聚乙烯亚胺介导NIH-3T3细胞体外转染pEGFP-N1系统优化丁爱军.DOCVIP

聚乙烯亚胺介导NIH-3T3细胞体外转染pEGFP-N1系统优化 丁爱军，任丽伟，许 峰，秦玉蓉，傅德智，李碧春 （225009江苏 扬州，扬州大学动物科学与技术学院动物遗传育种与繁殖实验室） [摘要]目的 优化PEI 介导的细胞转染以提高目的基因在细胞中的表达强度。方法 根据PEI/DNA不同的质量比利用凝胶阻滞分析的结合情况；不同质量比 PEI/DNA形成的复合物对NIH-3T3细胞的转染，通过细胞表达强度获得最佳PEI/DNA质量比；根据最佳PEI/DNA质量比，探讨重复转染的方法是否提高细胞表达强度。结果 ①通过凝胶阻滞分析 PEI/DNA能够稳定结合的质量比是1:1～10:1；②PEI/DNA复合物对NIH-3T3细胞进行转染其荧光表达强度当PEI/DNA(质量比)≤2时随着 PEI的增加而提高，当PEI/DNA(质量比)＞2时，荧光表达强度反而降低；③采用重复转染的方法试验组2细胞荧光表达强度明显高于试验组1和组3，差异显著（P＜0.05）。结论 在PEI/DNA(质量比)=2的条件下PEI/DNA 复合物转化NIH-3T3细胞的效果比较理想，重复转染可以提高细胞转染后荧光表达强度。 [关键词]聚乙烯亚胺;细胞转染;表达强度 [中图法分类号] [文献标志码]A Study of optimizng the system of transfecting NIH-3T3 cells with pEGFP-N1 by Polyethylenimine DING Ai-jun，Ren Li-wei，Xu Feng，Qin Yu-rong，Fu De-Zhi，LI Bi-chunAnimal Genetics and Breeding Laboratory ，College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China） [Abstract] Objective The objective of this study is to optimize the system of transfecting cells with aim genes by polyethylenimine (PEI) so as to increase cell expression percent of those. Methods On the basis of PEI/DNA at different quality ratios,the ability of PEI to combine and protect DNA was investigated by agarose gel electrophoresis retardation assay. Analyzing cell fluorescence expression percents of transfecting PEI/DNA complexes at different quality ratios into NIH-3T3 cells was to obtained the optimal quality ratio of PEI/DNA complex. Incresing cell expression percent was explored by the way of repeat transfection.Results 1.The quality ratios of forming stable PEI/DNA complexes by agarose gel electrophoresis retardation assay was in range of 1:1-10:1;2. Cell expression percent of PEI/DNA can be improved with the increase of PEI/DNA(W/W) ratios and reached highest at PEI/DNA=2,but began to decrease at PEI/DNA(W/W)＞2; 3.By the way of repeat transfection , cell expression percent in the test group 2 was significantly higher than that in the other test groups（P＜0.05）.Conclusion Cell expression percent can be