Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine
Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine
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DOI：
10.1007/s10616-010-9259-z
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发表时间：
2010-01-01
期刊：
影响因子：
2.2
通讯作者：
Yamaguchi, Naoto
中科院分区：
文献类型：
--
作者：
Fukumoto, Yasunori;Obata, Yuuki;Yamaguchi, Naoto
将遗传物质导入细胞是当前分子细胞生物学研究的基本前提。虽然用市售试剂进行基因转染会产生良好的基因表达，但其高昂的成本阻碍了大量或大规模的转染剂的实验。阳离子聚合物线性聚乙烯亚胺(Mw 25000)(PEI)是最具成本效益的载体之一，它通过形成多链体促进DNA紧凑，从而通过内吞作用将DNA有效地输送到细胞内。然而，PEI的使用仍然受到限制，因为它具有很强的细胞毒性，而且其稳定性较低，导致无法容忍的转染率下降。在这里，我们表明，酸化的PEI是重要的，它的转染活性。在0.2N盐酸中溶解PEI粉末可使PEI在4℃和-80A的温度下保存有较长的货架期，并且在pH值为4.0的乳酸缓冲盐水中，PEI与质粒DNA的复合形成最佳。此外，在转染后8-12h更换培养基可使PEI的细胞毒性降至最低，而不牺牲与商品化试剂相当的高转染率。与商业试剂相比，使用酸化PEI的每次测试成本大幅降低到约1：10,000。因此，我们得出结论，酸化PEI可以很好地实现低成本、高效率的转染法。
Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000) (PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and -80 A degrees C, and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing the culture medium at 8-12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately 1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective, high-efficiency transfection.