Hama-Inaba H, Nishimoto T, Ohtsubo M, Sato K, Kasai M
Division of Genetics, National Institute of Radiological Sciences, Chiba, Japan.
Nucleic Acids Symp Ser. 1988(19):149-52.
We have established a simple and efficient method of electroporation applicable to gene transfer in mammalian cells. It uses a single decaying pulse of around 1 ms at room temperature in the medium such as Saline G appropriate for repair of pulse-induced pores in the plasma membrane. Many types of cells (both floating and adherent) could be transformed efficiently by the electric field strengths between 1-2 kV/cm. For instance P3U1, mouse myeloma cell, could be transformed by a pulse at 1.2 kV/cm with the frequency of 10(-2) per viable cells and with survivals of 90%. We have applied these conditions to transform tsBN2 cell line of BHK21/13 by a cosmid clone (approximately 45 kb) carrying the human gene complementing to tsBN2 mutation. Significant levels of transformation were observed for this gene. Since this gene can only work as a whole size (approximately 30 kb), the results show that electroporation is useful to introduce cosmid or possibly genomic DNA to mammalian cells.
我们已经建立了一种简单有效的电穿孔方法，适用于哺乳动物细胞中的基因转移。它在室温下于诸如生理盐水G等适合修复脉冲诱导的质膜孔的培养基中使用约1毫秒的单个衰减脉冲。在1-2 kV/cm的电场强度下，许多类型的细胞（包括悬浮细胞和贴壁细胞）都能被高效转化。例如，小鼠骨髓瘤细胞P3U1可以在1.2 kV/cm的脉冲下以每活细胞10(-2)的频率进行转化，存活率为90%。我们已应用这些条件，通过携带与tsBN2突变互补的人类基因的黏粒克隆（约45 kb）来转化BHK21/13的tsBN2细胞系。观察到该基因有显著水平的转化。由于该基因只能以完整大小（约30 kb）起作用，结果表明电穿孔对于将黏粒或可能的基因组DNA导入哺乳动物细胞是有用的。
