JAX MorPhiC Data Production Center

项目摘要 该提案将建立细胞中的等位基因的分子表型（MorPhiC）第1阶段：数据生产 研究和开发中心完全位于杰克逊基因组医学实验室-JAX MorPhiC中心。JAX MorPhiC将由一个高度合作的科学家团队组成， 在哺乳动物基因敲除（KO）、干细胞移植、基因工程 和发育生物学，分子表型，单细胞分析，和先进的代谢组学， 脂质组学能力。我们将在该计划的第1阶段期间KO 250个蛋白质编码基因， 将这些科斯工程化到人诱导多能干细胞（iPSC）中， 两种细胞谱系，胚外和神经外胚层，在那里细胞将全面 表型分析我们选择这些血统的理由是，它们是两个最具进化分歧的 灵长类特异性是我们基因优先级的标准之一。此外，额外的- 胚胎谱系迅速发育成负责许多生物过程的细胞功能终点， 从而允许解释不同基因的功能，而早期神经外胚层谱系是相关的 神经发育障碍在目标1中，我们将优先考虑基因敲除;选择标准包括 在胚胎外或神经外胚层谱系中的表达，灵长类特异性特征， 功能，同时富集转录因子和与人类疾病有关的基因。在目标2中，我们 使用JAX中建立的高效协议和工作流程以高通量生成iPSC KO克隆 细胞工程核心我们将工程师KO克隆250个基因在良好的特点，稳定的人类 iPSC系KOLF2.1，同时考虑性别、遗传背景、可能的适应性/补偿性的影响， 反应和不同的KO策略，包括纳入条件/可逆和可扩展的方法。 在目标3中，将对从KO iPSC分化的衍生物进行全面的表型分析。我们有 选择了一种检测组合，以最大限度地整合一致性、可扩展性和功能信息性 帮助实现MorPhiC联盟的总体目标。这些包括成像，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 在行政目标4中进行协调，这将确保有效的项目管理和监督， 外部通信，以及向MorPhiC数据资源和行政部门的数据传播 协调中心（DRACC）。成功完成拟议的工作将解决几个主要障碍 阻碍了MorPhiC联盟的最终目标，即在功能上表征所有人类基因： 最有效的KO策略和最佳的表型分析技术，以及扩展的规模效率 这一计划将在未来的MorPhiC阶段进行。在这个过程中，我们将产生250人的宝贵资源， iPSC KO线将通过JAX现有的操作基础设施分发给科学界。

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.