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.

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.

The domesticated pig has emerged as an important tool for development of surgical techniques, advancement of xenotransplantation, creation of important disease models, and preclinical testing of novel cell therapies. However, germ line-competent pluripotent porcine stem cells have not yet been derived. This has been a major obstacle to genetic modification of pigs. The transcription factor Oct4 is essential for the maintenance of pluripotency and for reprogramming somatic cells to a pluripotent state. Here, we report the production of transgenic pigs carrying an 18 kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct) to allow identification of pluripotent cells by monitoring Oct4 expression by EGFP fluorescence. Eleven viable transgenic piglets were produced by somatic cell nuclear transfer. Expression of the EGFP reporter construct was confined to germ line cells, the inner cell mass and trophectoderm of blastocysts, and testicular germ cells. Reprogramming of fibroblasts from these animals by fusion with pluripotent murine embryonic stem cells or viral transduction with human OCT4, SOX2, KLF4, and c-MYC cDNAs resulted in Oct4-EGFP reactivation. The OG2 pigs have thus proved useful for monitoring reprogramming and the induction and maintenance of pluripotency in porcine cells. In conclusion, the OG2 transgenic pigs are a new large animal model for studying the derivation and maintenance of pluripotent cells, and will be valuable for the development of cell therapy.

Introduction:  The hyperacute rejection after porcine‐to‐human xenotransplantation can now be reliably overcome either by transgenic expression of human complement regulating factors or by knocking out the gene for α(1,3)‐galactosyltransferase in pigs. The next immunological hurdle is the acute vascular rejection (AVR) primarily caused by endothelial cell activation. Human hemeoxygenase‐I (hHO‐1) has anti‐apoptotic and cell protective properties. Thus, the expression of hHO‐1 on porcine endothelial cells could have beneficial effects in a xenotransplantation setting. Here, we report the generation and characterization of pigs transgenic for hHO‐1. Methods:  Fibroblasts were obtained by an ear punch from a female pig that showed a mosaic expression of hDAF and were cultured in vitro as described previously indoleamine 2,3‐dioxygenase [1]. Cells reaching confluency of 70–80%, were detached with EDTA/trypsin and subsequently transfected by electroporation at 450 V/350 μF with a vector coding for hHO‐I driven by the SV40 promoter. Transfected cells were selected for resistance against G418 (800 μg/ml) for 14 days. Resistant cell clones were screened for integration of the vector by PCR. One positive cell clone was used in somatic nuclear transfer. In total, 205 reconstructed embryos were transferred to two synchronized peripubertal German Landrace gilts which gave birth to nine live piglets with normal birth weights. The integration and expression of hHO‐1 and hDAF were determined by PCR, Southern blot and RT‐PCR. Cell cultures were established from a hHO‐1 transgenic piglet with no DAF‐integrant. Cells were used as donor cells in a recloning approach to produce a homogenous group of hHO‐1 transgenic animals and eleven genetically hHO‐1 transgenic offspring were obtained. Expression of hHO‐1 was determined in different organs by RT‐PCR, Northern and Western blot; and in endothelial cells and peripheral blood lymphocytes by flow cytometry and Western blot. Resistance of hHO‐1 transgenic PAECs against TNF‐α mediated (1 ng, 10 ng, 30 ng, 50 ng TNF‐α) apoptosis was detected with a caspase GLO assay (Promega, Germany) in a luminometer. Endothelial cell activation was measured by realtime PCR using primers for the adhesion molecules ICAM‐1, VCAM‐1 and E‐selectin. Results:  PCR and Southern blot analyses revealed that all of offspring had integrated the vector in their genome. Six transgenic animals were sacrificed for in depth characterization. However, albeit all animals were cloned from the same cell clone, variation in the expression pattern and levels was observed in RT‐PCR and Northern Blot. The recloned animals showed identical expression levels and patterns of hHO‐1. All animals showed weak expression of hHO‐1 in most of the xenorelevant organs like heart, kidney and liver. HO‐1 transgenic PAECs were significantly resistant to TNF‐α mediated apoptosis compared to wild type PAECs. To show that this protection was due to hHO‐1 over‐expression, transgenic cells were incubated with the specific hHO‐1 inhibitor ZnPP IX (...

The hyperacute rejection response (HAR) after porcine-to-human xenotransplantation can now be reliably overcome. The next immunological hurdle is the acute vascular rejection (AVR) primarily caused by endothelial cell activation followed by disseminated intravascular coagulopathy, increased apoptosis and inflammatory symptoms. Several genes have been proposed to show protective effects against AVR, including human heme oxygenase-I (hHO-1) and human A20 (hA20) gene. HHO-1 has primarily anti-apoptotic and cell protective properties. The hA20 molecule possesses protective features against inflammatory and apoptotic stimuli in endothelial cells. Thus transgenic expression of these genes in pigs may be promising to prolong survival of porcine xenografts. We used somatic cell nuclear transfer (SCNT) for production of transgenic pigs.We produced pigs transgenic for human heme oxygenase 1 (hHO-1) to evaluate the protective effects of that molecule and to compare it with other transgenes used to control of the hyperacute rejection response (HAR), e.g. the DAF transgenes which gave HAR protection in in vitro cell death assays. Importantly, hHO-1 transgenic porcine aortic endothelial cells were significantly better protected against TNF-a mediated apoptosis. In close collaboration with partners at the LMU Munich (Prof. Kupatt et al.) the transgenic pig lines were tested in an ischemia/reperfusion (I/R) circuit. After occlusion of the left anterior descending artery (LAD), hHO-1 transgenic hearts had significantly smaller infarct lesions and concomitantly significantly better global myocardial function than size-matched wild-type controls. In close collaboration with partners at Hannover Medical University (Prof. Winkler et al.), hHO-1 transgenic porcine kidneys were perfused with pooled human blood for the maximum period of 240 min without addition of C1-Inhibitor in an ex vivo perfusion circuit. In parallel, we produced and characterized pigs that express hHO-1 on a Gal -/background. Gal -/-/hHO-1 pig hearts were tested in the I/R circuit and preliminary results indicate a protective effect shown by decreased infarct size, less inflammation and improved global and regional myocardial function after LAD occlusion.Expression of hA20 from the CAGGS promoter was found in skeletal muscle, heart and PAECs. Cultured human A20-transgenic PAECs showed significantly reduced apoptosis when compared to their wild type counterparts. Only partial protection of hA20-transgenic pig hearts was observed after I/R. While infarct size was not different between the two groups after ischemic assault, hA20-transgenic pig hearts showed a significantly better hemodynamic performance (determined as SES) than the wild type porcine hearts. MPO activity was reduced in transgenic vs. wild type hearts. We also produced pigs carrying shRNA constructs directed against PERV expression. These animals showed significantly reduced PERV-expression for over 6 months compared to wild-type and sham controls. This approach could improve the safety of porcine xenog...