Transgenic Human Stem Cell Lines and Reagents

描述（由申请人提供）：人多能干细胞（hPSC），包括胚胎干细胞（ESC），特别是从患者组织中建立的诱导多能干细胞（iPSC），为理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，并为细胞治疗提供了有前途的来源。实现人类PSC这些潜力的关键一步是遗传修饰/基因靶向，如创造转基因动物，这彻底改变了生物医学研究和发现。与它们的小鼠对应物不同，hPSC的转基因是低效的，并且转基因表达通常是不稳定的，特别是在神经分化时，更不用说功能调节了。与容易获得的转基因动物相反，遗传修饰hPSC的技术困难和普遍需要的hPSC系的不可用性已经成为神经科学研究的重大障碍。本提案旨在通过构建和分配转基因hPSC和相关试剂来克服这些障碍。基于我们十年来在hPSC研究方面的经验，我们将首先使用TALEN或CRISPR/CAS介导的同源重组技术（Aim 1）在神经科学研究人员最常寻找的神经基因中建立具有（GFP）报告基因的hESC系。我们还将构建在神经启动子（Aim 2）控制下的嫉妒位点中具有功能调节剂的hPSC系，这将允许在体外询问人神经细胞的功能， in vivo.这些细胞系将通过WiCell Institute或NIH指定的分发中心（例如，Rutgers University Cell and DNA Repository [RUCDR]）。促进神经科学研究的广泛范围以及涉及专门的模型系统（例如，疾病iPSC），我们将进一步存款通过Addegene证明有效的转基因试剂用于传播（目的3）。这种hPSC生产线和工具的可用性将以具有成本效益的方式大大加快神经科学发现的下一波浪潮，并能够转化为临床神经科学。

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.