Genome Engineering & iPSC Center Core

项目摘要 基因组工程和患者来源的多能干细胞技术极大地改变了我们的生活。 理解疾病的途径。这两项技术是我们调查的最新补充。 但已经迅速渗透到生物医学科学的各个方面，提供了前所未有的力量， 来发现特定通路中的组分的扰动如何导致疾病。基因组工程 核心C由11名调查员和所有必要的设备、试剂和所需的专门知识组成 执行支持本PO 1更新中概述的3个项目的研究目标所需的实验 申请名称为“在α 1-AT缺乏症中用于肝纤维化和过度增殖的新疗法 (ATD)".我们的设施设在华盛顿大学基因组工程和IPSC中心（GEiC）内 由遗传学系建立并监督。它是为了方便 这些强大的新技术在华盛顿大学的实验室中的应用。所有项目 在此PO 1续订申请计划中建议广泛使用Core提供的服务。 这些实验主要针对ATD中修饰变体及其潜力的持续评估 开发这种疾病的新疗法。将使用的服务包括设计， 基因组编辑试剂（例如gRNA、供体质粒和Cas9衍生物）的构建和验证。 核心还使用基因组编辑技术生产修饰的细胞系，包括基因敲除， 变体引入、表位标记或基因置换。这些细胞系将用于更好地了解 ATD的生物学，并评估因其改变疾病的潜力而选择的变体的影响 促进药物开发。核心还将设计和制作所需的材料， 快速生产携带选定变体的动物模型。核心从两个皮肤产生iPSC 活检和肾小管上皮细胞存在于尿液样品中。iPSC的基因组工程现在是 在核心中常规进行，以引入新的变体并转化疾病相关的突变等位基因 恢复野生型或用作对照。将采集皮肤活检（成纤维细胞）或尿液（肾小管上皮细胞） 来自ATD患者或对照的细胞将被重编程以产生iPSC系。转基因 将使用基因组编辑技术产生患者来源的iPSC。此外， 核心为研究人员提供援助，在执行基因组工程，特别是在开发 新的技术和试剂，以及用于iPSC系的维持和分化。

Project Summary Genome engineering and patient-derived pluripotent stem cell technology have dramatically changed our abilities to understand disease pathways. These two technologies are recent additions to our investigative armamentarium but have rapidly permeated all aspects of biomedical science, providing unprecedented power to discover how perturbations of components in specific pathways lead to disease. The Genome Engineering Core C is composed of 11 investigators and all of the necessary equipment, reagents and expertise required to perform the experiments needed to support the research goals of the 3 projects outlined in this PO1 renewal application entitled “New therapies for liver fibrosis and hyperproliferation in alpha1-AT deficiency (ATD)”. Our facility is housed within the Washington University Genome Engineering and IPSC Center (GEiC) and was established and is overseen by the Department of Genetics. It was created to facilitate the implementation of these powerful new technologies in laboratories at Washington University. All projects proposed within this PO1 renewal application plan to extensively utilize the services provided by the Core. These experiments are largely aimed at the ongoing evaluation of modifying variants in ATD and their potential exploitation for development of new treatments for this disorder. The services to be utilized include the design, construction and validation of genome editing reagents (e.g. gRNAs, donor plasmids, and Cas9 derivatives). The Core also produces modified cell lines using genome editing technologies, including gene knockout, variant introduction, epitope tagging or gene replacement. These cell lines will be used to better understand the biology of ATD and to assess the impact of variants selected for their potential to modify disease progression and facilitate drug development. The Core will also design and generate materials needed for rapid production of animal models harboring selected variants. The Core produces iPSCs from both skin biopsies and the renal tubular epithelial cells present in urine samples. Genome engineering of iPSCs is now routinely performed in the Core to introduce new variants and to convert disease-associated mutant alleles back to wildtype or use as controls. Skin biopsies (fibroblasts) or urine (renal tubular epithelia) will be procured from ATD patients or controls and the cells will be reprogrammed to produce iPSC lines. Genetically modified patient-derived iPSCs will be generated using genome editing techniques. In addition, the personnel in the Core provide assistance for investigators in performing genome engineering, particularly in the development of new techniques and reagents, and for the maintenance and differentiation of iPSC lines.