Andalusian Stem Cell Bank, Centro de Investigaciones Biomédicas, Universidad de Granada, Parque Tecnológico Ciencias de la Salud, Granada, Spain.
PLoS One. 2011;6(8):e23734. doi: 10.1371/journal.pone.0023734. Epub 2011 Aug 18.
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.
慢病毒载体（LVs）被认为是最有前途的载体之一，可有效地将遗传信息传递用于基础研究和基因治疗方法。将 LVs 与药物诱导表达系统结合使用，应该可以通过最小化对相关靶细胞的副作用来实现对转基因表达的严格控制。1998 年，开发了一种基于原始 TetR 阻遏物的新的强力霉素调控系统，作为 TetR-VP16 嵌合体（tTA 和 rtTA）的替代物，以避免由于表达转录激活结构域而产生的次要影响。然而，以前描述的基于 TetR 的系统需要细胞克隆和/或抗生素选择四环素反应性细胞，以实现良好的调控。在本手稿中，我们构建了一个基于两个慢病毒载体的双 Tet-ON 系统，一个通过脾脏焦点形成病毒（SFFV）启动子（STetR）表达 TetR，另一个通过可调控的 CMV-TetO 启动子（CTetOE）表达 eGFP。使用这些载体，我们证明了 TetR 阻遏物，与反向转录激活剂（rtTA）相反，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生成可调控的大量细胞系（表达高水平的 TetR），这些细胞系可以通过改变强力霉素浓度和/或改变每个细胞的 CTetOE 载体基因组的数量来调节转基因表达。基于这些结果，我们开发了一种新的一体式慢病毒载体（CEST），通过 SFFV 启动子驱动 TetR 的表达，并通过强力霉素反应性 CMV-TetO 操纵子驱动 eGFP 的表达。该载体有效地产生了 Tet-ON 可调控的永生化（293T）和原代（人骨髓间充质干细胞和人原代成纤维细胞）细胞。大量强力霉素反应性细胞系在低浓度强力霉素的情况下表达高水平的转基因，并与其亲本细胞在表型上没有明显区别。
