Development of a Functional Reporter Gene HTS Assay for the Identification of mGluR7 Modulators
| Autor: | Angelo Giacometti, Georg C. Terstappen, Laura Aldegheri, Elisa Ballini |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
0301 basic medicine
DNA Complementary Drug Evaluation Preclinical Clone (cell biology) CHO Cells Biology Receptors Metabotropic Glutamate 01 natural sciences Biochemistry Analytical Chemistry Adenylyl cyclase 03 medical and health sciences chemistry.chemical_compound Genes Reporter Cricetinae Cyclic AMP Animals Luciferase Luciferases Reporter gene Forskolin Chinese hamster ovary cell Colforsin Molecular biology Recombinant Proteins Rats 0104 chemical sciences 010404 medicinal & biomolecular chemistry 030104 developmental biology chemistry Metabotropic glutamate receptor Luminescent Measurements Molecular Medicine Bioreporter Biotechnology |
| Zdroj: | SLAS Discovery. 5:255-261 |
| ISSN: | 2472-5552 |
| DOI: | 10.1177/108705710000500408 |
| Popis: | For the identification of modulators of the metabotropic glutamate receptor mGluR7, a functional cell-based high throughput screening (HTS) assay was developed. This assay utilizes the signal transduction pathway of mGluR7, which is negatively coupled to adenylyl cyclase. A cAMP-responsive luciferase reporter gene and rat mGluR7 cDNA were cotransfected into CHO-K1 cells by electroporation. Stable recombinant cells were selected by resistance to the antibiotic G418. Functional selection was carried out by analyzing the effect of the agonist glutamate to reduce elevated cAMP levels after forskolin stimulation. Out of 83 G418-resistant cell clones, the clone with the best functional characteristics was selected. This clone displayed the strongest reduction of forskolin-stimulated cAMP levels. Glutamate (10 mM) decreased cAMP levels, as monitored by luciferase expression, by about 50%, and the more potent agonist L-2-amino-4-phosphonobutyrate resulted in nearly complete reduction, exhibiting an EC(50) of 0.9 mM. The functional response of the clone did not change during cell passages, indicating the stability of this novel recombinant cell line. The luciferase reporter gene assay, which allows easy nonradioactive luminescence detection of mGluR7 activity, was optimized for its application in automated HTS. |
| Databáze: | OpenAIRE |
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