Nuclease-mediated double-strand break (DSB) enhancement of small fragment homologous recombination (SFHR) gene modification in human-induced pluripotent stem cells (hiPSCs).
Nuclease-mediated double-strand break (DSB) enhancement of small fragment homologous recombination (SFHR) gene modification in human-induced pluripotent stem cells (hiPSCs).
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DOI：
10.1007/978-1-62703-761-7_18
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发表时间：
2014
期刊：
影响因子：
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通讯作者：
Gruenert, Dieter C
中科院分区：
文献类型：
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作者：
Sargent, R Geoffrey;Suzuki, Shingo;Gruenert, Dieter C
使用序列特异性核酸内切酶和供体DNA特异性修饰基因组DNA的方法的最新进展为遗传性疾病的细胞和基因治疗的新治疗范例打开了大门。序列特异性核酸内切酶，特别是转录激活因子样（TAL）效应核酸酶（TALEN），已经与多核苷酸小/短DNA片段（SDF）偶联，以校正诱导多能干细胞（iPS）中囊性纤维化（CF）跨膜传导调节因子（CFTR）基因中最常见的突变，即密码子508处的3个碱基对缺失（delF 508）。本文呈现的研究描述了候选TALEN的产生及其与野生型（wt）CFTR-SDF共转染到对于delF 508突变纯合的CF-iPS细胞中。使用基于等位基因特异性PCR（AS-PCR）的循环富集方案，分离并扩增纠正的CF-iPS细胞的克隆群体。
Recent developments in methods to specifically modify genomic DNA using sequence-specific endonucleases and donor DNA have opened the door to a new therapeutic paradigm for cell and gene therapy of inherited diseases. Sequence-specific endonucleases, in particular transcription activator-like (TAL) effector nucleases (TALENs), have been coupled with polynucleotide small/short DNA fragments (SDFs) to correct the most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, a 3-base-pair deletion at codon 508 (delF508), in induced pluripotent stem (iPS) cells. The studies presented here describe the generation of candidate TALENs and their co-transfection with wild-type (wt) CFTR-SDFs into CF-iPS cells homozygous for the delF508 mutation. Using an allele-specific PCR (AS-PCR)-based cyclic enrichment protocol, clonal populations of corrected CF-iPS cells were isolated and expanded.