In situ proteomics for brain using genetically encoded probes.

总结 这一探索性的建议将发展一种交叉遗传学方法， 基于诱导的生物素连接酶断裂片段的哺乳动物脑蛋白质组学 二聚化。目前的蛋白质组数据库从高通量 在非生理环境中的实验，通常涉及高表达， 高度转化的细胞系或体外系统。发展一种更符合生理的方法 关键蛋白质网络的遗传复杂的疾病，我们将适应分裂生物素 连接酶法用于小鼠脑中原位诱导邻近标记我们将标记一个 目的内源性蛋白质与一个分裂生物素连接酶片段 在小鼠中进行编辑，并使用遗传杂交将这种融合基因与 从不同的基因座表达的互补分裂片段。二聚化以形成 活性酶通过重叠表达和雷帕霉素施用来控制。 这将使我们能够识别感兴趣的蛋白质的生理网络， 上下文作为初步的证明，我们将标记内源性Zfp804a，其人 同源基因是由一个复制良好的精神疾病风险基因座编码的。同时，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 亚细胞区室，并将方法扩展到其他神经发育风险 基因座和Zfp804a伴侣蛋白。

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.