decay-accelerating factor (DAF), product, or to predict the ability to overpass HAR. Here we describe objective procedures addressing this need. Organs and tissues from five transgenic pigs were collected and classified according to their xenotransplantation value. The ability to overcome HAR was assessed by classical complement pathway hemolysis assays. Quantitative PCR mRNA expression and Western blot protein level studies were performed. Real-time cytotoxicity assays (RTCA) on fibroblast cultures exposed to baboon and human sera informed on longer-term rejection dynamics. While greater /DAF expression correlated with better performance, the results obtained varied among specimens. Interestingly, the individual with highest mRNA and protein levels showed positive feedback for transcript after challenge with human and baboon sera. Moreover, expression correlated to DAF levels in the liver, lung and intestine, but not in the heart. Moreover, we found significant correlations among valuable and non-valuable tissues. In sum, the methodology proposed allows us to characterize the transgene functioning and performance. Moreover, the correlations found could allow us to predict /DAF expression in surrogate tissues, thus eliminating the need for direct biopsies, resulting in preservation of organ integrity before xenotransplantation.