Abstract
We report a step-by-step protocol describing how to develop and use a yeast-based bioassay for androgen-like compounds. Saccharomyces cerevisiae cells are genetically engineered to express the human androgen receptor (hAR) and the bioluminescent (BL) reporter gene luciferase (from Photinus pyralis) under the control of the androgen response element (ARE). In the presence of androgens, activated hAR binds to the ARE sequences and activates luciferase expression. After addition of D-luciferin, luciferase activity measurements can be performed, and the BL signal is proportional to the androgenic activity of the sample. Cytotoxic effects of the sample are monitored by the use of a control yeast strain that allows BL signal correction according to cell viability. After overnight culture of the recombinant strain, the assay can be accomplished in a 96-well microplate format in 1 working day with a detection limit of 0.05 nM for testosterone and intra- and interassay variability of 14% and 23%, respectively.
Original language | English |
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Pages (from-to) | 1895-1902 |
Number of pages | 8 |
Journal | Nature Protocols |
Volume | 3 |
Issue number | 12 |
DOIs | |
Publication status | Published - Nov 2008 |
Externally published | Yes |
Bibliographical note
Funding Information:ACKNOWLEDGMENTS The work was partly supported by the Italian Ministry for University and Research (MIUR), by Fondazione Cassa di Risparmio di Bologna, by Maj and Tor Nessling Foundation and by the Academy of Finland.