A suite of conditional mouse models for secretome labeling

一套用于分泌蛋白组标记的条件小鼠模型

• 批准号: 10640784
• 负责人: Jonathan Z Long
• 金额: $ 19.82万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10640784
• 关键词: Address; Albumins; Animal Model; Animals; Benchmarking; Biochemical; Biocompatible Materials; Biology; Biotin; Biotinylation; Cell Compartmentation; Cells; Chemicals; Collection; Communication; Communities; Complex; Cytosol; Data; Dependovirus; Deposition; Development; Disease; Dose; Endocrine; Environment; Enzymes; Equipment and supply inventories; Event; Fasting; Foundations; Funding Opportunities; Generations; Genetic Recombination; Genetic Transcription; Goals; Growth Factor; Health; Hormones; Human; In Vitro; Label; Laboratories; Liver; Mammals; Maps; Measurement; Measures; Mediating; Membrane; Messenger RNA; Methodology; Methods; Molecular; Mus; Nature; Organ; Pathway interactions; Performance; Physiological; Physiology; Plasma; Population; Process; Protein Secretion; Proteins; Proteome; Proteomics; Protocols documentation; Reagent; Regulation; Research; Research Personnel; Resolution; Role; Shotguns; Signal Pathway; Specificity; Streptavidin; System; Technology; Testing; Tissues; Validation; Viral; Virus Diseases; animal model development; body system; cell type; extracellular; human disease; improved; in vivo; innovation; intercellular communication; interest; intraperitoneal; mouse model; new technology; paracrine; polypeptide; public repository; subcellular targeting; tool; transduction efficiency

PROJECT SUMMARY. Secreted proteins and polypeptides mediate fundamental axes of cell and tissue crossltalk. In recent years, there has been a renewed interest in profiling the entire collection of secreted proteins and proteins (e.g., the secretome) from cells and organs in vivo. To address this challenge, we have recently described a high-sensitivity proximity labeling methodology using adeno-associated viruses (AAVs) to delivery the proximity labeling enzyme TurboID into distinct subcellular compartments for cell type-specific secretome profiling in vivo. Our methodology provides a direct biochemical readout of secretome composition and dynamics in a cell type-specific manner, directly in a living animal. Here, we will generate and validate a suite of conditional mouse models based on this secretome profiling methodology. This application and our proposed suite of conditional secretome labeling mice directly responds to the Funding Opportunity Announcement PAR-19-369 which encourages the development of improved animal models and novel technologies to better understand health and disease. These conditional mice are important to develop because many cell types still cannot be transduced efficiently or quantitatively with AAVs, or are rapidly turned over such that transient AAV infection does not result in stable labeling of those cellular populations. In Specific Aim 1, we will generate three conditional secretome labeling mouse lines with TurboID localized to the secretory pathway, cytosol, or membrane and use shotgun proteomics to benchmark their performance relative to our first-generation viral reagents. In Specific Aim 2, we will use these conditional mice to address a fundamental and well-defined question that had previously remained inaccessible experimentally: to what extent are the levels of secreted proteins determined transcriptionally in vitro? Successful completion of this proposal will provide investigators with a conditional animal model to answer previously inaccessible and fundamental questions regarding the identity of secreted protein and polypeptide factors mediating cell and tissue crosstalk in their experimental system of interest.

项目摘要。分泌的蛋白质和多肽介导细胞和组织的基本轴 串扰。近年来，人们对分析整个​​分泌蛋白集合产生了新的兴趣 以及来自体内细胞和器官的蛋白质（例如分泌组）。为了应对这一挑战，我们最近 描述了一种使用腺相关病毒 (AAV) 进行递送的高灵敏度邻近标记方法 邻近标记酶 TurboID 进入不同的亚细胞区室，用于细胞类型特异性分泌组 体内分析。我们的方法提供了分泌蛋白组组成和动力学的直接生化读数 以细胞类型特异性的方式，直接在活体动物中进行。在这里，我们将生成并验证一套条件 基于这种分泌蛋白组分析方法的小鼠模型。该应用程序和我们建议的套件 条件分泌蛋白组标记小鼠直接响应资助机会公告PAR-19-369 鼓励开发改进的动物模型和新技术，以更好地理解 健康和疾病。这些条件小鼠对于发育很重要，因为许多细胞类型仍然无法 用 AAV 有效或定量转导，或快速翻转，从而导致短暂的 AAV 感染 不会导致这些细胞群的稳定标记。在具体目标 1 中，我们将生成三个 使用 TurboID 定位于分泌途径、胞质溶胶或 膜并使用鸟枪蛋白质组学来衡量其相对于我们第一代病毒的性能 试剂。在具体目标 2 中，我们将使用这些条件小鼠来解决基本且明确的问题 以前通过实验仍然无法解决的问题：分泌的水平在多大程度上 体外转录确定的蛋白质？成功完成该提案将为研究人员提供 用有条件的动物模型来回答以前无法理解的基本问题 实验中介导细胞和组织串扰的分泌蛋白和多肽因子的身份 利益系统。