The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells
The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells
复制标题
DOI：
10.1038/sj.gt.3300745
复制
发表时间：
1998-10-01
期刊：
影响因子：
5.1
通讯作者：
Wagner, E
中科院分区：
文献类型：
--
作者：
Ogris, M;Steinlein, P;Wagner, E
在生理盐浓度下，与转铁蛋白缀合的或未修饰的聚乙烯亚胺（PEI，800 kDa）复合的质粒DNA形成巨大的（高达> 1000 nm）聚集体，除非各个组分以高度正的氮/磷酸盐（N/P）电荷比混合。然而，在低离子强度下，在宽的N/P比范围内形成平均尺寸为40 nm的小颗粒。有趣的是，在转染实验中，与在生理盐溶液中形成的大复合物相比，这些小颗粒导致荧光素酶基因表达效率降低10倍（B16 F10细胞）至超过100倍（Neuro 2A细胞，K562细胞）。小颗粒向细胞表面的有限运输是这种效应的一个可能原因。细胞内释放减少可能是转染效率降低的另一种解释;与氯喹孵育或将内体溶解肽INF 5掺入小复合物中可使基因表达增强约10倍。使用绿色荧光蛋白报告基因和流式细胞术在细胞水平上分析基因表达，揭示了总体基因表达的差异主要是由于每个表达细胞的不同强度，而表达细胞百分比的差异不太显著。
Under physiological salt concentration, plasmid DNA complexed with transferrin-conjugated or unmodified polyethylenimine (PEI, 800 kDa) forms huge (up to > 1000 nm) aggregates, unless the individual components are mixed at a highly positive nitrogen/phosphate (N/P) charge ratio. At low ionic strengths, however, small particles with an average size of 40 nm are formed over a broad range of N/P ratios. Interestingly, in transfection experiments these small particles result in a 10-fold (B16F10 cells) to more than 100-fold (Neuro2A cells, K562 cells) reduced luciferase gene expression efficiency in comparison to the large complexes formed in physiological salt solutions. Limited transport of the small particles to the cell surfaces is one possible reason for this effect. Application of the small particles in more concentrated form and over extended periods of time improves transfection activity Reduced intracellular release may be another explanation for the decreased transfection efficiency; incubation with chloroquine or incorporation of the endosomolytic peptide INF5 into the small complexes enhances gene expression approximately 10-fold. Analysis of gene expression at the cellular level using a green fluorescence protein reporter gene and flow cytometry revealed that the differences in overall gene expression largely result from different intensities per expressing cell while the difference in the percentage of expressing cells is less substantial.