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 vec tor. The altered plasmid contained 2 sequential estrogen response elements (ERE) coupled to a human phosphoglycerate kinase (PGK) promoter inserted up stream from a cDNA sequence encoding enhanced green fluorescent protein (GF P) with a 3'-polyadenylation signal. The plasmid was linearized and transfe cted into MCF-7 cells, a human breast cancer-derived line that expresses th e 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 17 beta-estradiol (E2). Stably transfected MCF-7 cells (MCF7-ERE) exh ibited 2000-3000 times more fluorescence at 488 nm excitation and 512 mm em ission than non-transfected cells. MCF7-ERE cells exhibited a linear increa se 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(-1 0) M E2. From the maximal level, GFP expression plateaued, and then decline d when E2 was increased to the highest concentration tested, 10(-7) M. 4-Hy droxytamoxifen (TFN-OH) treatment of cells produced a dose-dependent inhibi tion of E2-induced GFP expression, indicating the interaction of ER in the regulation of GFP gene expression. A series of estrogenic chemicals were ev aluated for their capacity to induce GFP expression in MCF7-ERE cells, show ing induced expression of GFP at concentrations 2-4 log units higher than t he E2 concentration giving maximal GFP expression. The ERE-PGK-GFP reporter gene system is capable of rapid GFP expression in the presence of low conc entrations of E2, and of quantifying estrogenicity of chemicals compared wi th a standard curve of the natural ligand, 17 beta-estradiol.