Microtubule dynamics and error correction

项目摘要 该项目要求资金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的具体目标， NIH资助R01GM145567。本项目的主要目标是了解 驱动蛋白相关马达和动力学的力产生的机制贡献 微管与染色体分离。为了实现这些目标， CRISPR细胞具有荧光标记的驱动蛋白马达，可以快速重新定位到 细胞的不同区域。我们已经发现，这些蛋白质的功能分析， 细胞比更常用的转染更具有生理相关性 和表达方法。这部分是因为蛋白质在适当的 部分原因是由于没有内源性污染活性， 蛋白质，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， 相关水平的蛋白质在细胞中成像具有挑战性。我们的隔离和分析 这些细胞是在显微镜下进行的， 设备.为了分离和功能评估这些细胞，我们使用定制修改的 高分辨率、光敏GE Healthcare显微镜系统。可惜 以前为其服务合同提供保险的公司（GE Healthcare/Cytiva）不再提供保险 是一家显微镜公司。因此，没有部件可用于覆盖仪器 失败截至2022年9月12日，仪器控制器出现故障，使我们无法获得钥匙 仪器强调了我们的生产力活细胞成像。我们设计了一种方法 把我们系统的工作部件拆下来，然后把联合收割机和一个最小的 尼康CREST X-Light V3旋转共焦光盘。该系统将有两个 优点：1）它将恢复我们的成像能力，因此，我们的科学 生产力; 2）它将使我们能够对扁平培养细胞和胚胎细胞进行成像 斑马鱼这将为我们提供额外的能力来调查 低表达的驱动蛋白（如Kif9和Kif25）对生物体的贡献 开发，根据我们批准的Danio rerio范围添加变更，并恢复我们的 分离和评估CRISPR工程化细胞系的能力。

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.