Andreason G L, Evans G A
Molecular Genetics Laboratory, Salk Institute, La Jolla, California 92138-9126.
Anal Biochem. 1989 Aug 1;180(2):269-75. doi: 10.1016/0003-2697(89)90429-6.
Electroporation can be a highly efficient method for introducing DNA molecules into cultured cells for transient expression of genes or for permanent genetic modification. However, effective transformation by electroporation requires careful optimization of electric field strength and pulse characteristics. We have used the transient expression of the firefly luciferase gene as a rapid and sensitive indicator of gene expression to describe the effects on transfection efficiency of altering electroporation field strength and shape. Using the luciferase assay, we investigated the correlation of cell viability with optimal transfection efficiency and determined the optimal parameters for a number of phenotypically distinct mammalian cell lines derived from the nervous and immune systems. The efficiency of electroporation under optimal conditions was compared with that obtained using DEAE-dextran or calcium phosphate-mediated transformation. Transfection by electroporation using square wave pulses, as opposed to exponentially decaying pulses, was found to be significantly increased by repetitive pulses. These methods improve the ability to obtain high efficiency gene transfer into many mammalian cell types.
电穿孔可以是一种将DNA分子导入培养细胞以实现基因瞬时表达或永久基因修饰的高效方法。然而，通过电穿孔进行有效的转化需要仔细优化电场强度和脉冲特性。我们使用萤火虫荧光素酶基因的瞬时表达作为基因表达的快速灵敏指标，来描述改变电穿孔场强和形状对转染效率的影响。通过荧光素酶测定，我们研究了细胞活力与最佳转染效率的相关性，并确定了一些源自神经和免疫系统的表型不同的哺乳动物细胞系的最佳参数。将最佳条件下的电穿孔效率与使用DEAE-葡聚糖或磷酸钙介导的转化所获得的效率进行了比较。发现与指数衰减脉冲相反，使用方波脉冲进行电穿孔时，重复脉冲可显著提高转染效率。这些方法提高了将高效基因转移到多种哺乳动物细胞类型中的能力。
