聚乙烯亚胺介导NIH.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 优化聚乙烯亚胺（polyethylenimine，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（0:1、0.5:1、1:1、2:1、3:1、4:1、5:1、6:1、7:1、8:1、9:1、10:1）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；比较实验组1（PEI/DNA质量比2:1）、实验组2（重复PEI/DNA质量比2:1)及实验组3（PEI/DNA质量比4:2）3种转染方案，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实验组2细胞荧光表达强度明显高于实验组1和实验组3（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 oOptimizationng of the system ofin vitro transfecting NIH-3T3 cells with pEGFP-N1 by polyethylenimine Ding Aijun，, Ren Liwei，, Xu Feng，, Qin Yurong，, Fu Dezhi，, Li Bichun （Laboratory of Animal Genetics and Breeding Laboratory，, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu Province, 225009, China） [Abstract] Objective To optimize the system of transfecting cells with aim taeget genes by polyethylenimine (PEI) so as to increase cell expression percent of those. Methods On the basis of PEI/DNA at different mass ratios（ (0:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1 ,6:1, 7:1, 8:1, 9:1, and 10:1）), the ability of PEI to combine and protect DNA was investigated by agarose gel electrophoresis retardation assay. Analyzing cCell fluorescence expression was analyzed after percents of transfecting PEI/DNA complexes at different massratios into NIH-3T3 cells wasin order to obtained the optimal masss ratio of PEI/DNA complex. Comparison of repeat PEI:DNA ratio of 2:1 (w/w) , PEI:DNA ratios of 2:1 and 4:2 (w/w) of three3 testexperiment groups of transfection,. Repeating transfection was used ot Iincreaseing cell expression. percent was explored by the way of repeat transfection.Results Agarose gel electrophoresis retardation assay① indicated that Tt