67 GENERATION OF PIGS TRANSGENIC FOR hCD59/DAF AND HUMAN THROMBOMODULIN BY SOMATIC NUCLEAR TRANSFER

Abstract

After a porcine–to–primate xenotransplantation, hyperacute rejection (HAR) destroys the transplanted organ within minutes. The HAR can be overcome either by a knockout of the gene for α–1,3–galactosyltransferase or by overexpression of human complement regulatory proteins such as hCD59 and DAF. When HAR can be controlled, the next hurdle is acute vascular rejection (AVR) which is primarily due to an incompatibility of human protein C and porcine thrombomodulin, both of which are important factors in the coagulation cascade. This incompatibility leads to thrombosis and finally to a disseminated intravascular coagulation (DIC), causing rejection of the xenotransplant. Human thrombomodulin is a good candidate gene for improving survival time of porcine organs after xenotransplantation and for overcoming AVR. Here, we transfected adult fibroblasts obtained from a double transgenic boar (hCD59/DAF) with a construct for human thrombomodulin (hTM). After selection with 800 μg/mL G418 for 14 days, cells were analyzed for integration of the construct by PCR and were visually selected for expression of the hTM–GFP fusion protein under ultraviolet light with a fluorescein isothiocyanate (FITC) filterset. A total of 39 positve clones were obtained, of which two were used in somatic nuclear transfer. Ovaries were collected from a local slaughterhouse, and follicles of 2–5 mm in diameter were aspirated. After 38–42 h of in vitro maturation oocytes were denuded and enucleated. For cell cycle synchronization, the donor cells were serum–starved for 48 h, subsequently trypsinized and placed into the perivitelline space of the enucleated oocytes. The complexes were fused and activated electrically followed by an incubation in DMAP for 3 h. Puberal gilts were synchronized by treatment with 5 mL Regumate® (Intervet UK, Ltd., Milton Keynes, Buckinghamshire, UK) for 13 days. At the end of treatment, the animals received 1000 IU pregnant mare serum gonadotropin (PMSG) intramuscularly followed by an injection of 500 IU hCG 72 h later. Cloned embryos were transferred surgically 20 h after hCG injection. Maintenance of pregnancy was supported by injections of 1000 IU PMSG on Day 11 and 500 IU hCG on Day 14 of the pregnancy. Out of 1409 reconstructed complexes, 1161 were fused (82.4%) successfully. In total, 1040 embryos were transferred to 8 recipients (∼130 embryos/gilt, range 70–162). Five of the eight recipients (62.5%) became pregnant as determined by ultrasound on Days 25, 36, and 51 and will farrow within the next weeks. These results show that cloning with triple transgenic adult donor cells is compatible with high pregnancy rates. Porcine multitransgenic organs will be used in perfusion experiments to test the effectiveness of this novel approach to overcoming the incompatibilities between the porcine and the human coagulation systems. This project is funded by the Deutsche Forschungsgemeinschaft (FOR 535). The hTM–construct was a gift of Dr. Wu which is gratefully acknowledged.