Developing an automated yeast dissection system for aging research

项目摘要 用于老化研究的酵母自动解剖系统的开发 老龄化是疾病的最大风险因素，而这些疾病是死亡的主要原因。的目标 衰老研究的目的是发现与衰老相关的关键基因和途径，这些基因和途径最终可能有助于 延缓衰老和延缓与年龄有关的疾病的发作。芽殖酵母 酿酒酵母一直是研究衰老的有力模型，并为我们的研究做出了重大贡献。 了解真核细胞衰老的基本机制。然而，传统的酵母老化试验， 包括显微切割方法，具有技术挑战;例如，这些方法是低通量的， 实验过程是费力的。一个实验通常持续几周或几个月， 将测定的细胞在冰箱中储存过夜以在整个实验过程中暂停复制。 这种繁琐的程序大大阻碍了衰老研究领域的进展。我们已经开发 一个创新的、高效的微流控平台，用于子细胞的连续和自动解剖 而不会干扰母细胞的萌芽。该平台允许自动化的全寿命跟踪， 单酵母细胞的时空分辨率和大规模量化，导致显著减少 劳动力、时间和成本。此外，高分辨率荧光成像的酵母细胞生长在恒定和 动态变化的环境提供了检查基因表达和信号传导的动态的能力 以高吞吐量的方式连接网络。这个平台获取的数据数量和类型是不可能的 与传统的分析方法。在此，我们建议开发自动化解剖平台， 酵母老化的快速和高通量研究。我们将验证所提出的可靠性和能力 平台我们建议验证原型自动解剖平台的两个重要的测定酵母 老化研究：以自动化和无人值守的方式确定突变体的复制寿命， 连续跟踪特定GFP融合蛋白在生长过程中的丰度和细胞定位的变化， 整个衰老过程。该提案的长期计划是将完全自动化的工作台商业化 用于细胞老化的高通量研究的系统。

Project Summary Developing an Automated Yeast Dissection System for Aging Research Aging is the single greatest risk factor for diseases that are principal causes of mortality. The objectives of aging research are to discover key genes and pathways related to aging that may eventually contribute to retardation of aging and a delay in the onset of age-associated diseases. The budding yeast Saccharomyces cerevisiae has been a powerful model for the study of aging and has enabled significant contributions to our understanding of basic mechanisms of aging in eukaryotic cells. However, traditional assays of yeast aging, including microdissection methods, have technical challenges; for instance, the methods are low-throughput and the experimental procedures are laborious. An experiment typically lasts several weeks or months, and requires overnight storage of the assayed cells at a refrigerator to pause replication throughout the course of experiment. This tedious procedure has substantially hindered progress in the field of aging research. We have developed an innovative and highly effective microfluidic platform for continuous and automatic dissection of daughter cells without disturbing mother cells as they bud. The platform allows an automated whole-lifespan tracking with high spatiotemporal resolution and large-scale quantification of single yeast cells, resulting in significant reduction of labor, time, and cost. In addition, the high-resolution florescence imaging of yeast cells grown in constant and dynamically changing environments offers the ability to examine the dynamics of gene expression and signaling networks in a high-throughput manner. The quantity and types of data acquired by this platform are impossible with the traditional assay methods. Herein, we propose to develop automated dissection platform that enable fast and high-throughput studies of yeast aging. We will validate the reliability and capability of the proposed platform We propose to validate the prototype automated dissection platform for two important assays in yeast aging research: determination of replicative lifespan for mutants in an automated and unattended fashion, and continuous tracking the changes in abundance and cellular localization for specific GFP fusion proteins during the entire aging process. Longer term plan of this proposal is to commercialize a fully automation benchtop system for high-throughput studies of cellular aging.