Genome Engineering & iPSC Center Core

项目摘要 基因组工程和患者衍生的多能干细胞技术极大地改变了我们的 理解疾病途径的能力。这两项技术是我们最近研究的新技术 但已迅速渗透到生物医学科学的方方面面，提供了前所未有的力量 以发现特定途径中的成分扰动如何导致疾病。基因组工程 核心C由11名调查人员以及所需的所有必要设备、试剂和专业知识组成 进行必要的实验，以支持本次PO1更新中概述的3个项目的研究目标 题为《Alpha1-AT缺乏症的肝纤维化和过度增殖的新疗法》的申请 (ATD)“。我们的设施位于华盛顿大学基因组工程和IPSC中心(GEIC)内 并由遗传学部建立和监督。它的创建是为了方便 在华盛顿大学的实验室中实施这些强大的新技术。所有项目 在此PO1续签申请计划中提出，以广泛利用核心提供的服务。 这些实验主要是为了对ATD的修饰变体及其潜力进行正在进行的评估 开发这种疾病的新治疗方法。要利用的服务包括设计、 基因组编辑试剂的构建和验证(例如，gRNA、供体质粒和Cas9衍生物)。 Core还使用基因组编辑技术生产改良的细胞系，包括基因敲除， 变异体引入、表位标记或基因替换。这些细胞系将被用来更好地理解 ATD的生物学和评估因其潜在的疾病修饰而被选择的变异的影响 发展和促进药物开发。核心还将设计和生成所需的材料 快速生产含有特定变种的动物模型。Core可以从两种皮肤上产生iPSCs 活检和尿样中的肾小管上皮细胞。IPSCs的基因组工程现在是 在核心中常规执行以引入新的变种并转换与疾病相关的突变等位基因 返回到通配类型或用作控件。将获得皮肤活检(成纤维细胞)或尿液(肾小管上皮细胞)。 来自ATD患者或对照组的细胞将被重新编程以产生IPSC系。转基因 患者来源的IPSCs将使用基因组编辑技术生成。此外，该中心的工作人员 CORE协助研究人员进行基因组工程，特别是在开发 新技术和试剂，以及IPSC品系的维护和分化。

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.