Microtubule dynamics and error correction

微管动力学和误差校正

基本信息

批准号：
10775393
负责人：
Linda Wordeman
金额：
$ 24.99万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-07 至 2026-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10775393
关键词：
Address Animal Model Biological Assay Biological Sciences CRISPR/Cas technology Cell Aging Cell Death Cell Line Cells Centrosome Chromosomal Instability Chromosome Segregation Chromosomes Classification Clustered Regularly Interspaced Short Palindromic Repeats Congresses Contract Services Coupled Cultured Cells Custom Decision Making Defect Development Disadvantaged Embryo Engineering Enhancers Equipment Failure Fluorescence Funding Generations Genes Geometry Goals Grant Human Image Impairment Kinesin Kinetochores Light MXD1 gene Marketing Methods Microscope Microscopic Microscopy Microtubules Mitosis Mitotic Modification Motor Pathway interactions Pharmaceutical Preparations Pharmacology Phosphotransferases Physiological Positioning Attribute Predisposition Process Productivity Proteasome Inhibitor Protein Analysis Proteins Recombinant Transgenes Resistance Resolution Role System Technology Testing Time Transfection Tubulin United States National Institutes of Health Vertebrates Visualization Zebrafish cellular imaging cost effective high resolution imaging imaging capabilities improved instrument live cell imaging mutant neurodevelopment novel overexpression small molecule targeted agent tool tumorigenesis zebrafish development

项目摘要

Project Summary This project requests funding to upgrade equipment to clone and analyze CRISPR/Cas9 transgenic recombinant cells. These cells are required to address the Specific Aims in NIH grant R01GM145567. The broad goal of this project is to understand the mechanistic contribution of force generation by kinesin-related motors and dynamic microtubules to chromosome segregation. To accomplish these goals we engineer CRISPR cells with fluorescently tagged kinesin motors that can be rapidly relocated to diverse regions of the cell. We have found that functional analysis of proteins in these cells is much more physiologically relevant than the more commonly used transfection and expression methods. This is in part because the proteins are at the appropriate level in the cell and in part because there is no contaminating activity from endogenous protein because the modifications for visualization and experimental manipulation are performed on the endogenous gene. One disadvantage is that the physiologically relevant levels of protein is challenging to image in cells. Our isolation and analysis of these cells is performed microscopically and critically dependent on our imaging equipment. To isolate and functionally evaluate these cells we use a custom-modified high resolution, light sensitive GEHealthcare microscopy system. Unfortunately, the company that previously covered its service contract (GEHealthcare/Cytiva) no longer exists as a microscope company. Thus, there are no parts available to cover instrument failures. As of 9/12/22 the instrument controller has failed leaving us without the key instrument underscoring our productivity for live cell imaging. We have devised a means to cannibalize the working parts of our system and combine it with a minimal version of the Nikon CREST X-Light V3 spinning disk confocal. This system will have two advantages: 1) it will reinstate our imaging capabilities and, accordingly, our scientific productivity; and 2) it will allow us to image both flat cultured cells and embryonic zebrafish. This will provide us with both additional capability to investigate the contribution of poorly expressed kinesins (such as Kif9 and Kif25) to organismal development, per our approved change of scope addition of Danio rerio, and restore our ability to isolate and evaluate CRISPR-engineered cell lines.

项目摘要该项目要求资助升级设备以克隆和分析CRISPR/CAS9 转基因重组细胞。这些细胞需要解决特定目标 NIH授予R01GM145567。该项目的广泛目标是了解动力蛋白相关电动机和动态的力量产生力的机械贡献微管与染色体分离。为了实现这些目标，我们要设计带有荧光标记的驱动蛋白电动机的CRISPR单元，可以迅速迁移到细胞的不同区域。我们发现这些蛋白质的功能分析细胞在生理上比更常用的转染更重要和表达方法。这部分是因为蛋白质是适当的细胞中的水平，部分是因为内源性没有污染活性蛋白质是因为可视化和实验操作的修改是在内源基因上进行。一个缺点是生理上相关蛋白质的水平对细胞中的图像具有挑战性。我们对这些单元在显微镜和严格的成像上进行设备。为了隔离并在功能上评估这些单元格，我们使用定制修饰高分辨率，光敏性gehealthcare显微镜系统。不幸的是，以前涵盖其服务合同（GeHealthcare/Cytiva）的公司不再存在作为显微镜公司。因此，没有可用的零件可以覆盖仪器失败。截至9/12/22，仪器控制器失败了，使我们没有钥匙仪器强调了我们的生产力成像生产率。我们设计了一种手段蚕食系统的工作部位，并将其与最小版本结合起来 Nikon Crest X-Light V3旋转磁盘共焦。该系统将有两个优点：1）它将恢复我们的成像功能，因此我们的科学生产率; 2）它将允许我们对扁平培养的细胞和胚胎形象成像斑马鱼。这将为我们提供调查的附加功能表达不良的驱动蛋白（例如KIF9和KIF25）对生物的贡献根据我们批准更改Danio Rerio的范围的变更，并恢复了我们的能够分离和评估CRISPR工程细胞系的能力。