REAL-TIME SINGLE-CELL RECORDING OF CIRCADIAN GENE EXPRESSION IN DROSOPHILA BRAIN

果蝇大脑昼夜节律基因表达的实时单细胞记录

• 批准号: 7722579
• 负责人: TATIANA B KRASIEVA
• 金额: $ 0.21万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-15 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722579
• 关键词: Arabidopsis; Automobile Driving; Bioluminescence; Brain; Cell Size; Cells; Chimeric Proteins; Chronobiology; Circadian Rhythms; Computer Retrieval of Information on Scientific Projects Database; Condition; Confocal Microscopy; Depth; Drosophila genus; Fire - disasters; Funding; Galactosidase; Gene Expression; Gene Expression Profiling; Genes; Goals; Grant; Green Fluorescent Proteins; Institution; Invasive; Length; Life; Light; Lighting; Luciferases; Mammals; Microscopy; Monitor; Mus; N-terminal; Neurons; Nuclear Translocation; Organism; Photons; Photoreceptors; Process; Proteins; Purpose; Rate; Reporter Genes; Reporting; Research; Research Personnel; Resources; Retina; Retinal Photoreceptors; Signal Transduction; Source; Stimulus; System; Time; Tissues; Transgenes; United States National Institutes of Health; Vertebrates; Zebrafish; animal tissue; charge coupled device camera; circadian pacemaker; cryptochrome; fly; immunoreactivity; multi-photon; promoter; red fluorescent protein; suprachiasmatic nucleus

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The ultimate goal of this study is to find out how neurons in the circadian clock circuit in Drosophila interact with each other and respond to environmental conditions such as light. In order to do this we would like to devise a way to monitor circadian gene expression of single cell in live tissues and animals. In chronobiology, the fire fly luciferase (luc) has been successfully used as a reporter gene to monitor real-time circadian gene expression in whole animals and tissues due to high turnover rate of luciferase's enzymatic activity. For this purpose, typically promoter from a clock gene or a clock-controlled gene is fused to luc to drive cycling gene expression. This has been done in several organisms such as Arabidopsis, Drosophila, mouse, and zebrafish. In vertebrates, this system was also used to record cycling gene expression of single cells via high sensitivity CCD cameras. However, it has been difficult to use this system for single-cell monitoring in Drosophila, because of small cell sizes. Alternative way to record single-cell gene expression is to use fluorescent proteins. In mouse, degradable GFP has been used to record single-cell gene expression in neurons in the suprachiasmatic nucleus. This is possible in mammals, because mammalian tissues other than the retina are insensitive to light, and light stimulus generated by illumination, excitation and emission of GFP cannot shift mammalian circadian clocks. This is not the case with Drosophila, because flies have blue-light photoreceptor cryptochrome in most clock cells, and clocks in those cells can be shifted independently from the retinal photoreceptor. Wave lengths for excitation and emission of GFP are too close to cryptochrome's sensitive wavelengths. Therefore, we would like to use the far red fluorescent protein mCherry to report circadian gene expression in Drosophila. Among all the far red fluorescent protein we chose mCherry, because it is monomeric, fast maturing (15min), long excitation/emission wavelengths (587/610), and it's commercially available (Clontech). We would like to use multi-photon excitation microscopy (MPM) or single-photon confocal microscopy to visualize mCherry signals in dissected fly brains or even partially dissected live flies, because MPM uses far-red to infrared light for excitation, which further reduces the possibility of shifting the clock. MPM is also good because it's non-invasive, and penetrates deep tissues. The transgenes we are currently planning to make are as follows: 1 Promoter from the Drosophila clock gene period (per) driving a fusion protein of PER and mCherry. We will make fusion protein of full-length PER and mCherry as well as N-terminal 2/3 of PER and mCherry. Both PER fragments fused to luc have been used for bioluminescence monitoring of gene expression. N-terminal 2/3 of PER has also been fused to B-galactosidase to make PER-Bgal fusion protein, which showed rhythmic B-gal activity as well as immunoreactivity. 2 We can also fuse per promoter directly to mCherry. In this case we need to make mCherry degradable with exogenous PEST sequence. We also need to fuse nuclear translocation signal to mCherry, since mCherry signals in neuronal processes could be hard to quantify.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 这项研究的最终目标是找出果蝇中昼夜节律时钟电路中的神经元如何相互作用并响应诸如光线之类的环境条件。为此，我们想设计一种方法来监测活组织和动物中单细胞的昼夜节律表达。 在年代生物学中，由于荧光素酶的酶活性高更高的转移率，火蝇荧光素酶（LUC）已成功用作记者基因，以监测全动物和组织中的实时昼夜节律基因表达。为此，通常是从时钟基因或时钟控制基因的启动子融合到LUC以驱动循环基因表达。这是在拟南芥，果蝇，小鼠和斑马鱼等几种生物中进行的。在脊椎动物中，该系统还用于通过高灵敏度CCD摄像机记录单细胞的循环基因表达。但是，由于小细胞尺寸，很难将该系统用于果蝇中的单细胞监测。记录单细胞基因表达的替代方法是使用荧光蛋白。在小鼠中，可降解的GFP已用于记录上核中神经元中的单细胞基因表达。在哺乳动物中，这是可能的，因为视网膜以外的哺乳动物组织对光不敏感，并且通过照明，激发和GFP的发射产生的光刺激无法改变哺乳动物的昼夜节律钟。果蝇不是这种情况，因为苍蝇在大多数时钟细胞中具有蓝光光感受器的隐性色素，并且这些细胞中的时钟可以独立于视网膜光感受器转移。激发和GFP发射的波长太接近了加密色素的敏感波长。 因此，我们想使用远红色荧光蛋白麦克利（MCHERRY）报告果蝇中的昼夜节律表达。在所有远红色荧光蛋白中，我们选择了MCHERRY，因为它是单体，快速成熟（15分钟），长激发/发射波长（587/610），并且是市售的（Clontech）。我们想使用多光子激发显微镜（MPM）或单光子共聚焦显微镜来可视化剖腹的苍蝇大脑中的MCHERRY信号，甚至是部分剖析的活蝇，因为MPM使用远红外光到红外光进行激发，从而进一步降低了移动时钟的可能性。 MPM也很好，因为它是无创的，并且可以穿透深层组织。 我们目前计划制作的转基因如下： 果蝇时钟基因周期（PER）的1个启动子驱动PER和MCHERRY的融合蛋白。我们将制作全长PER和MCHERRY的融合蛋白，以及n末端2/3的PER和MCHERRY。融合到LUC的两个片段已用于基因表达的生物发光监测。 N末端2/3的PER也已与B-半乳糖苷酶融合在一起，以制作每杆融合蛋白，该蛋白显示出节奏B-GAL活性以及免疫反应性。 2我们还可以将每个启动子直接融合到麦克里。在这种情况下，我们需要通过外源性害虫序列使麦克利降解。我们还需要将核转运信号融合到MCHERRY，因为在神经元过程中的MCHERRY信号很难量化。