Developing an automated yeast dissection system for aging research

项目摘要 一种用于老化研究的酵母自动解剖系统的研制 老龄化是疾病的最大风险因素，而疾病是死亡的主要原因。的目标 衰老研究是为了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病。芽生酵母菌 酿酒酵母一直是研究衰老的强大模型，并为我们的 了解真核细胞衰老的基本机制。然而，传统的酵母老化检测方法， 包括显微解剖方法，都有技术挑战；例如，这些方法是低通量和 实验程序费时费力。一个实验通常持续几周或几个月，需要 将被检测的细胞在冰箱中过夜保存，以在整个实验过程中暂停复制。 这种繁琐的程序在很大程度上阻碍了衰老研究领域的进展。我们已经开发出 一种创新高效的子细胞连续自动分离微流控平台 而不会在母细胞发芽时干扰它们。该平台支持自动全生命周期跟踪，具有高 单个酵母细胞的时空分辨率和大规模量化，导致显著减少 人力、时间和成本。此外，在恒温和恒温下生长的酵母细胞的高分辨率荧光成像 动态变化的环境提供了检测基因表达和信号的动态变化的能力 以高吞吐量方式部署网络。这个平台获取的数据数量和类型都是不可能的 与传统的检测方法进行比较。在此，我们建议开发自动化解剖平台，使其能够 酵母老化的快速高通量研究。我们将验证建议的可靠性和能力 平台我们建议对原型的自动分离平台进行验证，用于酵母中的两个重要检测 老化研究：以自动和无人值守的方式确定突变体的复制寿命，以及 持续跟踪特定GFP融合蛋白丰度和细胞定位的变化 整个老化过程。这项提议的较长期计划是将全自动化台式计算机商业化 用于高通量细胞老化研究的系统。

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.