UCLA High-Throughput Neuropsychiatric Disorder Phenotyping Center (UCLA HT-NPC)

项目摘要/摘要 人类遗传学研究已经确定了数百种有助于神经精神和神经发育的基因-- 精神疾病(NPD)风险。但对于大多数基因来说，它们的正常功能或缺失或 神经发育和神经功能的减少尚不清楚。在这里，我们建议解决实质性的 通过HIGH的发展来识别数百个NPD基因的潜在功能的挑战 吞吐量神经精神疾病表型中心(UCLA HT-NPC)，由9个高度活跃的 协作调查员(Aharoni、Bhadui、Damoiseaux、Geschind、Golshani、Kitai、Luo、Novich和Wells) 和两个实质性的核心设施(加州大学洛杉矶分校分子筛查共享资源和人类干细胞和 基因组工程中心)。通过分层方法，我们将高吞吐量和高价值相结合， 用干细胞工程学定量表型分析NPD基因的功能后果 敲除(零等位基因)，这是一个关键的初始步骤，将告诉我们对疾病途径的理解。在第一步， 我们将使用快速、高吞吐量为联盟选择的250个基因快速生成空等位基因 人类胚胎干细胞中基于慢病毒的系统。将对生存能力和神经诱导潜力进行评估，并量化 在所有品系上使用RNA-seq进行表型鉴定。那些通过活性和神经诱导测试的基因将 用于生产克隆缺失的HiPSC系(雄性和雌性)，用于下游表型和更广泛的 分发给社区。随后，我们将进行高通量、量化、多尺度的 基于2D和3D HiPSC模型的分子、形态和生理水平的表型 人类大脑皮层的发育。我们利用每个模型的相对优势和可扩展性来使我们能够 同时进行SnRNA和Bulk RNA-seq，测量神经元的成熟度、形态和突触密度 使用自动成像的细胞，包括多路复用的基于蛋白质的Codex(Phencle Cler)平台，以及 使用定制的微型显微镜通过光学记录表征神经元的活动和同步性 数组(STIMcope)。通过使用多种系统(例如，hESC/hiPSC；基因编辑、2D和3D培养)，我们测试 跨系统的生物再现性和通过复制实现的技术再现性。对.的使用 经过实验验证的跨多个分析范围的定量表型有助于数据共享和 与其他SSPsygene研究人员的比较，并为更广泛的领域提供了模板。

Project Summary/Abstract Human genetic studies have identified hundreds of genes contributing to Neuropsychiatric and Neurodevelop- mental Disease (NPD) risk. But for most genes, their normal function or the consequences of their absence or reduction on neurodevelopment and neural function are not known. Here, we propose to address the substantial challenges of discerning potential functions of hundreds of NPD genes through the development of a High Throughput Neuropsychiatric Disease Phenotyping Center (UCLA HT-NPC), driven by the activity of 9 highly collaborative investigators (Aharoni, Bhaduri, Damoiseaux, Geschwind, Golshani, Kitai, Luo, Novich, and Wells) and two substantial core facilities (UCLA Molecular Screening Shared Resource and the Human Stem Cell and Genome Engineering Center). Through a tiered approach, we combine high throughput and high value, quantitative phenotyping with stem cell engineering to characterize the functional consequences of NPD gene knockouts (null alleles), a key initial step that will inform our understanding of disease pathways. In the first step, we will rapidly generate null alleles for 250 genes chosen by the Consortium using a rapid, high throughput lentiviral based system in hESCs. Viability and neural induction potential will be assessed, and quantitative phenotyping conducted using RNA-seq on all lines. Those genes passing viability and neural induction tests will be used in the production of clonal null hiPSC lines (male and female) for downstream phenotyping and wider distribution to the community. Subsequently, we will perform high throughput, quantitative, multi-scale phenotyping at the molecular, morphological, and physiological levels in both 2D and 3D hiPSC-based models of human cortical development. We leverage the relative strengths and scalability of each model to enable us to perform both snRNA and bulk RNA-seq, measure the maturation, morphology, and synaptic density of neural cells using automated imaging, including the multiplexed, protein-based CODEX (Phenocycler) platform, and characterize neuronal activity and synchronization through optical recordings using custom-built mini-scope arrays (STIMscope). By using multiple systems (e.g. hESC/hiPSC; gene editing, 2D and 3D cultures), we test biological reproducibility across systems and technical reproducibility through replication. The use of experimentally validated, quantitative phenotypes across multiple scales of analysis facilitates data sharing and comparisons with other SSPsyGene investigators and provides a template for the field more broadly.