429 Generation of Oct4-EGFP Transgenic Pigs for Monitoring Reprogramming and Pluripotency

The Oct4 gene is an essential transcription factor for maintenance of pluripotency in mammals. Here, we report the production of cloned transgenic pigs carrying a genomic construct encompassing murine Oct4 regulatory regions and driving an enhanced green fluorescent protein (Oct4-EGFP) construct. We employed fetal porcine fibroblasts, stably co-transfected with neomycin and the mouse Oct4-EGFP construct, for somatic cell nuclear transfer to reconstruct transgenic embryos. The cloned embryos (811 embryos) were surgically transferred into the oviducts of 8 recipient animals. Two pregnancies were terminated at Day 25 for recovery of fetuses and the others delivered a total of 23 piglets, of which 11 survived the postpartum period. A detailed analysis showed that the Oct4-EGFP construct was active in cloned pig blastocysts from Days 5 to 6. EGFP fluorescence was found exclusively in the primordial germ cells of Day 25 fetuses, whereas somatic tissues did not express the transgene. We could also detect expression of Oct4-EGFP in individual cells of the postnatal testis. Testis-specific expression was confirmed by Northern blotting. We fused transgenic porcine fibroblasts with murine embryonic stem cells to analyze reactivation of the Oct4-EGFP transgene under experimental reprogramming conditions. The fused hybrids displayed stem cell morphology and a high proliferation rate and started to express EGFP fluorescence 72 h after fusion. In conclusion, we report the production of viable Oct4-EGFP transgenic piglets that express EGFP exclusively in germ line and pluripotent cells. This transgenic pig line is a valuable tool for derivation and maintenance of porcine embryonic stem cells and will be of utmost interest for reprogramming studies and for preclinical testing of stem cell therapies in a large animal model.