Generation of hESC reporter lines using improved gene targeting technology

描述（由申请人提供）：使用改进的基因靶向技术生成hESC报告系我们在这里提出开发hESC报告系，可以精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体内研究。为了将报告磁带导入hESC，我们将不使用随机整合的逆转录病毒载体，而是开发一种将报告磁带直接插入内源性基因的方法。由此产生的基因靶向方法不仅可以产生hESC报告系，还可以在不引入潜在有害基因（如病毒基因）的情况下修复hESC中的人类遗传缺陷。具体目标1：开发hESCs基因靶向所需的方法。小鼠esc的基因靶向方法已经非常成熟，在过去的四年中，早期的hESC基因靶向方法已经被报道。基于最近的工作，我们建议系统地解决每个人类esc特异性基因靶向问题，从靶向构建体的有效递送到保留其多能性的hESC克隆的有效分离和筛选。具体目标2：将报告基因盒插入相关内源基因的3'UTR中。我们建议使用Aim 1中开发的基因靶向方法，将荧光报告基因构建物引入基因的3'UTR中，使其表达受内源启动子驱动，同时保持内源蛋白的正常表达。我们将使用BAC重组来产生具有长同源性的靶向载体，以实现最高的靶向频率。首先，我们将瞄准OCT4/POU5F1，这是维持hESC未分化状态所必需的基因。报告信号（如eGFP）的缺失将表明细胞正在分化，不再具有多能性；报告基因也可用于在移植前标记或选择未分化细胞，或表明移植中存在未分化细胞。其次，我们将瞄准NKX2-5，这是一种心脏特异性转录因子，对心脏发育至关重要，如果时间足够，还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有助于HTP筛选诱导心肌细胞有效分化的因素，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：描述新的hESC报告细胞系。由于基因定位需要在潜在有害的条件下进行长时间的培养，因此在用于筛选和分析之前，对目标细胞系进行仔细的特征鉴定以确定潜在的问题是至关重要的。在Aim 2中开发的hESC细胞系将进行核型和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测试多能性。

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.