Next-generation Drosophila cell lines to elucidate the cellular basis of human diseases

项目摘要 本提案是对《融资机会公告》（PAR-16-369）“资源相关 动物模型和相关材料开发研究项目（R24）。我们的建议涉及 研究目标中规定的目标：“该补助金支持有助于 一个模型系统的知识，使系统更有用，更容易为研究界。 具体来说，我们将产生果蝇细胞系，标记和敲除资源，使细胞 对人类疾病中改变的基因和途径进行生物学分析，包括在疾病相关组织中 血统和遗传背景。近年来，我们目睹了对果蝇细胞的新的兴趣， 现在，果蝇研究人员经常在体内和细胞培养之间来回穿梭， 问题研究然而，果蝇细胞系的全部潜力，特别是用于疾病相关研究的潜力，还没有得到充分发挥。 由于可用的细胞系和标记物的多样性有限，为了克服这个瓶颈，我们 我建议使用令人兴奋的细胞系永生化和基因组工程的新进展， 这些试剂将使社区能够充分利用果蝇细胞系的力量来解决 与疾病相关的细胞生物学问题。具体而言，我们建议：目标1：从 特定的果蝇细胞谱系，这将使相关组织来源的细胞系的筛选和其他研究成为可能; 目的2：使用CRISPR基因组工程产生表达荧光标记物的细胞系资源 的亚细胞成分，这将使社会执行屏幕探索细胞器和细胞 目标3：使用CRISPR工程产生敲除细胞 用于合成致命筛选和其他应用的生产线。我们将传播信息和资源， 通过向社区提供详细的方案和质粒载体来创建细胞疾病模型， 以及新的细胞系和衍生物。重要的是，由于果蝇是一个已建立的研究系统， 由于人类疾病的多样性，我们将开发的资源和工具几乎与所有NIH研究所相关。 最后，我们记录了这些资源将有好处超越果蝇社区，因为他们可以 以许多创造性的方式用于揭示有关保守基因功能，蛋白质 复合物和相互作用，细胞器在正常或扰动条件下的功能，等等。

PROJECT SUMMARY This proposal is in response to the Funding Opportunity Announcement (PAR-16-369) “Resource-Related Research Projects for Development of Animal Models and Related Materials (R24).” Our proposal addresses the goals stated in the Research Objectives: “The grant supports research projects that contribute to the knowledge of a model system, making the system more useful and accessible to the research community.” Specifically, we will generate a resource of Drosophila cell lines, markers, and knockouts that will enable cell biological analysis of genes and pathways altered in human diseases, including in disease-relevant tissue lineages and genetic backgrounds. In recent years, we have witnessed renewed interest in Drosophila cell lines and it is now common for Drosophila researchers to go back and forth between in vivo and cell culture studies. However, the full potential of Drosophila cell lines, particularly for disease-related studies, has not yet been realized, as the diversity of cell lines and markers available is limited. To overcome this bottleneck, we propose to use exciting new advances in cell line immortalization and genome engineering to generate reagents that will enable the community to fully exploit the power of Drosophila cell lines to address disease-related cell biological questions. Specifically, we propose to: Aim 1: Generate new cell lines from specific Drosophila cell lineages that will enable screens and other studies in relevant tissue-derived cell lines; Aim 2: Use CRISPR genome engineering to generate a resource of cell lines expressing fluorescent markers of sub-cellular components that will enable the community to perform screens exploring organelles and cell biological events relevant to human disease; and Aim 3: Use CRISPR engineering to generate knockout cell lines for synthetic lethal screens and other applications. We will disseminate information and resources for creating cell disease models by making available to the community detailed protocols and plasmid vectors, as well as the new cell lines and derivatives. Importantly, as Drosophila is an established system for the study of diverse human diseases, the resource and tools that we will develop are relevant to nearly all NIH institutes. Finally, we document that these resources will have benefit beyond the Drosophila community as they can be used in a number of creative ways to uncover exciting new information about conserved gene function, protein complexes and interactions, organelle function under normal or perturbed conditions, and more.