Circadian rhythm generation mechanism : multi-reporter analysis of interlocked molecular feedback loops

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By applying bioluminescence and fluorescent reporter techniques, we monitored multiple gene expression and multiple functions in the living cells and tissues and verified the intracellular molecular clock composed of interlocked autoregulatory feedback loops involving several clock genes and their protein products.1.Real-time monitoring of two clock gene expression by dual bioluminescent reporter system :Vurgula luciferase (VL) and firefly luciferase (FL) cDNAs were inserted downstream of the promoter sequences of Per1 and Bmal1, respectively. We cultured various tissues of the transgenic mice expressing the dual reporter and monitored FL activity continuously for up to 40 days. Using perfusion system, VL activity in the culture medium was measured simultaneously with the intracellular FL activity, which is the first result of realtime monitoring of multiple gene expression.2.Simultaneous monitoring of clock gene expression and intracellular Ca^<2+> levels :Intracellular Ca^<2+> level was continuously monitored by FRET method in the cultured supracihasmatic nucleus transfected with Ca^<2+> binding protein Cameleon which is expressed under control of NSE promoter. We found both clock gene expression and Ca^<2+> levels exhibited circadian rhythm.3.Red luciferase to monitor multiple gene expression from single cells :Mutant luciferases emitting either red, orange and green luminescence were cloned and optimized for the reporter in mammalian cells. We constructed transgenic mouse lines expressing red luminescence under the control of Bmal1 promoter and successfully monitored red luminescent rhythms in the SCN and liver.