Androgen receptor transactivation assay using green fluorescent protein as a reporter.
For screening of a large number of samples for androgenic activity, a robust system with minimal handling is required. The coding sequence for human androgen receptor (AR) was inserted into expression plasmid YEpBUbi-FLAG1, resulting in the plasmid YEpBUbiFLAG-AR, and the estrogen response element (ERE) on the reporter vector YRpE2 was replaced by an androgen response element (ARE), resulting in the plasmid YRpE2-ARE. Thus, a fully functional transactivation assay system with beta-galactosidase as a reporter gene could be created. Furthermore, green fluorescent protein (GFP) was introduced as an alternative reporter gene that resulted in a simplification of the whole assay procedure. For evaluation of both reporter systems, seven steroidal compounds with known AR agonistic properties (5 alpha-dihydrotestosterone, testosterone, androstenedione, 17 alpha-methyltestosterone, progesterone, epitestosterone, and d-norgestrel) were tested, and their potencies obtained in the different assays were compared. Furthermore, potencies from the transactivation assays were compared with IC(50) values obtained in radioligand binding assays. The newly developed androgen receptor transactivation assay is a useful tool for characterizing compounds with androgenic activity.
Beck
Verena
V
Department of Biotechnology, University of Natural Resources and Applied Life Sciences, 1190 Vienna, Austria.
Reiter
Evelyne
E
Jungbauer
Alois
A
D001681:Biological Assay / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006801:Humans; D018613:Microscopy, Confocal; D010957:Plasmids; D011869:Radioligand Assay; D011944:Receptors, Androgen / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013256:Steroids / Q000737:chemistry; D015533:Transcriptional Activation / Q000235:genetics*
D011944:Receptors, Androgen; D013256:Steroids; D049452:Green Fluorescent Proteins