A Rapid and Sensitive Reporter Gene that Uses Green Fluorescent Protein Expression to Detect Chemicals with Estrogenic Activity

Abstract

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.

Online monitoring of stimulus-induced gene expression in pancreatic beta-cells.

“…Their use as dynamic and semiquantitative reporters to monitor stimulus-induced promoter activity in mammalian cells has been exploited sparsely (4,(13)(14)(15). The application of fluorescent proteins to monitor gene expression online has recently been discussed for micro-organisms (11).…”

Development of an in vitro model of excess intracellular reactive oxygen species

“…The expression vector, pPGKGFP, previously described by Miller et al (2000), with slight modification, was utilized for cellular transfection. Specifically, the original expression vector was modified by excision of the two-estradiol response elements.…”

“…Transfected cells were maintained at 37 -C under 5% CO 2 in standard minimal essential medium (MEM; Gibco BRL, Grand Island, NY) supplemented with 1 mM sodium pyruvate (Sigma, St. Louis, MO), 1 mg/ml insulin (Sigma), 1.25 mg/ml Fungizone \ (Gibco BRL), and 0.1 mg/ml gentamycin sulfate (Sigma). Electroporation of linearized pPGKGFP into MCF-7 cells was carried out as previously described (Miller et al 2000) utilizing a BTX Transfector 300 (Biotechnologies and Experimental Research, Inc., San Diego, CA), according to methods established by Ausubel et al (1994). Transfected cells were allowed to recover for a total of 40 min before being added to MEM (Gibco BRL, Grand Island, NY).…”

Development of a high-performance reporter plasmid for detection of chemicals with androgenic activity

“…All chemicals were dissolved in dimethylsulfoxide (DMSO; Nacalai Tesque, Kyoto, Japan) at concentrations of 10 mM, and the solutions were serially diluted in the same solvent at a common ratio of 1:10 with an automated pipetting device (Biomek 2000; Beckman Coulter (Miller et al 2000). The cells were passaged every 3-5 days at 60-80% confluence.…”