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

通过应用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回路组成的细胞内分子钟。通过双生物发光报告系统实时监测两个时钟基因的表达：将Vurgula luciferase （VL）和firefly luciferase (FL) cdna分别插入Per1和Bmal1的启动子序列下游。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。采用灌注系统，同时测定培养基中VL活性和细胞内FL活性，这是实时监测多基因表达的第一个结果。同步监测时钟基因表达和细胞内Ca^<2+>水平：用FRET法连续监测Ca^<2+>结合蛋白Cameleon在NSE启动子控制下表达的培养的核上核细胞内Ca^<2+>水平。我们发现时钟基因表达和Ca^<2+>水平均具有昼夜节律。红色荧光素酶用于监测单细胞中多个基因的表达：克隆并优化了在哺乳动物细胞中发出红色、橙色和绿色荧光素酶的突变体。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和肝脏中的红色发光节律。

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.