人胰岛素原基因的包装及增殖

目的:用PA317包装细胞包装突变人胰岛素基因原质粒,并检测其增殖情况.方法:用脂质体转染法将PLXSN-MPINS质粒转染至PA317包装细胞,用G418筛选出阳性PA317包装细胞克隆并测定胰岛素原基因浓度,用TRIZOL法提取未转染和转染后PA317细胞的基因组DNA,PCR检测突变人胰岛素基因的整合情况.结果:(1)用脂质体转染法获得G418阳性PA317包装细胞克隆;(2)该细胞克隆获高滴度的胰岛素原基因浓度;(3)PCR方法在转染后PA317基因组中检测到突变人胰岛素原基因.结论:脂质体转染法是一种操作简便且行之有效的转染方法.PA317包装细胞系可成功包装突变人胰岛素原基因质粒并可获得高滴度表达,为1型糖尿病基因治疗的进一步实验研究奠定了基础.

Package of recombinant retroviral vector carrying modified human proinsulin gene and identification of virus titer

Objective: To pack the plasmid carring modified human proinsulin gene with PA317,and to examine regeneration conditions. Methods:The PLXSN-MpINS plasmid was transfected into PA317 cells by using lipofectamine. We determined the virus titers and selected the cell clones producing high-titer virus. Integration of goal gene with genome DNA from PA317 cells was assayed by using genomic DNA PCR. Results:Obtain the PA317 resistant clones and infect NIH3T3 cells. The high-titer virus was determined. The integration of goal gene was identified. Conclusion:Transfecting exogenous DNA into mammalian cells mediated by lipofectamine is a convenient and feasible transfection method. PA317 is a packing cell line that can pack the plasmid carrying modified human proinsulin gene successfully.It can also produce high-titer virus after the supernatant virus is used to infect NIH3T3 cells.The modified human proinsulin gene is steadily integrated with the genome DNA from PA317 cells. The results provide a basis for further studies on genetic therapy of diabetes.