Transgenic Human Stem Cell Lines and Reagents

描述(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)，为了解疾病的发病机制提供了新的研究工具，为药物开发提供了一个通用的模板，并为细胞治疗提供了一个有前景的来源。实现人类PSCs这些潜力的关键一步是基因修饰/基因靶向，就像创造转基因动物一样，这使生物医学研究和发现发生了革命性的变化。与小鼠不同，hPSCs的转基因效率低，转基因表达往往不稳定，尤其是在神经分化方面，更不用说功能调节了。基因修饰hPSC的技术困难和普遍需要的hPSC株的缺乏，而不是现成的转基因动物，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hPSC和相关试剂来克服这些障碍。在我们十年hPSC研究经验的基础上，我们将首先利用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(GFP)中建立hESC系。我们还将在神经启动子(AIM 2)的控制下，在嫉妒部位建立具有功能调节的hPSC系，这将允许在体外和 在活体内。这些细胞系将通过Wicell研究所或NIH指定的分配中心(例如，罗格斯大学细胞和DNA存储库[RUCDR])进行分配。为了促进广泛的神经科学研究以及涉及专门的模型系统(例如疾病IPSCs)的研究，我们将进一步存放通过Addegene被证明有效的转基因试剂以供传播(目标3)。这种hPSC系列和工具的可获得性将大大加快下一波神经科学发现的速度，并使其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.