Timing live cell cycle length in diverse tissues

不同组织中活细胞周期长度的计时 摘要 细胞动力学是组织动态平衡的基础，它的异常定义了许多疾病状态。计时 细胞形成组织的速度最需要知道细胞的世代时间或细胞周期速度 方便地定义为两个连续有丝分裂之间的时间间隔。细胞周期速度的测定 因为深部组织中的细胞已经依靠S时相的活动来整合脉冲标记，或细胞分裂介导的- 饱和标签的稀释。因此，细胞周期比率只能作为群体平均数来计算 固定，或在标签被稀释到可检测范围内的特定水平之后，以及其他限制。 单个活细胞中能够分离它们的细胞周期速度的瞬时读数已经失效 伸手。响应肾、泌尿外科、肾内科催化工具和技术发展的号召 血液病，我建议开发一种新的基因编码的活细胞周期速度报告器。小白鼠 将建立表达新报告基因的菌株，用于检测广泛的细胞周期速度 荧光，原位或通过流式细胞术，以催化对不同组织中细胞动力学的研究。 我们将在最近成功开发出第一个活细胞周期速度记录器的基础上，通过 利用变色蛋白的不同半衰期，荧光计时器(FT)。我们选择了动力学 一种变体，当新合成约1.2小时后发出蓝色荧光，然后转化为红色 蛋白质在成熟过程中永久存在。以核心组蛋白H2 B融合蛋白的形式表达，细胞周期长度 单个细胞可以通过两种荧光之间的比率来确定：细胞周期越快，越蓝 此时将出现一个单元格。虽然这位第一位记者展示了原则证明和出色的表现 分解较短的细胞周期，如红系祖细胞和髓系祖细胞， 巨大的细胞类型以较慢的速度分裂是无法解决的。通过这项提案，我们将测试新的设计 功能，从而可以方便地获得与体内大多数哺乳动物细胞类型相关的活细胞周期速度 由单个基因编码的报告构建体H2 B-FTmHaloD2确定。考虑到自然存在的 在造血组织中广泛的细胞周期速度和我们自己在研究它方面的专业知识，我们将使用 将基线和损伤期间的造血干/祖细胞作为模型细胞类型进行测试、校准和 标准化工作流，了解如何使用新的比率测量仪来确定细胞动力学并进行排序 来自不同组织的活细胞。H2B-FTmHaloD2的新设计特征也应该很容易解决 缓慢的细胞周期存在于肾脏等固体组织中。

Timing live cell cycle length in diverse tissues Abstract Cellular dynamics underly tissue homeostasis and its abnormality defines many disease states. Timing how quickly cells form tissues requires knowing the cellular generational time, or cell cycle speed, most conveniently defined as the time interval between two consecutive mitoses. Determination of cell cycle speed for cells in deep tissues has relied on S-phase activity to incorporate pulsed labels, or cell division mediated- dilution of saturated labels. Thus, cell cycle rates have only been calculatable as a population average upon fixation, or after the label has been diluted to a certain level within the detectible range, among other limitations. An instantaneous readout of cell cycle speed in individual live cells that enable their isolation has been out of reach. In response to the call of Catalytic Tool and Technology Development in Kidney, Urologic, and Hematologic Diseases, I propose to develop new genetically encoded live cell cycle speed reporter. Mouse strain expressing the new reporter will be established for detecting a wide range of cell cycle speed by fluorescence, in situ or by flow cytometry, to catalyze the research on cellular dynamics in diverse tissues. We will build on our recent success in developing a first-of-its-kind live cell cycle speed reporter by exploiting the differential half-life of a color changing protein, the fluorescent timer (FT). We chose the kinetic variant that emits blue fluorescence when newly synthesized for ~1.2 hours, before converting into a red protein permanently during maturation. Expressed as a fusion protein to core histone H2B, cell cycle length of individual cells can be determined by the ratio between the two fluorescence: the faster the cell cycle, the bluer a cell appears. While this first reporter demonstrated the proof-of-principle and exceptional performance in resolving short cell cycles, such as those of the erythroid progenitors and myeloid-committed progenitors, the vast cell types dividing at slower rates were not resolvable. Through this proposal, we will test new design features so that the live cell cycle speed relevant for most mammalian cell types in vivo can be conveniently determined with a single genetically encoded reporter construct, H2B-FTmHaloD2. Given the naturally existing wide range of cell cycle speed in the hematopoietic tissues and our own expertise in studying it, we will use the hematopoietic stem and progenitor cells at baseline and during injury as model cell types to test, calibrate and standardize workflows for how a new ratiometric reporter can be used to determine cellular dynamics and sort for live cells from diverse tissues. The new design features of the H2B-FTmHaloD2 should also readily resolve the slow cell cycles present in solid tissues such as the kidney.