A modified TALEN-based system for robust generation of knock-out human pluripotent stem cell lines and disease models.
A modified TALEN-based system for robust generation of knock-out human pluripotent stem cell lines and disease models.
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DOI：
10.1186/1471-2164-14-773
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发表时间：
2013-11-09
期刊：
影响因子：
4.4
通讯作者：
Greber B
中科院分区：
文献类型：
--
作者：
Frank S;Skryabin BV;Greber B
转录激活因子样效应物核酸酶（TALEN）已经成为在困难系统（例如人类多能干细胞（hPSC））中实现靶向基因编辑和破坏的工具。TAL效应物的模块化结构理论上能够靶向任何基因组基因座，并且最近已经建立了用于定制TALEN组装的几种克隆系统。然而，缺乏适用于hPSC的通用TALEN表达系统。在这里，我们通过双组表达载体扩展了现有的TALE组装系统，以有效地将TALEN技术应用于hPSC。其特征在于改进的TALEN结构以及抗生素抗性和荧光报告盒，从而能够富集转染的细胞。通过HPRT 1基因的靶向破坏以创建Lesch-Nyhan-Syndrome的同基因疾病模型来证明组合系统的改进功能。使用雌性hPSC，HPRT 1的纯合破坏以高达15%的效率发生。将同基因敲除细胞分化为中枢神经系统（CNS）以及感觉样神经元，重现了先前基于患者特异性诱导的PSC描述的表型，并将这些发现分别扩展到非CNS神经元。组合的载体系统允许灵活且负担得起地产生敲除hPSC系，从而使得能够研究发育过程以及产生同基因疾病模型而不需要患者材料。
Transcription activator-like effector nucleases (TALENs) have emerged as a tool for enabling targeted gene editing and disruption in difficult systems, such as human pluripotent stem cells (hPSCs). The modular architecture of TAL effectors theoretically enables targeting of any genomic locus and several cloning systems for custom TALEN assembly have recently been established. However, there is a lack of versatile TALEN expression systems applicable to hPSCs. Here, we extend an existing TALE assembly system by a dual set of expression vectors for efficient application of TALEN technology in hPSCs. This is characterized by improved TALEN architecture as well as antibiotic resistance and fluorescent reporter cassettes, thus enabling enrichment for transfected cells. Improved functionality of the combined system was demonstrated by targeted disruption of the HPRT1 gene to create isogenic disease models of Lesch-Nyhan-Syndrome. Using female hPSCs, homozygous disruption of HPRT1 occurred at efficiencies of up to 15%. Differentiating isogenic knock-out cells both into central nervous system (CNS) as well as into sensory-like neurons recapitulated previously described phenotypes based on patient-specific induced PSCs and extended these findings to non-CNS neurons, respectively. The combined vector system allows for flexible and affordable generation of knock-out hPSCs lines, thus enabling investigation of developmental processes as well as the generation of isogenic disease models without the need for patient material.
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