title: Impacts of different promoters on the mammalian one-hybrid assay for detecting nuclear receptor agonists.
abstract: Nuclear receptors are a superfamily of ligand-activated transcription factors that play key roles in many biological processes, and have become one class of the most important targets in drug discovery. Mammalian one-hybrid system has been used to develop a cell-based functional transactivation high-throughput screening (HTS) assay for detecting nuclear receptors ligands. In the present study, we proved that different promoters used in the reporter vector had significant different impacts on the performance of HTS assays. The assay using the SV40 promoter in the reporter vector showed the characteristics of much higher signal/noise ratios, acceptable Z' factors (>0.6), low coefficient variation (<12.5%) and higher hits rate, which could be more robust, reproducible, and sensitive. In contrast, utilizing a TATA box promoter in the assay resulted in higher variance and low sensitivity. In addition, it was found that the assay using SV40 had longer signal decay time and was easier to be miniaturized in 384-well format. It has been confirmed that the choice of a promoter is a critical factor in developing a reporter gene HTS assay. However, the SV40 promoter used in the present study has been shown to be more adaptable than the minimal promoter TATA box in the Mammalian one-hybrid HTS assays for detecting nuclear receptor agonists.
authors.affiliation: College of Life Science, Hebei Normal University, Shijiazhuang, 050016, China.
authors.forename: Zhi-Hui
authors.initials: ZH
authors.lastname: Zheng
authors.forename: Xin-Hua
authors.initials: XH
authors.lastname: Lu
authors.forename: Hua
authors.initials: H
authors.lastname: Zhang
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Lv
authors.forename: Jian-Gong
authors.initials: JG
authors.lastname: He
authors.forename: Bao-Hua
authors.initials: BH
authors.lastname: Zhao
authors.forename: Shu-Yi
authors.initials: SY
authors.lastname: Si
mesh_terms: D000818:Animals; D001629:Bezafibrate / Q000032:analysis / Q000494:pharmacology; D002478:Cells, Cultured; D002635:Chenodeoxycholic Acid / Q000032:analysis / Q000494:pharmacology; D055808:Drug Discovery; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D006845:Hydrocarbons, Fluorinated / Q000032:analysis / Q000494:pharmacology; D008024:Ligands; D051379:Mice; D041681:NIH 3T3 Cells; D047492:Peroxisome Proliferator-Activated Receptors / Q000819:agonists*; D000077205:Pioglitazone; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011743:Pyrimidines / Q000032:analysis / Q000494:pharmacology; D000077154:Rosiglitazone; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000032:analysis / Q000494:pharmacology; D045162:Thiazolidinediones / Q000032:analysis / Q000494:pharmacology; D020798:Two-Hybrid System Techniques; D005466:Fluorocarbons; D000096926:Benzenesulfonamides
chemical_list: D001629:Bezafibrate; D002635:Chenodeoxycholic Acid; D006845:Hydrocarbons, Fluorinated; D008024:Ligands; D047492:Peroxisome Proliferator-Activated Receptors; D000077205:Pioglitazone; D011743:Pyrimidines; D000077154:Rosiglitazone; D013449:Sulfonamides; D045162:Thiazolidinediones; C423915:T0901317; D005466:Fluorocarbons; D000096926:Benzenesulfonamides; C006253:pirinixic acid