Core C: Cell Phenotyping and Molecular Imaging Core

核心 C：细胞表型和分子成像核心

基本信息

批准号：
10597206
负责人：
Jan M Skotheim
金额：
$ 23.44万
依托单位：
STANFORD UNIVERSITY
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：
2022-03-25 至 2027-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10597206
关键词：
Applications Grants Biochemical Biochemistry CRISPR/Cas technology Cancer Control Cell Cycle Cell Cycle Progression Cell Line Cell division Cells Cellular Assay Communities Computer software Cyclin D1 Data Dedications Development Dissociation Dyes Feedback Fluorescence G1/S Transition Gene Deletion Gene Expression Generations Genes Genetic Genetic study Goals Human Image Image Analysis In Vitro Laboratories Ligation Malignant Neoplasms Mass Spectrum Analysis Measurement Measures Methods Microscope Microscopy Mitotic Cell Cycle Molecular Mutate Mutation Pathway interactions Phenotype Phosphorylation Site Process Protein Overexpression Proteins Regulation Research Assistant Resolution Rest Science Site Spectrum Analysis System Technology Testing Therapeutic Time digital repositories experience experimental study fusion gene genome editing imaging facilities imaging software improved in vivo interest live cell imaging molecular imaging mutant novel programs protein protein interaction single cell analysis terabyte

项目摘要

PROJECT SUMMARY The goal of this program grant proposal is a better mechanistic understanding of the core Cyclin D-Cdk4/6-Rb- E2F pathway regulating the G1/S transition controlling cell division. The overarching hypothesis of the program is that an increased mechanistic understanding of G1/S control will provide fundamental advances in understanding the control of human cell division to facilitate development of novel cancer therapeutics. Our project aims all use biochemistry, genetics, and mass spectrometry to generate hypotheses for molecular mechanisms controlling interactions in the Cyclin D-Cdk4/6-Rb-E2F pathway. To test these hypotheses in vivo, all projects will rely on the cell phenotyping and molecular imaging core. Imaging is a key technology for testing molecular mechanisms in live cells. In particular, time lapse single cell analysis is the gold standard for assessing effects of mutations on cell cycle progression at the G1/S transition. In addition to time lapse imaging, Core C will provide access to fluorescence cross correlation spectroscopy methods (FCS and FCCS) that allow the measurement of protein concentrations in molar units, and the measurement of the dissociation constants of specific proteins in live cells (e.g., Rb and E2F). These capabilities will allow definitive testing of biochemical mechanisms for regulation along the Cyclin D-Cdk4/6-Rb-E2F axis generated by our in vitro and genetic studies in each Project. To serve the Projects, this core aims to 1) use CRIPSR/Cas9 genome editing to generate mutant cell lines; 2) image gene expression, cell growth, and cell cycle transition dynamics at single cell resolution; and 3) build and maintain software that analyzes the terabytes of time lapse imaging data. These cell phenotyping analyses will feedback and refine our mechanistic hypotheses, which we expect will lead to further genome editing and testing.

项目概要该计划拨款提案的目标是更好地理解核心 Cyclin D-Cdk4/6-Rb- E2F 通路调节 G1/S 转变控制细胞分裂。该计划的总体假设增加对 G1/S 控制的机械理解将带来根本性的进步了解人类细胞分裂的控制，以促进新型癌症疗法的开发。我们的项目目标全部使用生物化学、遗传学和质谱来生成分子假设控制 Cyclin D-Cdk4/6-Rb-E2F 通路中相互作用的机制。为了在体内测试这些假设，所有项目都将依赖于细胞表型分析和分子成像核心。成像是检测的关键技术活细胞中的分子机制。特别是，延时单细胞分析是评估的黄金标准突变对 G1/S 期细胞周期进展的影响。除了延时成像之外，Core C 将提供荧光互相关光谱方法（FCS 和 FCCS），允许以摩尔单位测量蛋白质浓度，以及测量解离常数活细胞中的特定蛋白质（例如，Rb 和 E2F）。这些功能将允许对生化进行明确的测试我们的体外和遗传学研究产生的 Cyclin D-Cdk4/6-Rb-E2F 轴调节机制在每个项目中。为了服务于该项目，该核心目标是 1) 使用 CRIPSR/Cas9 基因组编辑来生成突变体细胞系； 2) 以单细胞分辨率对基因表达、细胞生长和细胞周期转变动态进行成像；和 3) 构建和维护用于分析 TB 级延时成像数据的软件。这些细胞表型分析将反馈并完善我们的机制假设，我们预计这将导致进一步的基因组研究编辑和测试。