کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2085060 1545403 2007 9 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Functional characterization of cell lines for high-throughput screening of human neuromedin U receptor subtype 2 specific agonists using a luciferase reporter gene assay
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیوتکنولوژی یا زیست‌فناوری
پیش نمایش صفحه اول مقاله
Functional characterization of cell lines for high-throughput screening of human neuromedin U receptor subtype 2 specific agonists using a luciferase reporter gene assay
چکیده انگلیسی

We developed a functional cell-based high-throughput screening (HTS) assay to identify modulators of the human neuromedin subtype 2 receptor. This assay utilized the signal transduction pathway of hNMU2R, which is positively coupled to adenylyl cyclase and downstream calcium signal pathways. We describe in detail a robust, sensitive, and functional assay for the hNMU2R G-protein-coupled receptor expressed in human embryonic kidney (HEK)-293 cells, whose activity was reflected by a luciferase reporter gene transcriptionally regulated by a 3-repeat serum response element (SRE)-3 repeat multiple response element (MRE)-3 repeat cyclic AMP (cAMP) response element (CRE)-VIP mini promoter. The HEK 293 clonal cell line, stably co-transfected with the 3 × SRE/3 × MRE/3 × CRE/VIP mini promoter-driven luciferase and pCDNA3.1-NMU2R plasmid, was selected by active geneticin sulfate and their ability to express luciferase with a forskolin challenge following hNMU plus forskolin, known to activate intracellular signal transduction. Then the cell density, incubation time, dimethyl sulfoxide (DMSO) concentration used to screen the hNMU receptor subtype 2 specific agonist were optimized, and whether intrinsic luminescent substance of extracts isolated from traditional Chinese herbs disturbs luminescence of luciferase expressed in HEK293 cells was considered. The optimal incubation time was found to be between 8 and 9 h, the cell density and DMSO concentrations were optimized from 3 × 104 to 6 × 104, and less than 2%, respectively. Our data show that hNMU2R luci-HEK293 cells and their assay exhibit a low background and ideal model for high-throughput screening. These results demonstrate that this reporter gene assay is useful for pharmacological analysis, and is amenable to HTS for human NMU2R agonists.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: European Journal of Pharmaceutics and Biopharmaceutics - Volume 67, Issue 1, August 2007, Pages 284–292
نویسندگان
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