Mapping proteostasis in the lung

绘制肺部蛋白质稳态图

• 批准号: 9751146
• 负责人: Harris R Perlman
• 金额: $ 28.43万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9751146
• 关键词: Acute Lung Injury; Aging; Alveolar; Alveolar Macrophages; Animal Model; Animals; Attention; Biological Assay; Blinded; Bone Marrow; Breeding; Cell Culture Techniques; Cell Line; Cell Proliferation; Cells; Core Facility; Data; Disease; Ensure; Enterobacteria phage P1 Cre recombinase; Epithelial; Epithelial Cells; Fibroblasts; Flow Cytometry; Funding Opportunities; Gene Expression; Gene Expression Profiling; Generations; Genes; Genotype; High-Throughput Nucleotide Sequencing; Human; Human Resources; Immunologics; Inflammatory; Influenza A virus; Instruction; Intervention; Knock-out; Knockout Mice; Lung; Lung Inflammation; Mass Spectrum Analysis; Measurement; Methods; Modeling; Mus; Muscle; Muscle Cells; Muscle Fibers; Muscle function; Muscle satellite cell; Outcome Measure; Pathway interactions; Phenotype; Pneumonia; Population; Protein Isoforms; RNA analysis; Reagent; Reproducibility; Research; Research Personnel; Resolution; Resources; Sampling; Secondary Cell Culture; Skeletal Muscle; Sorting - Cell Movement; Standardization; Techniques; Technology; Tissues; Transcript; Translating; Universities; base; cell type; cost; cost effectiveness; experience; experimental study; indexing; influenza A pneumonia; interest; knockout animal; lung injury; macrophage; mouse model; next generation sequencing; novel; programs; proteostasis; satellite cell; synergism; transcriptome; transcriptome sequencing

Project Summary The Lung Phenotyping Core, designated as Core C, is a centralized facility that provides primary murine alveolar epithelial cells and macrophages from lungs, as well as satellite cells and muscle to each of the three projects and Core B. Core C also maintains secondary lung cell lines for the PPG. Centralization of the cell culture facilities will ensure that continuous supply of high-quality primary alveolar epithelial cells are available to each of the three projects and Core B. Core C will use advanced flow cytometry capabilities to quantify, phenotype and sort specific inflammatory and parenchymal cell populations from the murine lung. The sorting and isolation capabilities of the Core will allow cell type specific assessment of proteostasis networks via mass spectroscopy (as outlined by Core B). Core C will also isolate muscle satellite cells from mouse skeletal muscles; we have established the optimal culture conditions for cell proliferation and myotube formation of mouse satellite cells. The Core personnel have extensive experience in the isolation of primary alveolar epithelial cells and macrophages from human and mouse lungs, as well as general cell culture techniques. The consolidated cell culture facility provides an economical means of isolating and culturing primary and secondary cells. This translates into reduced overall costs (i.e., personnel, reagents, and animals) and more importantly maintains the quality of the cells used in research. The Core will also provide investigators with well-characterized mouse models of influenza A-induced pneumonia and quantitative measurements of lung inflammation, lung injury, and muscle function will be conducted by Core C according to the research plan outlined in Projects 1, 2, and 3. In addition, Core C will conduct RNA analysis to assess the relationship between specific gene expression and proteostasis using the Northwestern University Next-Generation Sequencing (NGS) Core Facility. High throughput sequencing technology will be used to study transcriptome dynamics. Additionally, the project investigators have proposed several murine strains, many of which will be bred with cell-type or tissue specific Cre recombinase lines to induce a tissue-specific knockout by Core C. Many of the strains will be used in more than one project, highlighting the synergy and economies of scale achievable by this Program Project.

项目概要 肺表型核心，指定为核心 C，是一个集中设施，提供初级小鼠 来自肺部的肺泡上皮细胞和巨噬细胞，以及这三个细胞中的每一个的卫星细胞和肌肉 项目和核心 B。核心 C 还维护 PPG 的次级肺细胞系。细胞的集中化 培养设施将确保持续供应高质量的原代肺泡上皮细胞 三个项目和核心 B 中的每一个。核心 C 将使用先进的流式细胞术功能来量化， 对小鼠肺部的特定炎症和实质细胞群进行表型分析和分类。排序 核心的分离能力将允许通过质量对蛋白质稳态网络进行细胞类型特异性评估 光谱（如核心 B 概述）。 Core C 还将从小鼠骨骼中分离出肌肉卫星细胞 肌肉；我们已经建立了细胞增殖和肌管形成的最佳培养条件 小鼠卫星细胞。核心人员在原发肺泡分离方面拥有丰富的经验 来自人和小鼠肺部的上皮细胞和巨噬细胞，以及一般细胞培养技术。这 综合细胞培养设施提供了一种经济的方法来分离和培养原代细胞和 二次细胞。这意味着总体成本（即人员、试剂和动物）的降低等等 重要的是保持研究中使用的细胞的质量。核心还将为调查人员提供 甲型流感引起的肺炎的良好表征的小鼠模型和肺的定量测量 炎症、肺损伤、肌肉功能等将由Core C根据研究计划进行 项目 1、2 和 3 中概述。此外，Core C 将进行 RNA 分析以评估关系 使用西北大学下一代技术在特定基因表达和蛋白质稳态之间进行研究 测序 (NGS) 核心设施。高通量测序技术将用于转录组研究 动力学。此外，项目研究人员还提出了几种小鼠品系，其中许多将被 用细胞类型或组织特异性 Cre 重组酶系培育，以诱导 Core C 进行组织特异性敲除。 许多菌株将用于多个项目，凸显协同效应和规模经济 本计划项目可实现。