306 GENERATION AND CHARACTERIZATION OF HUMAN A20 TRANSGENIC PIGS BY SOMATIC CELL NUCLEAR TRANSFER

Abstract

Xenotransplantation is considered a solution to diminish the acute shortage of human organs. Although the hyperacute rejection occurring instantly after xenotransplantation can already be reliably controlled, the following immunological defense like the acute vascular rejection (AVR) remains a major hurdle for long-term survival of xenografts in porcine-to-primate organ transplantation. AVR is primarily characterized by endothelial cell activation with severe consequences on coagulation. The human A20 (hA20) gene exhibits antiapoptotic and anti-inflammatory properties in endothelial cells (Ferran C 2006 Transplantation 82, 36–40) and could thus prevent endothelial cell activation leading to AVR and xenograft destruction. Here, hA20-transgenic pigs were produced by somatic cell nuclear transfer (SCNT) in order to examine the ability of hA20-expressing tissues and organs to modulate the AVR. Two hA20-expression vectors, containing the promoters CAGGS or EF1-α in addition to a neomycin resistance cassette, were transfected into porcine fetal fibroblasts. Transfection was accomplished by electroporation, and the cell clones were selected with G418 (800 μg mL^–1) for 14 days. Resistant cell clones were screened in PCR with hA20-specific primers. SCNT was performed as previously described (Hölker M et al. 2005 Cloning Stem Cells 7, 35–44). After 8 SCNT sessions with pCAGGSEhA20-transgenic cell clones, embryo transfer was carried out to 16 peripuberal recipients resulting in 12 pregnancies (75%). Sixteen fetuses were isolated after sacrificing the recipient sows, and 45 piglets were born. Six of 16 fetuses (37.5%) and 15 of 45 (33.3%) piglets were transgenic. Four SCNT sessions were completed with pEF1hA20-transgenic cell clones following embryo transfer to 6 sows and 5 pregnancies were established (83.3%). Five fetuses were isolated and 14 piglets born. Five of 5 fetuses (100%) and 9 of 14 (64.3%) piglets were transgenic. Expression analysis (RT-PCR and Northern blots) showed transcription of the hA20 gene in heart, muscle, and cultivated porcine aortic endothelial cells of the pCAGGSEhA20-transgenic animals. No transcription was detected in pEF1hA20-transgenic animals. Current results show that hA20-transgenic pigs develop physiologically compared to wildtype counterparts. Expressing tissues and organs were only found in pCAGGSEhA20-transgenic animals, which are now being tested regarding functionality of the hA20 transgene.

We would like to thank Prof. Beyaert from the University of Ghent, Belgium, for providing us with the A20-expression vector pCAGGSEhA20.