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

通过生物发光和荧光报告技术，我们监测了活细胞和组织中多个基因的表达和多种功能，并验证了由多个时钟基因及其蛋白产物组成的互锁自调节反馈环组成的细胞内分子时钟。1.双生物发光报告系统实时监测两个时钟基因的表达：在PER1和BMal1启动子序列的下游分别插入Vurula荧光素酶(VL)和萤火虫荧光素酶(FL)cDNA。我们培养了表达双报告基因的转基因小鼠的各种组织，并连续监测FL活性长达40天。2.时钟基因表达和细胞内钙离子水平的同时监测：在NSE启动子调控下表达的钙结合蛋白Cameleon，用FRET方法连续监测细胞内钙离子水平。我们发现Clock基因的表达和Ca^-lt；2+-gt；水平都符合昼夜节律。3.红色荧光素酶用于监测单个细胞的多个基因表达：在哺乳动物细胞中克隆和优化发出红色、橙色和绿色发光的突变荧光素酶。我们构建了在BMal1启动子控制下表达红色发光的转基因小鼠系，并成功地监测了SCN和肝脏的红色发光节律。

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.