Generation of hESC reporter lines using improved gene targeting technology

使用改进的基因打靶技术生成 hESC 报告系

• 批准号: 7588661
• 负责人: Tobias D. Raabe
• 金额: $ 27.43万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7588661
• 关键词: 3' Untranslated Regions; Address; Aging; Biological Assay; Blood; Brachyury protein; Cardiac; Cardiac Myocytes; Cell Line; Cell Therapy; Cells; Clone Cells; Collaborations; DNA Methylation; Degenerative Disorder; Disease; Frequencies; Gene Expression Profiling; Gene Targeting; Generations; Genes; Goals; Heart; Human; Human Genetics; Immune; In Vitro; Lung; Maintenance; Measurement; Measures; Mesoderm; Methods; Methylation; Modification; Mus; Mutation; Myocardial; Myosin ATPase; Pharmaceutical Preparations; Pilot Projects; Production; Proteins; Protocols documentation; Publishing; Reporter; Reporting; Research; Research Personnel; Retroviral Vector; Risk; SNP genotyping; Safety; Screening procedure; Signal Transduction; Sorting - Cell Movement; Stem cells; Stretching; Technology; Teratoma; Testing; Therapeutic; Therapeutic Uses; Time; Tissues; Transplantation; Undifferentiated; Untranslated Regions; Viral; Viral Genes; Work; antibiotic G 418; cardiogenesis; cell type; clinical application; experience; high throughput screening; human embryonic stem cell; improved; in vivo; novel; pluripotency; promoter; protocol development; public health relevance; repaired; response; stem cell differentiation; stem cell therapy; targeted delivery; transcription factor; vector

DESCRIPTION (provided by applicant): Generation of hESC reporter lines using improved gene targeting technology We propose here to develop hESC reporter lines that allow accurate real time measurement of the activity of tissue - specific promoters and that can be used for high-throughput screens and in vivo studies. To introduce reporter cassettes into hESC we will not use randomly integrating retroviral vectors but instead we will develop a method to insert reporter cassettes directly into endogenous genes. The resulting gene targeting method will not only allow production of hESC reporter lines but will also allow repair of human genetic defects in hESC without introducing potentially harmful genes, such as viral genes. Specific Aim 1: Develop methods required for gene targeting in hESCs. Methods for gene targeting in mouse ESCs are very well established, and early methods of gene targeting in hESC have been reported during the past four years. Building on this recent work we propose to systematically address each of the human ESC-specific gene targeting problems, from effective delivery of targeting constructs to efficient isolation and screening of hESC clones that retain their pluripotency. Specific Aim 2: Insert reporter cassettes into the 3'UTR of relevant endogenous genes. We propose to use the gene targeting method developed in Aim 1 to introduce fluorescent reporter constructs into the 3'UTR's of genes so their expression is driven by the endogenous promoters while preserving normal expression of the endogenous protein. We will use BAC recombineering to produce targeting vectors that have long stretches of homology, to achieve the highest possible targeting frequencies. First, we will target OCT4/POU5F1, a gene that is essential for maintenance of the undifferentiated state of hESC. Loss of a reporter signal (such as eGFP) will serve as an indication that the cells are differentiating and are no longer pluripotent; reporters can also be used to mark or select against undifferentiated cells before transplantation, or to indicate the presence of undifferentiated cells in transplants. Second, we will target NKX2-5, a cardiac-specific transcription factor that is essential for heart development and, if enough time remains, the mesoderm marker T/Brachyury and cardiac specific myosin MYH6/alpha-MHC. The resulting reporter lines will be useful in HTP screens for factors that induce effective differentiation of myocardial cells which should facilitate establishment of improved differentiation protocols, a major bottleneck in stem cell therapy. Specific Aim 3: Characterize the new hESC reporter lines. Because gene targeting requires lengthy culture under potentially harmful conditions, it is critical that the targeted lines are characterized carefully to identify potential problems before they are used in screens and assays. hESC lines developed in Aim 2 will be karyotyped and subjected to SNP genotyping and tested for pluripotency by gene expression profiling, DNA methylation profiling, in vitro differentiation and teratoma formation in mice. Public Health Relevance: This project will help improve a method to genetically alter human embryonic stem cells in order to faithfully and easily measure the activity of several heart - specific genes. This method, called gene targeting, will greatly facilitate development of protocols for differentiation of human embryonic stem cells into heart muscle cells to be used for transplantation into damaged human heart. Gene targeting works already very well for mice. It will completely avoid introduction of any viral or other foreign genes into the human cells to be used for this project and thus harbors no risk of immune rejection. Once established, it will work for any gene and can also be used for `clean ` and thus safe repair of almost any human genetic defect in stem cells and any tissue derived from stem cells.

