Aubin R A, Weinfeld M, Mirzayans R, Paterson M C
Health Canada, Life Sciences Division, Biotechnology, Sir F. G. Banting Research Centre, Ottawa, Ontario.
Mol Biotechnol. 1994 Feb;1(1):29-48. doi: 10.1007/BF02821509.
Polybrene/DMSO-assisted gene transfer is a simple and versatile transfection strategy capable of producing high numbers of stable transfectants from adherent monolayer cultures with low (nanogram) quantities of exogenous DNA. The procedure involves two stages: adsorption and internalization. The former is mediated by polybrene (a polycation polymer) and favors the uniform coating of target cells with polybrene-DNA complexes. Following adsorption, the cells are permeabilized by a brief exposure to dimethyl sulfoxide (DMSO) to facilitate the uptake of DNA complexes. Diverse cell types can be exposed to a wide range of polybrene concentrations without adverse effects. By contrast, the key determinant of success is the DMSO permeabilization regime, which must be configured independently for each cell line. Protocols optimized for gene transfer in murine and human fibroblasts are presented along with a guide for the rapid optimization of the method. The advantages and limitations of the method are also discussed.
聚凝胺/二甲基亚砜辅助基因转移是一种简单且通用的转染策略，能够以低（纳克）量的外源DNA从贴壁单层培养物中产生大量稳定的转染子。该过程包括两个阶段：吸附和内化。前者由聚凝胺（一种聚阳离子聚合物）介导，有利于用聚凝胺-DNA复合物均匀包被靶细胞。吸附后，通过短暂暴露于二甲基亚砜（DMSO）使细胞通透，以促进DNA复合物的摄取。多种细胞类型可以暴露于广泛的聚凝胺浓度而无不良影响。相比之下，成功的关键决定因素是DMSO通透方案，必须针对每个细胞系独立配置。文中介绍了针对小鼠和人成纤维细胞基因转移优化的方案以及该方法快速优化指南。还讨论了该方法的优缺点。
