Adenovirus enhancement of polyethylenimine-mediated transfer of regulated genes in differentiated cells
Adenovirus enhancement of polyethylenimine-mediated transfer of regulated genes in differentiated cells
复制标题
DOI：
10.1038/sj.gt.3300450
复制
发表时间：
1997-08-01
期刊：
影响因子：
5.1
通讯作者：
Forest, C
中科院分区：
文献类型：
--
作者：
MeunierDurmort, C;Grimal, H;Forest, C
有效的基因转移是分析转染启动子调控的先决条件。我们将阳离子聚合物聚乙烯亚胺（PEI）的DNA结合特性和PEI-DNA-腺病毒复合物中腺病毒的有效内吞活性相结合，从而在分化的培养细胞中提供有效的质粒递送。我们使用PEI和200 p.f.u.的组合，用含有与氯霉素乙酰转移酶（CAT）基因融合的猿猴病毒40启动子的构建体转染3 T3-F442 A脂肪细胞、C2.7肌细胞和FAO肝癌细胞。每个细胞的复制缺陷型5型腺病毒。所得CAT活性根据细胞类型而变化，对于3 T3-F442 A、FAO和C2.7细胞分别达到约0.6、8和38单位/mg蛋白。与单独使用PEI获得的转染效率相比，转染效率增加了140至300倍。然后，我们测试了生理调节的启动子：3 T3-F442 A或FAO细胞中的磷酸烯醇式丙酮酸羧激酶基因启动子和C2.7肌细胞中的己糖激酶II基因启动子。氯贝丁酯、地塞米松和胰岛素分别适当增加3 T3-F442 A、FAO和C2.7细胞的基因表达。因此，PEI和腺病毒的组合是一种简单、有效、廉价和通用的基因转移方法，其适用于几种分化的细胞并提供生理上一致的转基因调节。我们将此方法命名为PEI-腺转染。
Efficient gene transfer is a prerequisite for analysing regulation of transfected promoters. We combined the DNA binding property of the cationic polymer polyethylenimine (PEI) and the potent endocytic activity of advenovirus in a PEI-DNA-adenovirus complex which provided efficient plasmid delivery in differentiated cultured cells. We transfected 3T3-F442A adipocytes, C2.7 myocytes and FAO hepatoma cells with a construct containing the simian virus 40 promoter fused to the chloramphenicol acetyltransferase (CAT) gene, using a combination of PEI and 200 p.f.u. per cell of replication-deficient type 5 adenovirus. Resulting CAT activities varied according to the cell type reaching about 0.6, 8 and 38 units/mg protein for respectively 3T3-F442A, FAO and C2.7 cells. Increases in transfection efficiencies were 140- to 300-fold when compared with those obtained with PEI alone. Then we tested physiologically regulated promoters: the phosphoenolpyruvate carboxykinase gene promoter in 3T3-F442A or FAO cells and the hexokinase II gene promoter in C2.7 myocytes. Gene expression was appropriately increased by clofibrate, dexamethasone and insulin for 3T3-F442A, FAO and C2.7 cells, respectively. Thus, the combination of PEI and adenovirus is a simple, efficient, inexpensive and versatile method of gene transfer which is applicable to several differentiated cells and provides a physiologically coherent transgene regulation. We name this method PEI-adenofection.