A rapid and sensitive reporter gene that uses green fluorescent protein expression to detect chemicals with estrogenic activity.
A reporter gene sequence was constructed within a eukaryotic expression vector. The altered plasmid contained 2 sequential estrogen response elements (ERE) coupled to a human phosphoglycerate kinase (PGK) promoter inserted upstream from a cDNA sequence encoding enhanced green fluorescent protein (GFP) with a 3'-polyadenylation signal. The plasmid was linearized and transfected into MCF-7 cells, a human breast cancer-derived line that expresses the estrogen receptor (ER). No selectable marker was present in the plasmid, requiring stably transfected cells to be selected by fluorescence-activated cell sorting based on GFP expression after the cells were treated with 10(-9) M 17beta-estradiol (E2). Stably transfected MCF-7 cells (MCF7-ERE) exhibited 2000-3000 times more fluorescence at 488 nm excitation and 512 nm emission than non-transfected cells. MCF7-ERE cells exhibited a linear increase in GFP expression induced over a range of 10(-12) M E2, a concentration giving 2 times the background expression, to maximal expression at 3 x 0(-10) M E2. From the maximal level, GFP expression plateaued, and then declined when E2 was increased to the highest concentration tested, 10(-7) M. 4-Hydroxytamoxifen (TFN-OH) treatment of cells produced a dose-dependent inhibition of E2-induced GFP expression, indicating the interaction of ER in the regulation of GFP gene expression. A series of estrogenic chemicals were evaluated for their capacity to induce GFP expression in MCF7-ERE cells, showing induced expression of GFP at concentrations 2-4 log units higher than the E2 concentration giving maximal GFP expression. The ERE-PGK-GFP reporter gene system is capable of rapid GFP expression in the presence of low concentrations of E2, and of quantifying estrogenicity of chemicals compared with a standard curve of the natural ligand, 17beta-estradiol.
Miller
S
S
Department of Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843, USA.
Kennedy
D
D
Thomson
J
J
Han
F
F
Smith
R
R
Ing
N
N
Piedrahita
J
J
Busbee
D
D
D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004353:Drug Evaluation, Preclinical; D004958:Estradiol / Q000494:pharmacology; D045166:Estradiol Congeners / Q000494:pharmacology*; D005057:Eukaryotic Cells / Q000187:drug effects; D017930:Genes, Reporter / Q000187:drug effects* / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D010735:Phosphoglycerate Kinase / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011960:Receptors, Estrogen / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D014162:Transfection
D045166:Estradiol Congeners; D008164:Luminescent Proteins; D011960:Receptors, Estrogen; D013629:Tamoxifen; D049452:Green Fluorescent Proteins; C016601:afimoxifene; D012710:Serum Albumin, Bovine; D004958:Estradiol; D010735:Phosphoglycerate Kinase