Oct4-enhanced green fluorescent protein transgenic pigs: a new large animal model for reprogramming studies.

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.

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.