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: 7915400
• 负责人: Tobias D. Raabe
• 金额: $ 21.87万
• 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 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7915400
• 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)：使用改进的基因打靶技术生成hESC报告线我们在此建议开发hESC报告线，该报告线允许准确实时测量组织特异性启动子的活性，并可用于高通量筛选和活体研究。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，而是将开发一种将报告盒直接插入内源基因的方法。由此产生的基因打靶方法不仅可以生产hESC报告系，还可以修复hESC中的人类遗传缺陷，而不会引入潜在的有害基因，如病毒基因。具体目标1：开发在人类胚胎干细胞中进行基因打靶所需的方法。在小鼠胚胎干细胞中进行基因打靶的方法已经非常成熟，在过去的四年中已经报道了在人类胚胎干细胞中进行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特异的基因打靶问题，从有效的靶向构建物到有效分离和筛选保持其多能性的hESC克隆。具体目的2：将报告盒插入相关内源基因的3‘非编码区。我们建议使用目标1中发展的基因打靶方法，将荧光报告结构引入基因的3‘UTRs，使其在内源启动子的驱动下表达，同时保持内源蛋白的正常表达。我们将使用BAC重组工程来产生具有较长同源性的靶向载体，以实现尽可能高的靶向频率。首先，我们将针对OCT4/Pou5f1，这是维持hESC未分化状态所必需的基因。失去报告信号(如绿色荧光蛋白)将表明细胞正在分化，不再具有多能性；报告信号也可用于在移植前标记或选择未分化的细胞，或指示移植中存在未分化的细胞。其次，我们将针对NKX2-5，一种心脏特异的转录因子，对心脏发育至关重要，如果还有足够的时间，还将瞄准中胚层标记物T/brachyury和心脏特异的肌球蛋白MYH6/α-MHC。由此产生的报告系将用于HTP筛选诱导心肌细胞有效分化的因素，这应该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：描述新的hESC记者系列。因为基因打靶需要在潜在有害的条件下进行长时间的培养，所以在用于筛查和分析之前，仔细描述目标株的特征以识别潜在的问题是至关重要的。在AIM 2中开发的hESC株将进行核型分析和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。 公共卫生相关性：该项目将帮助改进一种对人类胚胎干细胞进行遗传改变的方法，以便忠实而容易地测量几个心脏特异基因的活性。这种被称为基因打靶的方法将极大地促进将人类胚胎干细胞分化为心肌细胞的方案的开发，用于移植到受损的人类心脏。基因打靶对小鼠来说已经很有效了。它将完全避免将任何病毒或其他外来基因引入用于该项目的人类细胞，因此没有免疫排斥的风险。一旦建立，它将对任何基因起作用，也可以用于“清洁”，从而安全修复几乎任何人类干细胞和来自干细胞的组织中的遗传缺陷。