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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.

摘要这一探索性的提议将发展一种原位遗传学的交叉方法。基于生物素连接酶裂解片段的哺乳动物脑蛋白质组学研究小鼠的二聚化。目前的蛋白质组数据库从高通量提取在非生理性环境中的实验，通常涉及在高度转化的细胞系或体外系统。开发一种更具生理学意义的方法对于基因复杂疾病的关键蛋白质网络，我们将改造一种分裂的生物素小鼠脑内诱导邻近原位标记的连接酶方法。我们将标记一个利用生殖系分离的生物素连接酶的一段内源目的蛋白在小鼠身上进行编辑，并使用遗传杂交将该融合基因与从一个不同的基因座表达的互补分裂片段。二聚化反应形成活性酶受重叠表达和雷帕霉素给药的控制。这将使我们能够识别感兴趣的蛋白质在生理上的物理网络背景。作为初始演示，我们将标记内源性Zfp804a，其人类同源基因由一个复制良好的精神疾病风险基因编码。同时，我们将开发基于细胞的模型来改进方法，将结果集中在选定的亚细胞隔区，并将该方法扩展到其他神经发育风险基因座和Zfp804a配对蛋白。