A sensitive recombinant cell-based bioluminescent assay for detection of androgen-like compounds

被引：24

作者：

Michelini, Elisa ^{[1
]}

Cevenini, Luca ^{[1
]}

Mezzanotte, Laura ^{[1
]}

Leskinen, Piia ^{[2
]}

Virta, Marko ^{[3
]}

Karp, Matti ^{[4
]}

Roda, Aldo ^{[1
,5
]}

机构：

[1] Univ Bologna, Dept Pharmaceut Sci, I-40126 Bologna, Italy

[2] Univ Turku, Dept Biol, FI-20014 Turku, Finland

[3] Univ Helsinki, Dept Appl Chem & Microbiol, FI-00014 Helsinki, Finland

[4] Tampere Univ Technol, Dept Chem & Bioengn, FIN-33101 Tampere, Finland

[5] Tampere Univ Technol, BCC, FIN-33101 Tampere, Finland

来源：

NATURE PROTOCOLS | 2008年 / 3卷 / 12期

基金：

芬兰科学院;

关键词：

D O I：

10.1038/nprot.2008.189

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：1895 / 1902

页数：8

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