A suite of conditional mouse models for secretome labeling

项目总结。分泌的蛋白质和多肽调节细胞和组织的基本轴 相声。近年来，对整个分泌蛋白质集合的分析重新引起了人们的兴趣 以及体内细胞和器官中的蛋白质(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进入细胞类型特异性分泌组的不同亚细胞室 在活体内进行分析。我们的方法学提供了分泌组组成和动力学的直接生化读数。 以特定细胞类型的方式，直接在活着的动物体内。在这里，我们将生成并验证一套条件 基于这种分泌组分析方法的小鼠模型。此应用程序和我们建议的套件 有条件的分泌组标记小鼠直接响应资助机会公告PAR-19-369 它鼓励改进的动物模型和新技术的发展，以更好地了解 健康和疾病。这些有条件的小鼠对发展很重要，因为许多细胞类型仍然不能 用AAVs有效地或定量地转导，或迅速翻转，从而使一过性AAV感染 不会导致对这些细胞群体进行稳定的标记。在具体目标1中，我们将生成三个 用TurboID定位于分泌途径、胞浆或 并使用鸟枪式蛋白质组学来测试它们相对于我们的第一代病毒的性能 试剂。在具体目标2中，我们将使用这些条件鼠标来解决基本的和定义明确的 以前在实验中仍然无法解决的问题：在多大程度上分泌了 体外转录测定的蛋白质？这项提案的成功完成将为调查人员提供 用一个有条件的动物模型来回答以前无法接触到的关于 分泌蛋白和多肽因子在其实验中介导细胞和组织串扰的一致性 利益体系。

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.