کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1177505 | 962021 | 2006 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Perfusion-culture-based secreted bioluminescence reporter assay in living cells
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موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
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چکیده انگلیسی
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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 354, Issue 1, 1 July 2006, Pages 15-21
Journal: Analytical Biochemistry - Volume 354, Issue 1, 1 July 2006, Pages 15-21
نویسندگان
Kazutoshi Yamagishi, Toshiteru Enomoto, Yoshihiro Ohmiya,