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Anal Biochem. 2006 Jul 1;354(1):15-21. Epub 2006 Apr 27.

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

Author information

  • 1Cell Dynamics Research Group, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Midorigaoka, Ikeda, Osaka 563-8577, Japan. kazutoshi@yahoo.co.jp

Abstract

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.

PMID:
16713985
[PubMed - indexed for MEDLINE]
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