An Comprehensive Analysis of Circadian Clock Controlled Genes in Central and Peripheral Clock Systems.

中枢昼夜节律振荡器位于哺乳动物大脑的视交叉上核(SCN)。SCN产生的电生理活动或体液因子的昼夜波动调节外周节律。要阐明产生昼夜节律的分子机制，需要对时钟和时钟控制基因进行全面分析。在这项研究中，我们建立了一个比个体水平更容易在细胞水平上筛选基因表达的系统。首先，我们构建了一种新的转基因大鼠，其中小鼠的PER1基因启动子与荧光素酶报告基因相连。MPer1：：Luc转基因大鼠SCN的光发射具有很强的节律性，并在静态培养中持续了32天，表明mPer1：：Luc转基因大鼠的启动子具有完整的功能。然后从转基因大鼠的胚胎脑中建立了几个SCN来源的细胞系。在584个克隆品系中，有12个品系表达NCAM，并存在超过8个周期的光输出昼夜节律。用Forsklin处理振荡最强的细胞系后，可实现节律的时相提前或延迟，并获得与光行为节律相一致的时相反应曲线。携带PER1：：Luc报告基因的稳定的Rat1转基因细胞在Forsklin、Dex或PMA的刺激下有昼夜节律，但这种节律不如我们从SCN来源的细胞系中记录的那样强烈，并且总是在培养约6个周期后减弱。因此，这两个细胞系中的每一个都可以被视为中枢或外周昼夜节律振荡器的细胞模型，并利用DNA芯片分析这些细胞中的基因表达谱。

The central circadian oscillator is located at the suprachiasmatic nucleus (SCN) of the mammalian brain. The circadian fluctuation of electrophysiological activities or humoral factors produced by the SCN regulates the peripheral rhythms. Elucidation of molecular mechanisms generating circadian rhythms requires comprehensive analyses of the clock and clock controlled genes. In this study, we have established an easier screening system of gene expression at the cellular level than that at the individual level. Firstly, we constructed a new transgenic rat in which the mouse Per1 gene promoter is linked to a luciferase reporter. Light emission from the SCN of the mPer1::luc transgenic rat line was robustly rhythmic, and persisted for up to 32 days in static culture, indicating that the promoter in the mPer1::luc transgene was fully functional. Then several SCN derived cell lines were established from the embryonic brain of the transgenic rat. Of 584 cloned lines, 12 lines expressed NCAM, in addition to the circadian rhythms of light output for more than 8 cycles. The time dependent phase advance or delay of the rhythm was accomplished after the treatment of a cell line with the most robust oscillation by forskolin, and the phase response curve obtained by forskolin was consistent with that obtained in the behavioral rhythm by light. Although a stable Rat1 transfectant with the Per1::luc reporter had circadian rhythms after the stimulation by forskolin, Dex, or PMA, the rhythms were not as robust as those we recorded from the SCN derived cell lines and always damped out after about 6 cycles in culture. Therefore, each of two lines could be regarded as the cellular models of the central or peripheral circadian oscillator, and the profiles of gene expression in these cells were analyzed by DNA microarrays..