Knutson J C, Yee D
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Anal Biochem. 1987 Jul;164(1):44-52. doi: 10.1016/0003-2697(87)90365-4.
Electroporation, the reversible breakdown of cell membranes caused by a high-voltage discharge, is a rapid, simple, and efficient method for introducing DNA into mammalian cells. An instrument for electroporation which permits the high-voltage discharge waveform to be varied with respect to rise time, peak voltage, and fall time is described. The uptake and expression of SV40 DNA following electroporation of two cell types, a human carcinoma-derived cell line, HEp-2, and a human lymphoblastoid cell line, 721, depended on the peak voltage and the fall time of the voltage discharge. The electronic parameters which produced optimum DNA transfer, however, differed for the two cell types. DNA as large as 150 kb was introduced into cells by electroporation. Cells can be electroporated in either phosphate-buffered saline or culture medium containing fetal bovine serum, and the efficiency of DNA transfer does not vary with cell densities from 10(6) to 2 X 10(7)/0.5 ml. Exposing the cells to multiple voltage discharges did not improve DNA transfer. DNA has been introduced by electroporation into all cell types tested, including human carcinoma-derived cell lines, human lymphoblastoid cell lines, human fibroblast strains, and primary human lymphocytes. To obtain maximal DNA transfer by this method, however, one must optimize the peak voltage and fall time of the discharge waveform for each cell type.
电穿孔是由高压放电引起的细胞膜可逆性击穿，是一种将DNA导入哺乳动物细胞的快速、简单且高效的方法。本文描述了一种电穿孔仪器，该仪器能够使高压放电波形在上升时间、峰值电压和下降时间方面有所变化。对两种细胞类型（一种源自人癌的细胞系HEp-2和一种人淋巴母细胞系721）进行电穿孔后，SV40 DNA的摄取和表达取决于电压放电的峰值电压和下降时间。然而，产生最佳DNA转移效果的电子参数在这两种细胞类型中有所不同。通过电穿孔可将长达150 kb的DNA导入细胞。细胞可在磷酸盐缓冲盐溶液或含有胎牛血清的培养基中进行电穿孔，并且DNA转移效率在细胞密度从10(6)至2×10(7)/0.5 ml范围内不会发生变化。使细胞暴露于多次电压放电并不能提高DNA转移效果。通过电穿孔已将DNA导入所有测试的细胞类型，包括源自人癌的细胞系、人淋巴母细胞系、人成纤维细胞株以及原代人淋巴细胞。然而，要通过这种方法获得最大的DNA转移效果，必须针对每种细胞类型优化放电波形的峰值电压和下降时间。
