In vivo biotinylation for analysis of nuclear and protein dynamics

描述（由申请人提供）：识别与人类健康相关的分子不仅可以提高新的治疗方法的机会，而且可以揭示对潜在疾病的病因学的关键见解。脊椎动物的前向遗传筛选极大地帮助了我们对生物过程和疾病的理解，我们的目标是扩大斑马鱼群落筛选对发育和功能至关重要的成分的能力。我们建议采用两种新的筛选方法“BioID”和“隔离细胞核标记在特定细胞类型”（完好无损）用于斑马鱼。这两种技术都涉及在处理样品之前在体内发生的细胞核或蛋白质的组织特异性生物素化，并且这两种技术都比目前的方法有几个优点。对于BioID方法，我们将生成Tol2 Gateway载体的版本，可以容纳任何感兴趣的启动子和/或蛋白质。对于完好无损的方法，我们的目标是产生稳定的转基因系，使社区能够使用已经可用的Gal4系作为实现组织特异性的手段。我们还将为那些希望使用特定启动子的实验室生成一个稳定的、无处不在表达生物素连接酶的细胞系。对于这两种方法，我们将使用斑马鱼胚胎和幼虫作为纯化和鉴定新因子的材料来源，为体内后表达步骤制定简单的方案。

DESCRIPTION (provided by applicant): The identification of molecules relevant to human health not only improves the chances for new therapeutic approaches, but can also reveal critical insight into the etiology underlying disease. Forward genetic screens in vertebrates have greatly aided in our understanding of biological processes and disease, and we aim to expand the ability of the zebrafish community to screen for components critical to development and function. We propose to adapt two new screening methods 'BioID' and 'Isolation of Nuclei TAgged in specific Cell Types' (INTACT) for use with zebrafish. Both techniques involve tissue-specific biotinylation of nuclei or proteins that occurs in vivo before processing of samples, and both techniques offer several advantages over current methods. For the BioID method, we will generate versions of the Tol2 Gateway vector that can accommodate any promoter and/or protein of interest. For the INTACT method, our aim is to generate stable transgenic lines that would enable the community to use Gal4 lines already available as a means of achieving tissue specificity. We will also generate a stable line that ubiquitously expresses the biotin ligase for those laboratories that wish to use a specific promoter. For both approaches, we will develop simple protocols for post-in vivo expression steps using zebrafish embryos and larvae as the source of material for purification and identification of novel factors.