GFP-reporter for a high throughput assay to monitor estrogenic compounds.
In vitro test systems using yeast cells are a useful tool for the determination of the estrogenic activity of estrogens, phyto- and xeno-estrogens and can be used for monitoring large sample numbers in a routine analysis procedure. Our conventional transactivation assay functions with an expression plasmid expressing estrogen receptor alpha (ERalpha) under the control of a copper-inducible CUP1 promoter and a reporter plasmid expressing beta-galactosidase under the control of the vitellogenin estrogen response element (ERE). In the novel yeast screen system the lacZ gene in the reporter plasmid was substituted by a gene for green fluorescent protein (GFP). Incubation of yeast with various concentrations of estrogenically active substances led to expression of the reporter gene product GFP in a dose dependent manner. The yeast transactivation assay was further down-scaled to be performed in a microplate scale, which is an important step to facilitate handling of large sample numbers. The sensitivity and reproducibility of the novel test system could be confirmed by analysis of the potencies of various estrogenically active substances. Thus, the newly developed yeast estrogen screen using GFP as a reporter can substitute the assay that has been used for a period of several years.
Beck
Verena
V
Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
Pfitscher
Angelika
A
Jungbauer
Alois
A
D001681:Biological Assay / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism; D004967:Estrogens / Q000032:analysis* / Q000494:pharmacology; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae; D012680:Sensitivity and Specificity
D047628:Estrogen Receptor alpha; D004967:Estrogens; D049452:Green Fluorescent Proteins