Long duration electroporation for achieving high level expression of glucocorticoid receptors in mammalian cell lines.
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-1 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.
Bodwell
J
J
Department of Physiology, Dartmouth Medical School, Lebanon, NH 03756, USA. jack.e.bodwell@dartmouth.edu
Swiff
F
F
Richardson
J
J
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004274:DNA, Recombinant / Q000008:administration & dosage* / Q000235:genetics*; D018274:Electroporation / Q000379:methods*; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006854:Hydrocortisone / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D004274:DNA, Recombinant; D008164:Luminescent Proteins; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases; D006854:Hydrocortisone