Establishment of a Reporter System to Monitor Silencing Status in induced Pluripotent Stem Cell Lines
Establishment of a Reporter System to Monitor Silencing Status in induced Pluripotent Stem Cell Lines
复制标题
建立监测诱导多能干细胞系沉默状态的报告系统
DOI：
10.1016/j.ab.2013.08.014
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发表时间：
2013
期刊：
影响因子：
2.9
通讯作者：
Nishimori K and Fukuda T
中科院分区：
文献类型：
--
作者：
Donai K;Kuroda K;Guo Y;So K-H;Sone H;Kobayashi M;Nishimori K and Fukuda T
诱导多能干细胞（iPS）已被证明是再生医学中的有效技术;然而，低效率的重编程是成功产生iPS细胞系的主要障碍。高质量iPS细胞系的最重要特征之一是由逆转录病毒衍生的长末端重复启动子驱动的转基因失活。在这项研究中，我们建立了一个新的标记系统，包含三种蛋白质：分泌型荧光素酶（MetLuc），copepodPontellina plumatagreen荧光蛋白（copGFP），和一个抗病基因产物（Neor）。MetLuc-copGFP-Neorin小鼠胚胎成纤维细胞（MEF）的引入使我们能够监测报告基因表达的变化，作为重编程期间沉默状态的指标。iPS细胞的转化诱导报告基因活性显着降低，表明成功检测到逆转录病毒沉默。我们的系统使我们能够监测转基因的沉默状态，并有效地选择可用于进一步应用的iPS细胞系。
Induced pluripotent stem (iPS) cells have proven to be an effective technology in regenerative medicine; however, the low efficiency of reprogramming is a major obstacle to the successful generation of iPS cell lines. One of the most important characteristics of a high-quality iPS cell line is the inactivation of transgenes driven by a retrovirus-derived long terminal repeat promoter. In this study, we established a novel marker system containing three kinds of proteins: secreted-type luciferase (MetLuc), copepodPontellina plumatagreen fluorescent protein (copGFP), and an antibiotic-resistant gene product (Neor). The introduction of MetLuc–copGFP–Neorin mouse embryonic fibroblasts (MEFs) allowed us to monitor the reporter expression changes as an indicator of the state of silencing during reprogramming. Transformation of iPS cells induced a remarkable reduction in reporter activity, indicating that the retroviral silencing was detected successfully. Our system enables us to monitor the silencing status of transgenes and to efficiently select iPS cell lines that can be used for further applications.