基于爱泼斯坦-巴尔病毒的质粒在猪细胞中延长基因表达的机制。

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

作者信息

Son Jung Kyu, Oh Sang Taek, Cho Sung Kyu, Yoon Kun Ho, Lee Suk Kyeong

机构信息

Research Institute of Immunobiology, Department of Biomedical Sciences, The Catholic University of Korea, Seoul, Korea.

出版信息

Xenotransplantation. 2006 Nov;13(6):560-5. doi: 10.1111/j.1399-3089.2006.00350.x.

DOI:10.1111/j.1399-3089.2006.00350.x

PMID:17059583

Abstract

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.

摘要

背景

我们之前表明，一种基于爱泼斯坦-巴尔病毒（EBV）的质粒pEBVGFP在猪新生胰腺细胞簇（NPCCs）中能实现长时间的基因表达。在本研究中，对其潜在机制进行了探究。

方法

通过荧光激活细胞分选（FACS）分析和共聚焦显微镜观察，分析用pEBVGFP转染的猪细胞中的绿色荧光蛋白（GFP）表达情况。通过Southern印迹分析pEBVGFP整合到染色体DNA中的可能性。通过聚合酶链反应（PCR）研究基于EBV的质粒在猪细胞中的自我复制情况。对NPCCs进行免疫染色以鉴定用pEBVGFP转染的细胞。