In situ proteomics for brain using genetically encoded probes.

使用基因编码探针进行大脑原位蛋白质组学。

基本信息

批准号：
10576153
负责人：
BRUCE A HAMILTON
金额：
$ 23.7万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
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：
2022-09-20 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10576153
关键词：
Anatomy Avidity Base of the Brain Biological Process Biotin Bipolar Disorder Brain Brain region Cell Culture Techniques Cell Line Cell Nucleus Cell model Cells Chimeric Proteins Complex Cytoplasm Databases Development Dimerization Disease Enzymes Foundations Genes Genetic Genetic Crosses Genetic Risk Genetic Transcription Genetic Translation Health Human In Situ In Vitro Investigation Label Ligands Ligase Mental disorders Messenger RNA Methods Modeling Molecular Mus Neurodevelopmental Disorder Neurons Peptides Physiological Population Proteins Proteomics RNA Regulation Reporting Risk Factors Role Schizophrenia Sensitivity and Specificity Sirolimus Site Specificity Synapses System Testing Tissues Transformed Cell Line Work Zinc Fingers base cell type combinatorial design developmental toxicity experimental study fusion gene genetic approach genetic regulatory protein genome editing genome-wide interest neuropsychiatric disorder protein transport risk variant translational potential

项目摘要

Summary This exploratory proposal will develop an intersectional genetics approach to in situ proteomics in a mammalian brain based on a split biotin ligase fragments with induced dimerization in mice. Current proteomic databases draw from high-throughput experiments in non-physiological settings that typically involve high expression in a highly transformed cell line or in vitro system. To develop a more physiological approach to key protein networks for genetically complex disorders, we will adapt a split biotin ligase approach for inducible proximity labeling in situ in mouse brain. We will tag an endogenous protein of interest with one fragment of the split biotin ligase by germline editing in mice and use genetic crosses to pair this fusion gene with one for the complementary split fragment expressed from a distinct locus. Dimerization to form the active enzyme is controlled by overlapping expression and rapamycin administration. This will allow us to identify the protein of interest’s physical network in physiological context. As an initial demonstration we will tag endogenous Zfp804a, whose human cognate is encoded by a well-replicated risk locus for psychiatric illness. In parallel, we will develop cell-based models to refine the approach, focus results on selected subcellular compartments, and extend the approach to other neurodevelopmental risk loci and Zfp804a partner proteins.

概括该探索性提案将开发出一种相互遗传学方法来原位哺乳动物大脑中的蛋白质组学基于诱导的分裂生物素连接酶片段小鼠的二聚化。当前的蛋白质组学数据库从高通量在非生理环境中的实验，通常涉及A 高度转化的细胞系或体外系统。开发更物理的方法对于遗传复杂疾病的关键蛋白质网络，我们将适应分裂的生物素小鼠大脑中原位的诱导近接近标记的连接酶方法。我们将标记感兴趣的内源性蛋白质，分裂生物素连接酶的一个片段在小鼠中进行编辑，并使用遗传杂交将此融合基因与一个融合基因配对完全从不同的基因座表达的完全分裂的片段。二聚化以形成活性酶通过重叠的表达和雷帕霉素的给药来控制。这将使我们能够在生理中识别感兴趣的物理网络语境。作为初始演示，我们将标记内源性ZFP804A，其人类 Cognate是由精神病的良好风险基因座编码的。并行，我们将开发基于细胞的模型来完善方法，将结果集中在选定的结果上亚细胞隔室，并将方法扩展到其他神经发育风险 LOCI和ZFP804A合作蛋白。