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Analyses of circadian clock mechanisms by an efficient and multidirectional functional screening

通过高效、多向的功能筛选来分析生物钟机制

基本信息

批准号：
16590184
负责人：
HAYASAKA Naoto
金额：
$ 1.98万
依托单位：
Kinki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590184/
关键词：
Neuroscience Brain, Neuron Circadian clock Gene function ウイルス視交叉上核概日リズム Rgs16 レンチウイルス Hmg4 RNAi スライスカルチャー肝臓

项目摘要

We have previously identified circadianly regulated genes in the suprachiasmatic nucleus (SCN) and liver by Gene Chip analyses. To elucidate their functions in efficient and multidimensional ways, we have established methods using a lentiviral vector system to analyze gene functions both in vivo and in vitro. First, we constructed lentiviral vectors for overexpression or knockdown (siRNA) of candidate genes. Then we developed a technique to inject lentivirus into the mouse SCN using a stereotaxic instrument with a syringe pump. The method enabled us to analyze circadian phenotype (e.g., locomotor activity) in vivo right after injection of the virus in the SCN and made it possible to analyze functions of several candidate genes in a short term. Next, we further applied the virus infection to brain slices. We established a method to efficiently introduce a transgene into the SCN, which had been quite difficult like in other tissues. Using this new method, we studied roles of the candidate genes in circadian system by measuring different circadian outputs. By making SCN slices from transgenic rats/mice carrying Per2::luciferase, which demonstrate circadian rhythmicity of luciferase activity, we examine an effect of the candidate gene expression on the circadian rhythms of the Per2::luc. We also analyzed circadian activity of the SCN neurons in the same slices by electrophysiology. Lastly, we could also study roles of the candidate genes using dissociated neurons/glias infected with the lentivirus. Using these methods mentioned above, so far we have analyzed two candidate genes, Rgs16 and Hmg4 and are preparing to publish papers

我们之前已经通过基因芯片分析确定了视交叉上核（SCN）和肝脏中的昼夜调节基因。为了以高效和多维的方式阐明它们的功能，我们建立了使用慢病毒载体系统来分析体内和体外基因功能的方法。首先，我们构建了候选基因过表达或敲低（siRNA）的慢病毒载体。然后，我们开发了一种使用带有注射泵的立体定向仪器将慢病毒注射到小鼠SCN中的技术。该方法使我们能够在SCN中注射病毒后立即分析体内的昼夜节律表型（例如，运动活动），并使在短期内分析几个候选基因的功能成为可能。接下来，我们进一步将病毒感染应用于脑切片。我们建立了一种方法，可以有效地将转基因引入SCN，这与其他组织一样是相当困难的。利用这种新方法，我们通过测量不同的昼夜节律输出来研究候选基因在昼夜节律系统中的作用。通过制作携带Per2:：荧光素酶的转基因大鼠/小鼠的SCN切片，研究了候选基因表达对Per2:：荧光素酶昼夜节律的影响。我们还通过电生理学分析了同一切片中SCN神经元的昼夜活动。最后，我们还可以利用被慢病毒感染的解离神经元/胶质细胞来研究候选基因的作用。利用上述方法，目前我们已经分析了Rgs16和Hmg4两个候选基因，并准备发表论文