4D Confocal Imaging of Endogenous Proteins Tagged by CRISPR/Cas9 Genome Editing

CRISPR/Cas9 基因组编辑标记的内源蛋白的 4D 共聚焦成像

• 批准号: RTI-2017-00558
• 负责人: Moss, Thomas
• 金额: $ 10.85万
• 依托单位: Université Laval
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=616710
• 关键词: 4D; Confocal; Imaging; Endogenous; Proteins

The application is to finance a significant and very urgent upgrade of our multi-channel Leica SP5-II scanning confocal microscope, an essential and key instruments in the programs of each of the 8 applicants. The fact that this is the third consecutive application to NSERC for this upgrade, our 2016 application having missed funding by only one place, attests to its urgency and to the lack of any other funding sources. The applicants’ research groups use a broad range of biological systems, Xenopus, Drosophila and mouse, and human, mouse and insect cell and stem cell culture to elucidate fundamental mechanisms in Cellular and Molecular Biology. All use cutting-edge technologies in Genomics, Bioinformatics and Proteomics, CRISPR/Cas9 Genome Editing, and especially extensive High Resolution Live-, Fixed-Cell and Tissue Subcellular Imaging. Our strong research grouping, therefore, encompasses a very wide range of expertise, and hence provides a truly exceptional training environment. The requested upgrade is to provide a critically important increase in sensitivity to the SP5-II confocal microscope and to provide near Super-Resolution imaging. CRISPR/Cas9 gene editing has provided us with a simple and efficient means to insert in-phase epitope and fluorescent gene tags into the genes of complex animal genomes. Our groups have successfully established this technology in order to study endogenous proteins under their normal cellular control and at truly physiological abundance. However, the imaging of tagged proteins at natural abundance requires highly sensitive imaging systems. Our Leica SP5-II point scanning confocal microscope presently lacks the sensitivity to allow us to take advantage of endogenous protein tagging, whether epitope or fluorescent. Further, our research involves imaging subcellular detail and we are continuously pushing the limits of optical confocal microscopy. Both the need for enhanced sensitivity and for enhanced resolution can be fulfilled by the cost effective addition to the SP5-II of photo-counting HyD detectors, HyVolution™ deconvolution and an adapted immersion objective. With its standard and resonance modes, our Leica SP5-II instrument provides all the advantages of a scanning microscope, e.g. true confocal resolution and the availability of techniques such as FRET and FRAP, with the extremely low photo-toxicity of spinning disk systems and is fully equipped for live-cell studies. It allows simultaneous monitoring of 4 or 5 freely selectable fluorescence channels in all imaging modes. The SP5-II is also optically and mechanically equal to the best systems available. With the installation of Hybrid (HyD) detectors, a task-adapted objective and improved deconvolution software the SP5-II will fulfill our pressing needs for increased sensitivity and for near Super-Resolution imaging.

该申请是为了资助我们的多通道Leica SP 5-II扫描共聚焦显微镜的重大和非常紧迫的升级，这是8名申请人中每个人的计划中的重要和关键仪器。事实上，这是连续第三次向NSERC申请升级，我们2016年的申请只错过了一个地方的资金，证明了它的紧迫性和缺乏任何其他资金来源。 申请人的研究小组使用广泛的生物系统，非洲爪蟾，果蝇和小鼠，以及人类，小鼠和昆虫细胞和干细胞培养物来阐明细胞和分子生物学中的基本机制。所有这些都使用基因组学、生物信息学和蛋白质组学、CRISPR/Cas9基因组编辑以及特别是广泛的高分辨率活细胞、固定细胞和组织亚细胞成像的尖端技术。因此，我们强大的研究小组涵盖了非常广泛的专业知识，因此提供了一个真正卓越的培训环境。 所要求的升级是提供一个至关重要的增加灵敏度的SP 5-II共聚焦显微镜，并提供近超分辨率成像。CRISPR/Cas9基因编辑为我们提供了一种简单有效的手段，将同相表位和荧光基因标签插入复杂动物基因组的基因中。我们的团队已经成功地建立了这项技术，以研究在正常细胞控制下和真正的生理丰度下的内源性蛋白质。然而，天然丰度的标记蛋白质的成像需要高度灵敏的成像系统。我们的Leica SP 5-II点扫描共聚焦显微镜目前缺乏灵敏度，使我们能够利用内源性蛋白质标记，无论是表位还是荧光。此外，我们的研究涉及亚细胞细节成像，我们正在不断推动光学共聚焦显微镜的极限。通过在SP 5-II中添加具有成本效益的光计数HyD探测器、HyVolution™反卷积和改进的浸没物镜，可以满足对增强灵敏度和增强分辨率的需求。 Leica SP 5-II仪器具有标准和共振模式，提供了扫描显微镜的所有优点，例如真正的共聚焦分辨率和FRET和FRAP等技术的可用性，以及旋转盘系统极低的光毒性，并完全配备活细胞研究。它允许在所有成像模式下同时监测4或5个自由选择的荧光通道。SP 5-II在光学和机械方面也与现有的最佳系统相当。通过安装混合（HyD）探测器，任务适应物镜和改进的反卷积软件，SP 5-II将满足我们对提高灵敏度和近超分辨率成像的迫切需求。