Single-cell sampling and analysis

单细胞采样和分析

• 批准号: 288223-2008
• 负责人: Audet, Julie
• 金额: $ 2.11万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2012
• 资助国家: 加拿大
• 起止时间: 2012-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=496798
• 关键词: Single; cell; sampling; analysis

Understanding the molecular mechanism underlying cellular function is a major goal in science. Unfortunately natural cell populations obtained from the blood or from various tissues are often difficult to study, as they can only be obtained in heterogeneous mixtures. For instance, stem cells found in adult tissues have a great potential in medicine for replacing damaged or diseased tissues. However, they are rare and cannot be harvested or grown to the large numbers needed for conventional bulk biochemical assays. Thus, single cell assays are essential for studying and understanding cell behaviour and the development of stem cell-based technologies and therapies. Capillary electrophoresis (CE), performed in fused-silica columns or on microchip channels, is a separation technology that is capable of high resolution separations of analytes based on size and charge from small volumes (< 10^-9 L). As such CE combined with the enhanced detection sensitivity of a laser-induced fluorescence detection scheme represents a powerful approach for the analysis of single mammalian cells. The overall objective of this proposal is to develop and optimize strategies for the biochemical analysis of single-cell by capillary and microchip electrophoresis. The first specific objective is to maximize the recovery of analytes from specific cell compartments by optimizing the method of cell sampling and the fabrication of the separation column. The second objective is to demonstrate the utility of single-cell CE to analyse posttranslational modifications of a membrane-tethered transcription factor important in stem cell fate decision, Notch. Single-cell electrophoresis in capillaries or on microchips is emerging as the next revolution in tools for biological discovery. Improved sampling and the ability to estimate losses will allow more sensitive and precise quantitation of fluorescent analytes in cells. Furthermore, once optimized, single-cell CE will provide a powerful means to study proteins that have complex posttranslational regulation such as the Notch protein. Consequently, single-cell CE has the potential to become an essential tool in development of drug, gene or cellular therapies targeting Notch and stem cells in general.

了解细胞功能背后的分子机制是科学研究的一个主要目标。不幸的是，从血液或各种组织中获得的自然细胞群通常难以研究，因为它们只能在异质混合物中获得。例如，在成人组织中发现的干细胞在医学上具有替代受损或患病组织的巨大潜力。然而，它们是罕见的，不能收获或种植到常规批量生化分析所需的大量。因此，单细胞分析对于研究和理解细胞行为以及基于干细胞的技术和疗法的发展至关重要。毛细管电泳（CE）在熔融硅胶柱或微芯片通道上进行，是一种分离技术，能够根据小体积（< 10^-9 L）的尺寸和电荷对分析物进行高分辨率分离。因此，CE与激光诱导荧光检测方案的增强检测灵敏度相结合，代表了分析单个哺乳动物细胞的有力方法。本提案的总体目标是开发和优化毛细管电泳和微芯片电泳的单细胞生化分析策略。第一个具体目标是通过优化细胞取样方法和分离柱的制作，最大限度地从特定的细胞区室中回收分析物。第二个目标是证明单细胞CE分析在干细胞命运决定中重要的膜系转录因子Notch的翻译后修饰的效用。毛细管或微芯片上的单细胞电泳正在成为生物发现工具的下一个革命。改进的采样和估计损失的能力将使细胞中荧光分析物的定量更加敏感和精确。此外，一旦优化，单细胞CE将为研究具有复杂翻译后调控的蛋白质（如Notch蛋白）提供强有力的手段。因此，单细胞CE有潜力成为开发靶向Notch和干细胞的药物、基因或细胞疗法的重要工具。