Electroporation: parameters affecting transfer of DNA into mammalian cells.
Electroporation: parameters affecting transfer of DNA into mammalian cells.
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DOI：
10.1016/0003-2697(87)90365-4
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发表时间：
1987-07
影响因子：
2.9
通讯作者：
Joyce C. Knutson;Daniel Yee
中科院分区：
文献类型：
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作者：
Joyce C. Knutson;Daniel Yee
电穿孔是由高压放电引起的细胞膜的可逆击穿，是一种将 DNA 引入哺乳动物细胞的快速、简单且有效的方法。描述了一种电穿孔仪器，其允许高压放电波形随上升时间、峰值电压和下降时间而变化。两种细胞类型（人癌细胞系 HEp-2 和人淋巴母细胞系 721）电穿孔后 SV40 DNA 的摄取和表达取决于峰值电压和电压放电的下降时间。然而，两种细胞类型产生最佳 DNA 转移的电子参数不同。通过电穿孔将大至 150 kb 的 DNA 引入细胞中。细胞可以在磷酸盐缓冲盐水或含有胎牛血清的培养基中进行电穿孔，并且DNA转移的效率不随细胞密度从106ml到2×1070.5ml而变化。将细胞暴露于多次电压放电并不能改善 DNA 转移。 DNA 已通过电穿孔导入所有测试的细胞类型中，包括人癌衍生细胞系、人淋巴母细胞系、人成纤维细胞株和原代人淋巴细胞。然而，为了通过这种方法获得最大的 DNA 转移，必须优化每种细胞类型的放电波形的峰值电压和下降时间。
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 106to 2 × 1070.5 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.