Tur-Kaspa R, Teicher L, Levine B J, Skoultchi A I, Shafritz D A
Mol Cell Biol. 1986 Feb;6(2):716-8. doi: 10.1128/mcb.6.2.716-718.1986.
A method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.
本文描述了一种在分离的肝细胞中导入并表达克隆基因的方法。通过胶原酶灌注分离得到的原代大鼠肝细胞，用电穿孔法在悬浮状态下用质粒pSV2CAT进行转染。48小时后，转染肝细胞的可溶性提取物显示出氯霉素乙酰转移酶活性，与通过磷酸钙或DEAE-葡聚糖转染在大鼠肝癌细胞系H4AzC2中获得的活性相当。由于这些试剂具有细胞毒性，后两种方法不能成功用于原代肝细胞。这表明电穿孔是在原代上皮细胞（如大鼠肝细胞，在细胞培养中难以维持）中获得外源基因瞬时表达的一种有用方法。
