Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression.
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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DOI：
10.3390/ijms18081805
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发表时间：
2017-08-19
影响因子：
5.6
通讯作者：
Ieda M
中科院分区：
文献类型：
--
作者：
Umei TC;Yamakawa H;Muraoka N;Sadahiro T;Isomi M;Haginiwa S;Kojima H;Kurotsu S;Tamura F;Osakabe R;Tani H;Nara K;Miyoshi H;Fukuda K;Ieda M
在再生医学中，直接重编程是一种很有前途的方法。过表达心脏转录因子Gata4、Mef2c和Tbx5 （GMT）或GMT + Hand2 （GHMT）可直接将成纤维细胞重编程为心肌细胞样细胞（iCMs）。然而，转基因表达的关键时间和心脏重编程的分子机制仍不清楚。传统的多西环素（Dox）诱导的瞬时转基因表达系统需要同时转导两个载体（pLVX-rtTA/pLVX-cDNA），分别包含逆转录四环素反激活因子（rtTA）和四环素应答元件（TRE）控制的转基因，导致心脏重编程效率低下。在此，我们开发了一种基于单结构的多顺反子dox诱导载体（pDox-cDNA），表达rtTA和trex控制的转基因。荧光活化细胞分选（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使心脏重编程增加了三倍。四周后，pdox - gmt诱导的iCMs表达多种心脏基因，产生肌肉结构，并自发跳动。与单独使用pMX-GMT相比，pDox-Hand2与逆转录病毒pMX-GMT共转导使心脏重编程增加了3倍。时间Dox给药显示Hand2转基因表达在心脏重编程的前两周至关重要。微阵列分析表明，Hand2抑制细胞周期促进基因并增强心脏重编程。因此，我们开发了一个有效的时间转基因表达系统，这在心脏重编程的研究中可能是无价的。
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.
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影响因子：
16.6
作者：
Zhao Y;Londono P;Cao Y;Sharpe EJ;Proenza C;O'Rourke R;Jones KL;Jeong MY;Walker LA;Buttrick PM;McKinsey TA;Song K
通讯作者：
Song K
影响因子：
4.8
作者：
Dai, YS;Cserjesi, P;Molkentin, JD
通讯作者：
Molkentin, JD
影响因子：
64.8
作者：
Qian, Li;Huang, Yu;Srivastava, Deepak
通讯作者：
Srivastava, Deepak
通过对心脏转录因子的非肌细胞进行重编程通过重编程。
影响因子：
64.8
作者：
Song, Kunhua;Nam, Young-Jae;Luo, Xiang;Qi, Xiaoxia;Tan, Wei;Huang, Guo N.;Acharya, Asha;Smith, Christopher L.;Tallquist, Michelle D.;Neilson, Eric G.;Hill, Joseph A.;Bassel-Duby, Rhonda;Olson, Eric N.
通讯作者：
Olson, Eric N.
影响因子：
64.5
作者：
Takahashi, Kazutoshi;Yamanaka, Shinya
通讯作者：
Yamanaka, Shinya