Lentiviral vectors: Regulated gene expression
Lentiviral vectors: Regulated gene expression
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DOI：
10.1006/mthe.2000.0083
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发表时间：
2000-06-01
影响因子：
12.4
通讯作者：
Verma, IM
中科院分区：
文献类型：
--
作者：
Kafri, T;van Praag, H;Verma, IM
慢病毒载体可以在多种分裂和非分裂细胞中传递和表达基因。这些细胞包括终末分化的神经元、肌管、肝细胞和造血干细胞。我们现在描述慢病毒载体的产生，在其中转基因的表达可以被调节。我们已经开发了一种可诱导的慢病毒载体系统，它包含了H.Bujard和他的同事开发的整个四环素(Tet)调控系统。新载体分别在四环素可诱导启动子和人CMV启动子的控制下表达GFP报告基因和四环素反式激活因子。在体外转导人293细胞后，在效应物质多西环素存在的情况下，GFP的基础表达非常低。停用多西环素后，转基因表达增加了500多倍。“开关”转基因表达并不会改变诱导的动力学或程度。药物对GFP基因转录的抑制作用在24 h内达到最大值，但由于GFP的周转速度较慢，经多西环素处理后10天仍可检测到绿色荧光细胞。重组慢病毒转导大鼠脑后，在终末分化的神经元中可以观察到多西环碱调节的GFP表达。具体地说，通过从大鼠饮用水中添加或取消多西环素，可以在体内调节对GFP表达的诱导和抑制。这些研究表明，可诱导的慢病毒载体可以在体内传递和调节转基因表达。我们认为，受调控的基因表达是成功的基因治疗方法的重要工具。
Lentiviral vectors can deliver and express genes in a wide variety of dividing and nondividing cells. These include terminally differentiated neurons, myotubes, hepatocytes, and hematopoietic stem cells. We now describe the generation of lentiviral vectors in which the expression of the transgene can be regulated. We have developed an inducible lentiviral vector system that contains the entire tetracycline (Tet)-regulated system developed by H. Bujard and colleagues. The novel vector expresses the GFP reporter gene and the tetracycline transactivator under the control of the tetracycline-inducible promoter and the human CMV promoter, respectively. In vitro transduction of human 293 cells resulted in a very low basal expression of GFP in the presence of the effector substance doxycyline. Withdrawal of doxycyline induced a more than 500-fold increase in transgene expression. Switching transgene expression "off and on" did not change either the kinetics or the magnitude of induction. Maximal suppression of GFP mRNA transcription was achieved within 24 h of addition of the drug; however, due to the slow turnover rate of GFP, green fluorescent cells could be detected up to 10 days following doxycyline treatment. Following transduction of rat brain with recombinant lentiviruses, doxycyline-regulated GFP expression could be observed in terminally differentiated neurons. Specifically, by adding or withdrawing doxycyline from the rats' drinking water, induction and suppression of GFP expression could be regulated in vivo. These studies show that an inducible lentiviral vector can deliver and regulate transgene expression in vivo. We believe that regulated gene expression is an essential tool for successful gene therapy approaches.