Understanding Cell Division

摘要 本设备申请补充的家长拨款(1R35GM126980)有权 “了解细胞分裂。”该奖项涵盖了 戈尔布斯基实验室，分析染色体运动的机制，调节 有丝分裂细胞周期的研究，以及影响细胞分裂的因素和条件的特征 导致染色体不稳定。我们的大部分研究涉及高分辨率成像 表达荧光标记蛋白的培养细胞和RNA的操纵 干扰或CRISPR基因敲除和突变。Mizar Tilt灯片提供 高分辨率成像，光毒性最小。实验室中使用的方法 询问蛋白质功能包括siRNA或将degron/GFP标签敲入内源 基因。我们还使用了“修剪”技术，通过这种技术， 抗体和Trim13 E3泛素连接酶显微注射到活细胞中，可以快速 在没有遗传操作的情况下内源蛋白质的降解。所请求的 升级到我们的Mizar Tilt高分辨率光片，最近推出的Tilt-RM 共振镜，显微镜将减少光片上的固有缺陷 显微镜。这一改进将极大地增强系统的成像能力， 尤其是用于定量荧光测量。

Summary The parent grant (1R35GM126980) for this equipment request supplement is entitled “Understanding Cell Division.” This award covers the primary research focus of the Gorbsky laboratory, analyzing the mechanics of chromosome movement, the regulation of the mitotic cell cycle, and the characterization of factors and conditions that contribute to chromosome instability. Much of our research involves high resolution imaging of cultured cells expressing fluorescently tagged proteins and are manipulated with RNA interference or CRISPR gene knockout and mutation. The Mizar Tilt light sheet provides high resolution imaging with minimal phototoxicity. The approaches used in the lab to interrogate protein function include siRNA or knocking degron/GFP tags into endogenous genes. We are also using the “Trim-away” technique, through which a combination of antibody and Trim13 E3 ubiquitin ligase, microinjected into living cells, allows rapid degradation of endogenous proteins without genetic manipulations. The requested upgrade to our Mizar Tilt high resolution light sheet, the recently introduced Tilt-RM resonance mirror, microscope will reduce “striping” an inherent defect in light sheet microscopy. This modification will greatly enhance the imaging capabilities of the system, particularly for quantitative fluorescence measurements.