Long duration electroporation for achieving high level expression of glucocorticoid receptors in mammalian cell lines

被引:18
作者
Bodwell, J [1 ]
Swift, F [1 ]
Richardson, J [1 ]
机构
[1] Dartmouth Med Sch, Dept Physiol, Lebanon, NH 03756 USA
关键词
D O I
10.1016/S0960-0760(98)00162-9
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A method is presented that utilizes long duration electroporation (LDE) to more efficiently introduce DNA into mammalian cell lines than standard electroporation techniques. With SV40-based vectors, more than 550,000 glucocorticoid receptors (GRs) per cell could be obtained in COS-7 cells with good cell survival. In experiments with a CMV-driven vector expressing an enhanced Green Fluorescent Protein (EGFP), 54% of the cells were transfected, and 77% of EGFP positive cells expressed EGFP at moderate to high levels. In cell lines not containing the large T antigen, a CMV-driven vector for the GR was superior to the SV40-based vector. In EDR3, DG44, and CV-I cell lines approximately 220,000, 190.000 and 150,000 GRs/cell were obtained, respectively. Transfection efficiency of the EGFP vector ranged from 44 to 55% for the three cell lines. Cortisol treatment of COS-7 and DG44 cultures cotransfected with Vectors expressing the GR and a GRE driven luciferase gene produced 4 to 12 times more enzyme activity per plate with LDE than conventional electroporation protocols. LDE allows transient overexpression of proteins in COS-7 cells at the high levels generally achieved by mammalian overexpression systems only in stable cell lines. (C) 1999 Elsevier Science Ltd. All rights reserved.
引用
收藏
页码:77 / 82
页数:6
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