Perfusion-culture-based secreted bioluminescence reporter assay in living cells

• Published in: Analytical biochemistry 2006-07, Vol.354 (1), p.15-21
• Main Authors: Yamagishi, Kazutoshi, Enomoto, Toshiteru, Ohmiya, Yoshihiro
• Format: Article
• Language: English
• Subjects: Animals; Anthracenes - pharmacology; ARNTL Transcription Factors; Basic Helix-Loop-Helix Transcription Factors - antagonists & inhibitors; Basic Helix-Loop-Helix Transcription Factors - genetics; Basic Helix-Loop-Helix Transcription Factors - metabolism; Cell Line; Circadian clock; Circadian Rhythm - drug effects; Cypridina luciferase; Cypridina noctiluca; Dexamethasone - pharmacology; Enzyme Inhibitors - pharmacology; Gene Expression - drug effects; Gene Expression Regulation; Genes, Reporter; JNK Mitogen-Activated Protein Kinases - antagonists & inhibitors; JNK Mitogen-Activated Protein Kinases - metabolism; Luciferases - analysis; Luciferases - chemistry; Luciferases - genetics; Luminescent Measurements - methods; Mifepristone - pharmacology; Perfusion - methods; Perfusion culture; Pharmacological assay; Promoter Regions, Genetic; Rats; Secreted bioluminescence reporter
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2006.03.031

Bioluminescence reporter proteins have been widely used in the development of tools for monitoring biological events in living cells. In this study, we describe the development of a reporter system with secreted Cypridina noctiluca luciferase (CLuc) for a pharmacological assay that is based on targeted promoter activity. A model cell line was established with Rat-1 fibroblasts expressing CLuc driven by the promoter of a circadian clock gene, Bmal1. To accurately assay for temporally secreted CLuc activity, a perfusion culture in which the promoter activity was sequentially monitored by the reporter activity in the perfusate was adopted. By pulsing with dexamethasone (DEX), a glucocorticoid (GC) analog, the profile of the reporter activity successfully showed diurnal fluctuation, which is a canonical expression pattern of the Bmal1 gene. Trial studies illustrated that the DEX-pulsed circadian oscillation was reasonably attenuated by RU486, a GC receptor antagonist. Moreover, SP600125, a c-Jun N-terminal kinase inhibitor, caused phase shifting of the rhythmicity. We conclude that the CLuc reporter assay in combination with perfusion culture is a suitable pharmacological tool for drug discovery.