A suite of conditional mouse models for secretome labeling

项目摘要。分泌的蛋白质和多肽介导细胞和组织的基本轴 相声近年来，人们重新对分泌蛋白的整个集合进行分析 和蛋白质（例如，分泌物组）从体内细胞和器官中分离。为了应对这一挑战，我们最近 描述了使用腺相关病毒（AAV）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。我们的方法提供了一个直接的生化读出分泌组的组成和动力学 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 小鼠模型的基础上，这种分泌组分析方法。这个应用程序和我们提出的一套 条件分泌体标记小鼠直接响应资助机会公告PAR-19-369 鼓励开发改良的动物模型和新技术，以更好地了解 健康和疾病。这些条件性小鼠的发育很重要，因为许多细胞类型仍然不能发育。 有效或定量地用AAV转导，或快速地被翻转，使得瞬时AAV感染 并不导致这些细胞群体的稳定标记。在具体目标1中，我们将生成三个 用定位于分泌途径、胞质溶胶或胞浆的TurboID标记条件性分泌组的小鼠系， 膜，并使用鸟枪蛋白质组学来基准他们的表现相对于我们的第一代病毒 试剂在具体目标2中，我们将使用这些条件小鼠来解决一个基本的和明确的问题。 一个以前在实验上无法解决的问题：在多大程度上， 体外转录确定的蛋白质？成功完成此提案将为调查人员提供 有条件的动物模型来回答以前无法理解的基本问题， 实验性细胞和组织串扰中介导细胞和组织串扰分泌蛋白和多肽因子的鉴定 兴趣系统。

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.