Development of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein
Bovee, T.F.H. ; Helsdingen, R.J.R. ; Koks, P.D. ; Kuiper, H.A. ; Hoogenboom, L.A.P. ; Keijer, J. - \ 2004
Gene 325 (2004). - ISSN 0378-1119 - p. 187 - 200.
cyc1 gene - receptor-alpha - s-cerevisiae - assay - transcription - disruption - activation - screen - mice - beta
The aim of this study was to develop an estrogen transcription activation assay that is sensitive, fast and easy to use in the routine screening of estrogen activity in complex matrices such as agricultural products. Recombinant yeast cells were constructed that express the human estrogen receptor ¿ (ER¿) and ß-Galactosidase (ßGal), Luciferase (Luc) or yeast Enhanced Green Fluorescence Protein (yEGFP) as a reporter protein. Compared to other yeast assays, these new cells contain both the receptor construct as well as the reporter construct stably integrated in the genome with only one copy of the reporter construct. Dose-response curves for 17ß-estradiol (E2) obtained with the ßGal assay were similar to those reported and the calculated EC50 of 0.2 nM was even slightly better. However, 5 days of incubation were required before the chlorophenol red product could be measured. The Luc assay was as sensitive as the ßGal assay and gave an EC50 of 0.2 nM, but the signals were rather low and, although the assay can be performed within 1 day, the procedure is laborious and caused variability. The yEGFP revealed an EC50 of 0.4 nM, but compared to the ßGal and the Luc assay, the response was much better. This yEGFP assay can be performed completely in 96 well plates within 4 h and does not need cell wall disruption nor does it need the addition of a substrate. This makes the test sensitive, rapid and convenient with high reproducibility and small variation. These qualities make that this yEGFP assay is suited to be used as a high throughput system