Mechanism of prolonged gene expression by Epstein?Barr virus-based plasmid in porcine cells

Background: We previously showed that an Epstein-Barr virus (EBV)-based plasmid, pEBVGFP, exerts prolonged gene expression in porcine neonatal pancreatic cell clusters (NPCCs). In this study, the mechanism underlying this was investigated. Methods: GFP expression was analyzed in porcine cells transfected with pEBVGFP by FACS analysis and confocal microscopy. The possible integration of pEBVGFP into the chromosomal DNA was analyzed by Southern blot. Self-replication of the EBV-based plasmid in porcine cells was investigated by PCR. The NPCCs were immunostained to characterize cells transfected with pEBVGFP. Results: The EBV based plasmid provided prolonged GFP expression in porcine cells and duct cells were the main cells transfected among NPCCs. Southern blot showed that the transfected pEBVGFP stayed for a long time as an episome rather than integrating into the chromosomal DNA. pEBVGFP isolated from the transfected porcine cells had methylated CpG suggesting that they self-replicated in those cells. Conclusions: The EBV-based plasmid may be useful for genetically manipulating porcine cells to enhance their value as xenotransplantation sources.