Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli.

BACKGROUND: Porcine organs with transgenic expression of anti-apoptotic and anti-inflammatory genes like the human A20 gene (hA20), a tumor necrosis factor-alpha (TNF-alpha)-inducible gene, may control the acute vascular rejection (AVR) of porcine xenografts. The human A20 molecule possesses protective features against inflammatory and apoptotic stimuli in various cell types including endothelial cells, rendering it a promising candidate for transgenic pig production in the context of xenotransplantation. Here, we produced pigs transgenic for hA20 and investigated whether hA20-transgenic porcine aortic endothelial cells (PAECs) were resistant against the induction of apoptosis in vitro and to what extent hA20-transgenic porcine hearts were protected against ischemia/reperfusion (I/R) injury. METHODS: Porcine fetal fibroblasts (PFFs) were transfected with the vector pCAGGSEhA20-IRESNEO containing a chicken beta-actin/rabbit beta-globin (CAGGS)-promoter element, known to provide ubiquitous gene expression in both mice and pigs. Transfected PFFs were then used in somatic cell nuclear transfer (SCNT). Three hA20-transgenic pigs were killed for PAEC isolation and organ mRNA and protein expression analysis by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern and Western Blotting. PAECs were tested for susceptibility to apoptosis after TNF-alpha challenging and triggering of the CD95(Fas)/CD95Ligand pathway. Five transgenic and three wild type animals were subjected to an I/R experiment followed by measurement of infarct size, myeloperoxidase (MPO) activity and subendocardial segmental shortening (SES) to assess protective effects of hA20 in the porcine myocardium. RESULTS: The hA20-transgenic pigs developed normally and expression of hA20 was found in skeletal muscle, heart and PAECs. Cultured human A20-transgenic PAECs showed significantly reduced apoptosis when compared to their wild type counterparts and were less susceptible to the induction of cell death by CD95(Fas)L. Only partial protection of hA20-transgenic pig hearts was observed after I/R. While infarct size did not differ between the two groups after ischemic assault, hA20-transgenic porcine hearts showed significantly lower MPO activity and better hemodynamic performance (determined as SES) than their wild type counterparts. CONCLUSIONS: The hA20 gene was for the first time functionally expressed in transgenic pigs. Although the CAGGS is a ubiquitous promoter element, expression was restricted to heart, skeletal muscle and PAECs of transgenic animals. Cultivated hA20-transgenic PAECs were protected against TNF-alpha-mediated apoptosis, and partially protected against CD95(Fas)L-mediated cell death; cardiomyocytes were partially protected in I/R. These findings reveal hA20 as a promising molecule for controlling AVR in multi-transgenic pigs for xenotransplantation studies.

Development and validation of a highly efficient protocol of porcine somatic cloning using preovulatory embryo transfer in peripubertal gilts.

The efficiency of porcine somatic nuclear transfer (born piglets/transferred embryos) is low. Here, we report a highly efficient protocol using peripubertal gilts as recipients synchronized to ovulate approximately 24 h after transfer of cloned embryos. Retrospectively, we compared the efficiency of two different synchronization protocols: In group 1, recipient animals were synchronized to ovulate approximately 6 h prior to surgical embryo transfer while in group 2 the animals were treated to ovulate 24 h after embryo transfer. In total, 1562 cloned embryos were transferred to 12 recipients in group 1; two of them became pregnant (16.7%). One pregnancy was lost on day 32, the second pregnancy went to term, and led to the birth of one healthy piglet after Cesarean section. In group 2, 1531 cloned embryos were transferred to 12 recipients. Nine recipients (75.0%) became pregnant as determined by ultrasound scanning on day 25. All pregnancies went to term and delivered a total of 47 live-born piglets. The cloning efficiency of both groups differed significantly (group 1: 0.1%, group 2: 3.1%, p < 0.05). This modified protocol was then applied in subsequent experiments using different types of transgenic and nontransgenic donor cells with similar success rates. Results show that this protocol is robust and highly reproducible, and can thus be employed for routine production of cloned pigs.