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首页> 外文期刊>Journal of Turbulence >Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression
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Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression

机译：单型构建聚四屈霉素 - 诱导型载体改善直接心脏重编程，可用于识别转基因表达的临界时序

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摘要

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming.
机译：直接重新编程是再生医学中有希望的方法。心脏转录因子的过度表达GATA4，MEF2C和TBX5（GMT）或GMT加上HAMP2（GHMT）将成纤维细胞直接重新编程成心肌细胞样细胞（ICMS）。然而，转基因表达的临界时序和心脏重编程的分子机制仍不清楚。常规的十氧胞菌素（DOX） - 剩余的时间转基因表达系统需要分别同时转导（PLVX-RTTA / PLVX-cDNA），其分别涉及反向四环蛋白转移剂（RTTA）和四环响应元素（TRE） - 控制转基因的转基因效率低下心脏重编程。在此，我们开发了一种表达rTTA和TRE控制转基因的单构体基的多函数DOX诱导载体（PDOX-cDNA）。荧光活性细胞分选（FACS）分析，定量RT-PCR和免疫染色显示，与传统的PLVX-RTTA / PLVX-GMT相比，PDOX-GMT增加了3倍的心脏重编程。四周后，Pdox-GMT诱导的ICMS表达了多种心脏基因，产生了SARCOMERIC结构，并自发地击败。与单独的PMX-GMT相比，用逆转录病毒PMX-GMX-GMT的PDOX-HAND2的共转导增加了3倍。颞X管理显示，在心脏重新编程的前两周内，Hand2转基因表达至关重要。微阵列分析证明了Hand2压制细胞周期促进基因并增强心脏重编程。因此，我们已经开发出一种有效的颞型转基因表达系统，这在心脏重新编程的研究中可能是非常宝贵的。

著录项

  • 来源
    《Journal of Turbulence》 |2017年第8期|共14页
  • 作者

    Umei Tomohiko C.; Yamakawa Hiroyuki; Muraoka Naoto; Sadahiro Taketaro; Isomi Mari; Haginiwa Sho; Kojima Hidenori; Kurotsu Shota; Tamura Fumiya; Osakabe Rina; Tani Hidenori; Nara Kaori; Miyoshi Hiroyuki; Fukuda Keiichi; Ieda Masaki;

  • 作者单位

    Keio Univ Dept Cardiol Sch Med Shinjuku Ku 35 Shinanomachi Tokyo 1608582 Japan;

    Keio Univ Dept Cardiol Sch Med Shinjuku Ku 35 Shinanomachi Tokyo 1608582 Japan;

    Keio Univ Dept Cardiol Sch Med Shinjuku Ku 35 Shinanomachi Tokyo 1608582 Japan;

    Keio Univ Dept Cardiol Sch Med Shinjuku Ku 35 Shinanomachi Tokyo 1608582 Japan;

    Keio Univ Dept Cardiol Sch Med Shinjuku Ku 35 Shinanomachi Tokyo 1608582 Japan;

    Keio Univ Dept Cardiol Sch Med Shinjuku Ku 35 Shinanomachi Tokyo 1608582 Japan;

    Keio Univ Dept Cardiol Sch Med Shinjuku Ku 35 Shinanomachi Tokyo 1608582 Japan;

    Keio Univ Dept Cardiol Sch Med Shinjuku Ku 35 Shinanomachi Tokyo 1608582 Japan;

    Keio Univ Dept Cardiol Sch Med Shinjuku Ku 35 Shinanomachi Tokyo 1608582 Japan;

    Keio Univ Dept Cardiol Sch Med Shinjuku Ku 35 Shinanomachi Tokyo 1608582 Japan;

    Keio Univ Dept Cardiol Sch Med Shinjuku Ku 35 Shinanomachi Tokyo 1608582 Japan;

    Keio Univ Dept Cardiol Sch Med Shinjuku Ku 35 Shinanomachi Tokyo 1608582 Japan;

    Keio Univ Dept Physiol Sch Med Shinjuku Ku 35 Shinanomachi Tokyo 1608582 Japan;

    Keio Univ Dept Cardiol Sch Med Shinjuku Ku 35 Shinanomachi Tokyo 1608582 Japan;

    Keio Univ Dept Cardiol Sch Med Shinjuku Ku 35 Shinanomachi Tokyo 1608582 Japan;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 流体力学;
  • 关键词

    reprogramming; cardiomyocyte; doxycycline-inducible; cell cycle; fibroblast;

    机译：重新编程;心肌细胞;十氧环素诱导;细胞周期;成纤维细胞;

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