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

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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