A Doxycycline-Inducible System for Genetic Correction of iPSC Disease Models.
A Doxycycline-Inducible System for Genetic Correction of iPSC Disease Models.
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DOI：
10.1007/7651_2014_179
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Gadue P
中科院分区：
文献类型：
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作者：
Sim X;Cardenas-Diaz FL;French DL;Gadue P
患者来源的诱导多能干细胞（iPSC）是发育生物学和疾病建模研究的宝贵工具。在这两种应用中，患者iPSC的遗传校正是了解基因在发育或疾病状态中的具体贡献的有力方法。在这里，我们描述了一个多西环素诱导的转基因表达系统的安全港网站在iPSCs的靶向整合协议。我们的基因靶向策略使用锌指核酸酶（ZFN）来增强AAVS 1安全港位点处的同源重组，从而增加组成强力霉素诱导系统的两个靶向载体的位点特异性整合的效率。重要的是，在我们的系统中使用双药物选择将双靶向克隆的阳性选择效率提高到> 50%，从而减少了筛选过程的劳动力。如果需要，该方案还可以被修改以允许使用组织特异性启动子来驱动基因表达，而不是多西环素诱导型启动子（TRE）。此外，该方案还与使用转录激活因子样效应物核酸酶（TALEN）或重复的规律间隔短回文重复序列（CRISPR）-Cas9系统代替ZFN兼容。
Patient-derived induced pluripotent stem cells (iPSCs) are valuable tools for the study of developmental biology and disease modeling. In both applications, genetic correction of patient iPSCs is a powerful method to understand the specific contribution of a gene(s) in development or diseased state(s). Here, we describe a protocol for the targeted integration of a doxycycline-inducible transgene expression system in a safe harbor site in iPSCs. Our gene targeting strategy uses zinc finger nucleases (ZFNs) to enhance homologous recombination at the AAVS1 safe harbor locus, thus increasing the efficiency of the site-specific integration of the two targeting vectors that make up the doxycycline-inducible system. Importantly, the use of dual-drug selection in our system increases the efficiency of positive selection for double-targeted clones to >50 %, permitting a less laborious screening process. If desired, this protocol can also be adapted to allow the use of tissue-specific promoters to drive gene expression instead of the doxycycline-inducible promoter (TRE). Additionally, this protocol is also compatible with the use of Transcription-Activator-Like Effector Nucleases (TALENs) or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system in place of ZFNs.