描述（由申请人提供）：使用改进的基因靶向技术生成hESC报告系我们在此建议开发hESC报告系，其允许准确实时测量组织特异性启动子的活性，并且可用于高通量筛选和体内研究。为了将报告盒引入 hESC，我们不会使用随机整合的逆转录病毒载体，而是开发一种将报告盒直接插入内源基因的方法。由此产生的基因靶向方法不仅可以生产 hESC 报告系，还可以修复 hESC 中的人类遗传缺陷，而不会引入潜在有害的基因，例如病毒基因。具体目标 1：开发 hESC 基因靶向所需的方法。小鼠 ESC 中的基因打靶方法已经非常成熟，并且在过去四年中已经报道了 hESC 中的基因打靶的早期方法。在最近这项工作的基础上，我们建议系统地解决每个人类 ESC 特异性基因靶向问题，从有效递送靶向构建体到有效分离和筛选保留其多能性的 hESC 克隆。具体目标2：将报告盒插入相关内源基因的3'UTR。我们建议使用目标 1 中开发的基因打靶方法将荧光报告构建体引入基因的 3'UTR，以便它们的表达由内源启动子驱动，同时保留内源蛋白的正常表达。我们将使用 BAC 重组工程来产生具有长同源性的靶向载体，以实现尽可能高的靶向频率。首先，我们将靶向 OCT4/POU5F1，这是维持 hESC 未分化状态所必需的基因。报告信号（例如 eGFP）的丢失将表明细胞正在分化并且不再具有多能性；报告基因还可用于在移植前标记或选择未分化细胞，或指示移植物中未分化细胞的存在。其次，我们将瞄准 NKX2-5，这是一种对心脏发育至关重要的心脏特异性转录因子，如果还有足够的时间，我们将瞄准中胚层标记物 T/Brachyury 和心脏特异性肌球蛋白 MYH6/α-MHC。由此产生的报告系将可用于 HTP 筛选诱导心肌细胞有效分化的因子，这将有助于建立改进的分化方案，这是干细胞治疗的主要瓶颈。具体目标 3：表征新的 hESC 报告系。由于基因打靶需要在潜在有害的条件下进行长时间培养，因此在用于筛选和检测之前仔细表征靶标系以识别潜在问题至关重要。目标 2 中开发的 hESC 系将进行核型分析、SNP 基因分型，并通过基因表达谱、DNA 甲基化谱、体外分化和小鼠畸胎瘤形成来测试多能性。 公共健康相关性：该项目将有助于改进对人类胚胎干细胞进行基因改造的方法，以便准确、轻松地测量多个心脏特定基因的活性。这种称为基因靶向的方法将极大地促进将人类胚胎干细胞分化为心肌细胞以用于移植到受损的人类心脏中的方案的开发。基因靶向对于小鼠来说已经非常有效。它将完全避免将任何病毒或其他外来基因引入到用于该项目的人体细胞中，因此不存在免疫排斥的风险。一旦建立，它将适用于任何基因，也可用于“清洁”，从而安全修复干细胞和任何源自干细胞的组织中的几乎所有人类遗传缺陷。