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

项目总结 这项提议是对《筹资机会公告》(PAR-16-369)《与资源有关 发展动物模型及相关材料的研究项目(R24)。我们的建议解决了 研究目标中所述的目标：“该基金支持对 了解模型系统，使该系统更有用，研究界也更容易接触到该系统。 具体地说，我们将产生果蝇细胞系、标记和敲除的资源，这将使细胞 对包括疾病相关组织在内的人类疾病中改变的基因和途径的生物学分析 血统和遗传背景。近年来，我们看到人们对果蝇细胞重新产生了兴趣 现在，果蝇研究人员在体内和细胞培养之间来回穿梭是很常见的 学习。然而，果蝇细胞系的全部潜力，特别是在与疾病相关的研究中，还没有完全发挥出来 由于可用的细胞系和标记的多样性有限，因此已经实现了这一点。为了克服这一瓶颈，我们 建议利用细胞系永生和基因组工程方面令人兴奋的新进展来产生 试剂，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。具体地说，我们建议：目标1：从 特定的果蝇细胞系，将使相关组织来源细胞系的筛选和其他研究成为可能； 目的2：利用CRISPR基因组工程技术建立表达荧光标记的细胞系资源 亚细胞组件，使社区能够进行探索细胞器和细胞的筛选 与人类疾病有关的生物事件；和目标3：利用CRISPR工程产生基因敲除细胞 用于合成致命屏幕和其他应用的生产线。我们将传播信息和资源，以 通过向社区提供详细的协议和质粒载体来创建细胞疾病模型，如 以及新的细胞系和衍生品。重要的是，由于果蝇是研究果蝇的成熟系统 我们将开发的各种人类疾病、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会带来比果蝇社区更多的好处 以多种创造性的方式用于发现有关保守基因功能、蛋白质等令人兴奋的新信息 复合体和相互作用，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.