Exploitation of Near-Haploid Human Cells for Functional Gene Discovery

描述(由申请人提供) 摘要：几十年来，经典的遗传模式生物已经提供了一种公正和全面的方法来对基因组进行功能注释--将基因和表型联系起来。尽管付出了巨大的努力，但在人体模型系统中进行体细胞基因筛查仍然是遥不可及的。然而，最近，基于RNA干扰的功能筛选的发现为人类细胞提供了一个类似的发现平台，这个平台继续革命我们对基因组功能的理解，因为它与正常的细胞生物学和人类疾病有关。尽管如此，尽管仍将其视为无价资产，但基于RNA干扰的筛选昂贵且充满人工制品，因此永远无法达到在酵母、果蝇、C-elegans和斑马鱼中实现的全面功能注释。该方案的主要目标是在人类细胞中实现体细胞遗传筛选平台。我们将通过建立和利用我们实验室中存在的稳定的近单倍体人类细胞来实现这一目标。这些细胞的逆转录病毒插入突变提供了一种快速和廉价地识别人类基因组中单独和一致控制表型的功能单位的方法。突变的近单倍体细胞的克隆衍生和归档将产生一个基因受损的人类细胞系的文库，类似于酵母缺失文库。我将利用我在蛋白质组和信号转导功能基因组分析方面的现有专业知识来验证单倍体筛选方法。由此产生的功能数据与蛋白质-蛋白质相互作用网络、全基因组关联数据和转录签名的系统级集成将揭示诊断、预后和治疗价值的靶点。成功有望对人类细胞进行快速、公正和廉价的完全功能丧失表型筛选，因此将改变在基础科学和应用科学中采取的实验策略。 公共卫生相关性：医学科学中的一个重大挑战是识别在特定细胞和疾病过程中起作用的全部基因。尽管这样的基因组注释很容易在经典的遗传模型系统中实现，但在人类系统中使用类似的方法是昂贵的，并且充满了错误的发现。我们已经专门为哺乳动物细胞设计了一个廉价且无人工制品的功能基因组发现平台，一个将功能归因于特定细胞行为背景下的每个人类基因的平台，更重要的是一个承诺增强我们发现的翻译潜力的平台。

DESCRIPTION (Provided by the applicant) Abstract: For decades, classic genetic model organisms have provided an unbiased and comprehensive means to functionally annotate the genome-to connect genotype with phenotype. Despite immense efforts, somatic cell genetic screens in human model systems have remained unattainable. Recently, however, the discovery of RNA interference-based functional screening provided an analogous discovery platform for human cells, one that continues to revolutionize our understanding of genome function, both as it relates to normal cellular biology and human disease. That said and while still appreciating it as an invaluable asset, RNA interferencebased screens are expensive and fraught with artifact, and therefore will never reach the comprehensive functional annotation achieved in yeast, Drosophila, C-elegans and zebrafish. The principle goal of this proposal is to realize a somatic cell genetic screening platform in human cells. We will accomplish this goal through the establishment and employment of stable near-haploid human cells, which exist in our laboratory. Retroviral insertional mutagenesis of these cells provides a means to quickly and inexpensively identify functional units within the human genome that individually and concertedly control phenotype. The clonal derivation and archiving of mutagenized near-haploid cells will yield a library of gene-compromised human cell lines, analogous to that of the yeast deletion libraries. I will leverage my existing expertise in proteomic and functional genomic analysis of signal transduction to validate the haploid screening approach. A systems-level integration of the resulting functional data with protein-protein interaction networks, genome-wide association data and transcriptional signatures will reveal targets of diagnostic, prognostic and therapeutic value. Success promises rapid, unbiased and inexpensive complete loss-of-function phenotypic screening of human cells, and therefore will transform the experimental strategies taken in both basic and applied sciences. Public Health Relevance: A significant challenge in medical science is identifying the full complement of genes that functionally contribute to specific cellular and disease processes. Although such genome annotation is easily achieved in classic genetic model systems, analogous approaches in human systems are expensive and fraught with false discoveries. We have designed an inexpensive and artifact-free functional genomic discovery platform specifically for mammalian cells, one that ascribes function to every human gene in the context of specific cellular behaviors, and more importantly one which promises to enhance the translational potential of our discoveries.