Transgenic Human Stem Cell Lines and Reagents

描述（由申请人提供）：人类多能干细胞（hPSCs），包括胚胎干细胞（ESCs），特别是从患者组织中建立的诱导多能干细胞（iPSCs），为了解疾病发病机制提供了新的研究工具，为药物发现提供了多功能模板，也是细胞治疗的一个有希望的来源。实现人类PSCs这些潜力的关键一步是基因修饰/基因靶向，如转基因动物的创造，这已经彻底改变了生物医学研究和发现。与小鼠不同，人乳头瘤干细胞的转基因效率低下，转基因表达往往不稳定，尤其是在神经分化时，更不用说功能调控了。与现成的转基因动物相比，基因修饰人类造血干细胞的技术困难和缺乏普遍需要的人类造血干细胞系已经成为神经科学研究的重大障碍。目前的建议旨在通过构建和分发转基因人造血干细胞和相关试剂来克服这些障碍。基于我们在hPSC研究方面十年的经验，我们将首先在神经科学研究人员使用TALEN-或CRISPR/ cas介导的同源重组技术（Aim 1）最常寻找的神经基因中建立带有（GFP）报告基因的hESC系。我们还将在神经启动子的控制下，在嫉妒位点建立具有功能调节剂的hPSC系（Aim 2），这将允许在体外和体外对人类神经细胞的功能进行询问

DESCRIPTION (provided by applicant): Human Pluripotent Stem cells (hPSCs), including embryonic stem cells (ESCs) and especially, induced pluripotent stem cells (iPSCs) established from patient's tissues, offer a new research tool for understanding disease pathogenesis, a versatile template for drug discovery, and a promising source of cell therapy. A key step in realizing these potentials of human PSCs is genetic modification / gene targeting, as in the creation of transgenic animals that has revolutionized biomedical research and discovery. Unlike their mouse counterparts, transgenesis of hPSCs is inefficient and the transgene expression is often not stable, especially upon neural differentiation, let alone functional regulation. Technical difficulties in genetically modifying hPSCs and unavailability of commonly needed hPSC lines, as opposed to readily available transgenic animals, have become a significant barrier to neuroscience research. The present proposal aims to overcome these hurdles by building and distributing transgenic hPSCs and related reagents. Building upon our decade's experience in hPSC research, we will first establish hESC lines with a (GFP) reporter in neural genes that are most frequently sought by investigators in neuroscience using the TALEN- or CRISPR/CAS-mediated homologous recombination technology (Aim 1). We will also build hPSC lines with a functional regulator in an envy site under the control of a neural promoter (Aim 2), which will allow interrogation of the function of human neural cells in vitro and in vivo. These cell lines will be distributed through the WiCell Institute or an NIH designated distribution center (e.g., Rutgers University Cell and DNA Repository [RUCDR]). To promote neuroscience research in a broad spectrum as well as that involving specialized model systems (e.g., disease iPSCs), we will further deposit the transgenic reagents that are proven effective through Addegene for dissemination (Aim 3). Availability of such hPSC lines and tools will substantially speed up the next wave of neuroscience discovery in a cost effective manner and enable translation to clinical neuroscience.