Yamagishi Kazutoshi, Enomoto Toshiteru, Ohmiya Yoshihiro
Cell Dynamics Research Group, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Midorigaoka, Ikeda, Osaka 563-8577, Japan.
Anal Biochem. 2006 Jul 1;354(1):15-21. doi: 10.1016/j.ab.2006.03.031. Epub 2006 Apr 27.
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.
生物发光报告蛋白已广泛应用于开发监测活细胞中生物事件的工具。在本研究中，我们描述了一种基于靶向启动子活性的药理学检测方法的报告系统的开发，该系统使用分泌型海萤荧光素酶（CLuc）。利用大鼠-1成纤维细胞建立了一个模型细胞系，该细胞系由生物钟基因Bmal1的启动子驱动表达CLuc。为了准确检测随时间分泌的CLuc活性，采用了一种灌注培养方法，通过灌注液中的报告活性来连续监测启动子活性。通过用糖皮质激素（GC）类似物地塞米松（DEX）脉冲处理，报告活性的图谱成功显示出昼夜波动，这是Bmal1基因的典型表达模式。试验研究表明，GC受体拮抗剂RU486可合理减弱DEX脉冲诱导的昼夜节律振荡。此外，c-Jun氨基末端激酶抑制剂SP600125导致节律发生相移。我们得出结论，CLuc报告检测与灌注培养相结合是一种适用于药物发现的药理学工具。
