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可回复性永生化的基本原理是：先将永生化基因SV40T(猿猴病毒40大T抗原)导入原代细胞以获得永生化细胞株,使其获得体外增殖能力,然后再以特异性位点重组技术切除SV40T 基因,使细胞株回复到永生化前的状态,这一技术已被用于多种哺乳动物细胞的永生化。本研究在以往研究的基础上,构建了包括雌激素受体(ER)与重组酶Cre的融合基因Cre—ER、永生化基因SV40T、加强型绿色荧光蛋白(EGFP)与胸苷激酶 (TK)的融合基因EGFP-TK以及重组位点loxP序列的可回复性永生化逆转录病毒载体,旨在为肝细胞或其他细胞的可回复永生化奠定基础。一、材料与方法 1．主要材料：逆转录病毒载体pLLTSN为本研究小组前期研究成果,该质粒含有SV40T基因序列；质粒pIRES2- EGFP和pLNCX2购自美国Clontech公司；pHLOX由瑞士日内瓦大学医学部Didier Trono博士惠赠；pPDF15由英国圣安德鲁斯大学Pablo de Felipe博士惠赠；pCre-ER由法国路易斯巴斯德大学Pierre Chambon教授惠赠；pBABE-
The basic principle of recoverable immortalization is that the immortalized gene SV40T (simian virus 40 large T antigen) is first introduced into primary cells to obtain an immortalized cell line to obtain the proliferation ability in vitro, and then recombination with specific sites The technique of excising the SV40T gene and restoring the cell line to its pre-immortalized state has been used to immortalize a variety of mammalian cells. In this study, based on the previous studies, we constructed Cre-ER, SV40T, EGFP and TK, including ER and recombinase Cre, Of EGFP-TK fusion gene and recombination site loxP sequence of retroviral immortalized retroviral vector designed to lay the foundation for the recovery of immortalized hepatocytes or other cells. First, materials and methods 1. The main material: The retroviral vector pLLTSN is the early research group of the research team, which contains the SV40T gene sequence; Plasmids pIRES2-EGFP and pLNCX2 were purchased from Clontech, USA; pHLOX was donated by Dr. Didier Trono, University of Geneva, Switzerland; pPDF15 was donated by St. Benefit from Dr. Pablo de Felipe, Andrews University; pCre-ER, gift from Professor Pierre Chambon, University of Luis-Pasteur, France; pBABE-