JAX MorPhiC Data Production Center

项目总结 这项提议将建立细胞中空等位基因的分子表型(形态)阶段1：数据生产 完全设在杰克逊基因组医学实验室的研发中心--JAX 形态中心。Jax Moric将在同一屋檐下组成一个高度合作的科学家团队， 互补的技能组合和数十年在哺乳动物基因敲除(KO)、干细胞方面的积累经验 和发育生物学、分子表型、单细胞分析和高级代谢组学 脂质组学能力。我们将在这一倡议的第一阶段期间克隆250个蛋白质编码基因，并将 在人类诱导的多能干细胞(IPSCs)中设计这些KO，随后将其分化为 两种细胞谱系，胚胎外细胞和神经外胚层，其中的细胞将全面 表型。我们选择这些血统的理由是，他们是进化上分歧最大的两个 在物种之间，灵长类的专一性是我们基因优先排序的标准之一。此外，额外的- 胚胎谱系迅速发展成为负责许多生物过程的细胞功能终点， 因此允许解释不同基因的功能，而早期的神经外胚层谱系是相关的 神经发育障碍。在目标1中，我们将对基因敲除进行优先排序；选择标准包括 在胚胎外或神经外胚层谱系中的表达，灵长类动物特有的特征，广泛的类别 功能，同时丰富转录因子和与人类疾病有关的基因。在目标2中，我们将 使用JAX中建立的高效协议和工作流以高吞吐量生成IPSC KO克隆 细胞工程核心。我们将在特征良好、稳定的人类体内设计250个基因的KO克隆 IPSC系，KOLF2.1，同时考虑性别、遗传背景、可能的适应/补偿的影响 应对措施和不同的KO战略，包括纳入有条件的/可逆的和可扩展的方法。 在目标3中，将对不同于KO IPSCs的衍生品进行全面的表型鉴定。我们有 选择了多种分析组合，最大限度地整合了一致性、可扩展性和功能信息性 以帮助实现Moric联盟的总体目标。包括成像、单细胞 转录组学、单核表观基因组学和代谢组学/脂质组学。我们的研究目标将是 协调行政目标4，这将确保有效的项目管理和监督，内部和 向形态数据资源和管理部门进行外部通信和数据传播 协调中心(DRACC)。拟议工作的成功完成将解决几个主要障碍 阻碍形态联盟从功能上表征所有人类基因的最终目标：识别 最有效的KO策略和最佳表型技术，以及扩展的扩展效率 这一计划将迈向未来的形态阶段。在这个过程中，我们将创造出250人的宝贵资源 将通过日本宇宙航空研究开发机构现有的业务基础设施向科学界分发IPSC KO线路。

PROJECT SUMMARY This proposal will establish a Molecular Phenotypes of Null Alleles in Cells (MorPhiC) Phase 1: Data Production Research and Development Center based entirely at The Jackson Laboratory for Genomic Medicine – the JAX MorPhiC Center. JAX MorPhiC will comprise, under one roof, a team of highly collaborative scientists with complementary skill sets and decades of cumulative experience in mammalian gene knockouts (KO), stem cell and developmental biology, molecular phenotyping, single cell analysis, and advanced metabolomics and lipidomics capabilities. We will KO 250 protein-coding genes over the Phase 1 period of this initiative and will engineer these KOs in human induced pluripotent stem cells (iPSCs) that will subsequently be differentiated into two cell lineages, the extra-embryonic and the neuroectodermal, where cells will then be comprehensively phenotyped. Our rationale for selecting these lineages is that they are two of the most evolutionary divergent between species, and primate-specificity is one of our criteria for gene prioritization. In addition, the extra- embryonic lineage rapidly develops into a cellular functional endpoint responsible for many biological processes, thus permitting interpretation of function of diverse genes, whereas the early neuroectodermal lineage is relevant to neurodevelopmental disorders. In Aim 1, we will prioritize genes for knockout; selection criteria include expression in extra-embryonic or neuroectodermal lineages, primate-specific features, broad classes of functions while enriching for transcription factors, and genes implicated in human disease. In Aim 2, we will generate iPSC KO clones in high-throughput using high-efficiency protocols and workflows established in JAX's Cellular Engineering core. We will engineer KO clones for 250 genes in the well-characterized, stable human iPSC line, KOLF2.1, while considering the effects of sex, genetic background, possible adaptive/compensatory responses, and different KO strategies including incorporation of conditional/reversible and scalable approaches. In Aim 3, will carry out comprehensive phenotyping of derivatives differentiated from KO iPSCs. We have selected a combination of assays to maximally integrate consistency, scalability, and functional informativeness to help achieve the overall objectives of the MorPhiC Consortium. These include imaging, single cell transcriptomics, single nucleus epigenomics, and metabolomics/lipidomics. Our research Aims will be coordinated in administrative Aim 4, which will ensure efficient project management and oversight, internal and external communications, and data dissemination to the MorPhiC Data Resource and Administrative Coordination Center (DRACC). Successful completion of the proposed work will address several main barriers hampering the ultimate goal of the MorPhiC Consortium to functionally characterize all human genes: identifying the most effective KO strategies and optimal phenotyping technologies, as well as scaling efficiencies to expand this program towards future MorPhiC Phases. In the process, we will generate a valuable resource of 250 human iPSC KO lines that will be distributed to the scientific community via existing operational infrastructure at JAX.