Department of Biomedical Sciences, University of Windsor, Windsor, ON, Canada.
Methods Mol Biol. 2022;2482:353-371. doi: 10.1007/978-1-0716-2249-0_24.
Circadian rhythms are daily oscillations in physiology and gene expression that are governed by a molecular feedback loop known as the circadian clock. In Drosophila melanogaster, the core clock consists of transcription factors clock (Clk) and cycle (cyc) which form protein heterodimers that activate transcription of their repressors, period (per) and timeless (tim). Once produced, protein heterodimers of per/tim repress Clk/cyc activity. One cycle of activation and repression takes approximately ("circa") 24-h ("diem") and repeats even in the absence of external stimuli. The circadian clock is active in many cells throughout the body; however, tracking it dynamically represents a challenge. Traditional fluorescent reporters are slowly degraded and consequently cannot be used to assess dynamic temporal changes exhibited by the circadian clock. The use of rapidly degraded fluorescent protein reporters containing destabilized GFP (dGFP) that report transcriptional activity in vivo at a single-cell level with ~1-h temporal resolution can circumvent this problem. Here we describe the use of circadian clock reporter strains of Drosophila melanogaster, Clock and Clock, to track clock transcriptional activity using the intestine as a tissue of interest. These methods may be extended to other tissues in the body.
昼夜节律是指生理和基因表达的日常波动,由一个称为生物钟的分子反馈环控制。在黑腹果蝇中,核心生物钟由转录因子 clock (Clk) 和 cycle (cyc) 组成,它们形成蛋白异二聚体,激活其抑制剂 period (per) 和 timeless (tim) 的转录。一旦产生,per/tim 蛋白异二聚体抑制 Clk/cyc 活性。一个激活和抑制的周期大约需要 24 小时(“diem”),即使没有外部刺激也会重复。生物钟在体内许多细胞中都很活跃;然而,动态跟踪它是一个挑战。传统的荧光报告基因会缓慢降解,因此不能用于评估生物钟表现出的动态时间变化。使用快速降解的荧光蛋白报告基因,其中包含不稳定的 GFP(dGFP),可以在单细胞水平上以约 1 小时的时间分辨率报告转录活性,从而可以避免这个问题。在这里,我们描述了使用黑腹果蝇的 Clock 和 Clock 昼夜节律报告基因株系,通过将肠道作为感兴趣的组织来跟踪时钟转录活性。这些方法可以扩展到体内的其他组织。