Development of an All-in-One Lentiviral Vector System Based on the Original TetR for the Easy Generation of Tet-ON Cell Lines
Development of an All-in-One Lentiviral Vector System Based on the Original TetR for the Easy Generation of Tet-ON Cell Lines
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DOI：
10.1371/journal.pone.0023734
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发表时间：
2011-08-18
期刊：
影响因子：
3.7
通讯作者：
Martin, Francisco
中科院分区：
文献类型：
--
作者：
Benabdellah, Karim;Cobo, Marien;Martin, Francisco
慢病毒载体被认为是为基础研究和基因治疗方法有效传递遗传信息的最有前途的载体之一。将LV与药物诱导表达系统相结合，应该可以在对相关靶细胞的副作用最小的情况下严格控制转基因表达。1998年，在原有的TetR抑制子的基础上开发了一个新的多西环素调控系统，作为TetR-VP16嵌合体(TTA和RTTA)的替代品，以避免由于反式激活器域的表达而产生的二次效应。然而，以前描述的基于TetR的系统需要细胞克隆和/或四环素反应细胞的抗生素选择才能实现良好的调节。在本论文中，我们构建了一个基于两个慢病毒载体的双重Tet-on系统，一个通过脾病灶形成病毒(SFFV)启动子(STetR)表达TetR，另一个通过可调节的CMV-Teto启动子(CTetOE)表达EGFP。利用这些载体，我们证明了与反向反式激活因子(RTTA)相反，TetR阻遏子可以过量表达，以结合和调节大量的Teto操纵子。我们还表明，这种双载体系统可以产生可调控的批量细胞系(表达高水平的TetR)，能够通过改变多西环素浓度和/或通过改变每个细胞的CTetOE载体基因组的数量来调节转基因的表达。基于这些结果，我们开发了一种新的一体化慢病毒载体(CEST)，通过SFFV启动子驱动TetR的表达，通过多西环素应答的CMV-Teto操纵子驱动EGFP的表达。该载体有效地产生了Tet-On可调节永生化(293T)和原代(人间充质干细胞和人原代成纤维细胞)细胞。对多西环素敏感的细胞系大量表达高水平的转基因和少量的多西环素，并且与其亲本细胞的表型不同。
Lentiviral vectors (LVs) are considered one of the most promising vehicles to efficiently deliver genetic information for basic research and gene therapy approaches. Combining LVs with drug-inducible expression systems should allow tight control of transgene expression with minimal side effect on relevant target cells. A new doxycycline-regulated system based on the original TetR repressor was developed in 1998 as an alternative to the TetR-VP16 chimeras (tTA and rtTA) to avoid secondary effects due to the expression of transactivator domains. However, previously described TetR-based systems required cell cloning and/or antibiotic selection of tetracycline-responsive cells in order to achieve good regulation. In the present manuscript we have constructed a dual Tet-ON system based on two lentiviral vectors, one expressing the TetR through the spleen focus forming virus (SFFV) promoter (STetR) and a second expressing eGFP through the regulatable CMV-TetO promoter (CTetOE). Using these vectors we have demonstrated that the TetR repressor, contrary to the reverse transactivator (rtTA), can be expressed in excess to bind and modulate a high number of TetO operons. We have also showed that this dual vector system can generate regulatable bulk cell lines (expressing high levels of TetR) that are able to modulate transgene expression either by varying doxycycline concentration and/or by varying the amount of CTetOE vector genomes per cell. Based on these results we have developed a new all-in-one lentiviral vector (CEST) driving the expression of TetR through the SFFV promoter and the expression of eGFP through the doxycycline-responsive CMV-TetO operon. This vector efficiently produced Tet-ON regulatable immortalized (293T) and primary (human mesenchymal stem cells and human primary fibroblasts) cells. Bulk doxycycline-responsive cell lines express high levels of the transgene with low amount of doxycycline and are phenotypically indistinct from its parental cells.