Cell and Molecular Biology Core

细胞和分子生物学核心

• 批准号: 8710196
• 负责人: Dieter C Gruenert
• 金额: $ 17.16万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8710196
• 关键词: Animals; Antigens; Autologous Transplantation; Bone Marrow; Cell Line; Cells; Cellular biology; Clinic; Coculture Techniques; Databases; Development; Documentation; Economic Burden; Engineering; Exposure to; Fingerprint; Gene Expression; Generations; Genes; Genotype; Globin; Goals; Healthcare Systems; Hematopoietic; Hematopoietic stem cells; Hemoglobinopathies; Human; Human Cell Line; In Vitro; Individual; Instruction; Karyotype; Location; Methods; Methylation; MicroRNAs; Microsatellite Repeats; Molecular; Molecular Analysis; Molecular Biology; Molecular Profiling; Monitor; Mus; Patients; Process; Quality of life; Reagent; Research; Research Project Grants; Retroviral Vector; Safety; Services; Sickle Cell Anemia; Somatic Cell; Staging; Staining method; Stains; Stromal Cells; Technology; beta Globin; beta Thalassemia; gene correction; improved; in vivo; induced pluripotent stem cell; mutant; pathogen; precursor cell; programs; promoter; social; therapeutic development; vector

The purpose of Core B is to provide critical support for molecular/cellular analyses, ceil and vector banking, and generation of animal-product-free reagents fbr research needs performed under the three Projects and Core C. The specific service aims of Core B are: Aim 1: To provide cellular analysis of iPS and other cell lines. Core B will be responsible for providing expertise, assistance and coordination of immunohistochemical staining for expression antigens in iPS and differentiated cell lines, and for analysis of karyotype. Aim 2: To provide molecular analysis of iPS and other cell lines. Core B will provide molecular analyses of cell lines to validate their status as IPS, primary, or differentiated cell lines. This will include gene expression profiles, promoter methylation, and microRNA analyses. The core will be responsible for confirming B-globin genotype for mutant and corrected cell lines and microsatellite fingerprint profiles. One goal is to define a molecular expression profile or signature to monitor and validate in vitro differentiation of IPS to hematopoietic precursor cells competent to repopulate bone marrow in vivo. Aim 3: To provide cell and vector banking and evaluate cells for eventual therapeutic development. In anticipation that materials produced may be transitioned to the clinic, we will establish a centralized location and database to document and maintain key ceil lines, vectors, reagents, and other ancillary materials. This will be done in conjunction with Core A. Aim 4: To engineer a human cell line to replace mouse 0P9 stromal cells used for differentiation of IPS cells into hematopoietic cell lines. One of the most effective methods used to date has employed co-culture of IPS and hES cells with the mouse stromal 0P9 cell line. Since exposure to animal derived products might introduce pathogens with potential adverse safety consequences for patients, development of a human stromal cell line to replace 0P9 will be one component of creating animal-product free processes for generating gene-corrected HSCs. Aim 5: To perform routine reprogramming of primary cell lines into IPS ceil lines for use in early stage research for projects 2 and 3. Somatic cells will be reprogrammed to IPS cells for the gene connection (Project 2) and HCS differentiation (Project 3) studies will initially be performed using retroviral vectors carrying 4 reprogramming genes (0CT4, S0X2, KLF4, and cMYC). As new reprogramming technologies are developed in Project 1 they will be applied for the generation of IPS cells.

核心B的目的是为分子/细胞分析、细胞和载体银行，以及在三个项目和核心C下进行的无动物产品试剂FBR研究需求的产生提供关键支持。核心B的具体服务目标是：目标1：提供iPS和其他细胞系的细胞分析。核心B将负责提供专业知识，协助和协调免疫组织化学染色在iPS和分化细胞系中的表达抗原，并进行核型分析。目的2：对诱导性多发性硬化等细胞系进行分子分析。CORE B将提供细胞系的分子分析，以验证它们作为IPS、原代或分化细胞系的状态。这将包括基因表达谱、启动子甲基化和microRNA分析。该核心将负责确认突变和校正细胞系的B-珠蛋白基因以及微卫星指纹图谱。一个目标是定义一个分子表达谱或签名，以监测和验证IPS向有能力在体内重新填充骨髓的造血祖细胞的体外分化。目的3：为最终的治疗开发提供细胞库和载体库并评估细胞。预计生产的材料可能会转移到临床，我们将建立一个集中的位置和数据库，以记录和维护关键的细胞系、载体、试剂和其他辅助材料。这将与Core A.一起完成。目标4：设计一种人类细胞系，以取代用于将IPS细胞分化为造血细胞系的小鼠0P9基质细胞。迄今为止使用的最有效的方法之一是使用IPS和HES细胞与小鼠基质0P9细胞系共培养。由于接触动物源性产品可能会引入病原体，给患者带来潜在的不良安全后果，开发一种取代0P9的人类基质细胞系将是创造无动物产品的过程以产生经基因修正的HSCs的一个组成部分。目的：将原代细胞系常规重编程为IPS细胞系，用于项目2和项目3的早期研究。体细胞将被重编程为IPS细胞用于基因连接(项目2)和人巨细胞瘤分化(项目3)最初将使用携带4个重编程基因(0CT4、S0X2、KLF4和cMYC)的逆转录病毒载体进行研究。随着新的重新编程技术在项目1中的开发，它们将被应用于产生IPS单元。