High-Throughput Screening to Identify Small Molecules for Improving Homologous Re

描述(申请人提供)：本项目旨在进行高通量筛选(HTS)，以确定可以提高人类诱导多能(IPS)细胞同源重组(HR)效率的小分子。IPS细胞技术的出现对再生医学领域产生了巨大的影响。然而，要成功地利用iPS细胞进行基因纠正治疗或其他基于细胞的治疗，我们首先需要克服人iPS细胞HR效率低的问题。我们建议首先为我们的HTS工作建立一个高效可靠的遴选平台。考虑到困难 为了保持人iPS细胞的单细胞集落，我们将首先建立适合于同源定向重组(HDR)实验的人成纤维细胞。我们将使用慢病毒载体转导成纤维细胞。慢病毒载体由两个拷贝的突变绿色荧光蛋白(GFP)组成，两个拷贝均不能表达功能性GFP，其中一个含有限制性内切酶I-SCEI重组区。含有两个GFP突变拷贝的成纤维细胞被称为HDR-成纤维细胞。当通过单独的慢病毒转导引入I-SCEI时，两个突变的GFP拷贝之间可能发生HR，之后功能性GFP将被表达。因此，可以通过测量GFP阳性细胞的百分比来确定HR频率。由于本项目的最终目标是提高人iPS细胞的HR效率，所以第一阶段工作的目标2是从hdr-iPS成纤维细胞中分离出hdr-iPS细胞。我们计划建立一个或多个这样的iPS细胞系，其特征是具有典型的iPS形态、多潜能细胞标记和基因表达谱以及正常倍体。我们还将使用RS-1验证这些细胞的HDR检测，RS-1已知可以提高哺乳动物细胞的HR效率。这些HDR-IPSCs将用于第二阶段的大规模小分子高温超导。

DESCRIPTION (provided by applicant): The present project aims to perform high-throughput screening (HTS) to identify small molecules that could improve the homologous recombination (HR) efficiency in human induced pluripotent (iPS) cells. The emergence of the iPS cell technology has dramatically impacted the field of regenerative medicine. However, to successfully utilize iPS cells for gene correction therapy or other cell based therapies in human, we first need to overcome the low efficiency of HR in human iPS cells. We propose to first establish an efficient and reliable selection platform for our HTS work. Considering the difficulty to maintain single cell colonies of human iPS cells, we will first establish the human fibroblasts that are suitable for homology directed recombination (HDR) assay. We will use lentiviral vectors to transduce the fibroblast cells. The lentiviral vector consists of two copies of mutated Green Fluorescent Protein (GFP), neither of which can express functional GFP and one of them contains a reorganization site for restrictive enzyme I-SceI. The fibroblast cells containing the two mutated copies of GFP are designated "HDR-fibroblasts". Upon introduction of I-SceI via a separate lentiviral transduction, HR may take place between the two mutated GFP copies, after which functional GFP will be expressed. Therefore, the HR frequency can be determined through measuring the percentage of GFP positive cells. As the ultimate goal of the present project is to improve HR efficiency in human iPS cells, Aim 2 of the Phase I work is to derive HDR-iPS cells from the HDR- fibroblasts. We plan to generate one or more such iPS cell lines that are characterized with typical iPS morphology, pluripotent cell markers and gene expression profiles, as well as normal ploidy. We will also validate these cells for HDR assay using RS-1, which is known to enhance HR efficiency in mammalian cells. These HDR-iPSCs will be used in Phase II for large scale HTS of small molecules.