Vogel Roland, Amar Lahouari, Thi Anh Do, Saillour Paulette, Mallet Jacques
Laboratoire de Génétique Moléculaire de la Neurotransmission et des Processus Neurodégénératifs (LGN), CNRS-UMR 7091, Hôpital de la Pitié Salpêtrière, 83 Boulevard de l'Hôpital, 75013 Paris, France.
Hum Gene Ther. 2004 Feb;15(2):157-65. doi: 10.1089/104303404772679968.
A single lentivirus vector allowing doxycycline-regulated expression of transgenes in the brain was generated by incorporating the tetracycline (Tet)-dependent regulatory system into the backbone of the vector. Two distinct expression cassettes were inserted upstream and downstream from the central Flap sequence that provides for enhanced transduction of nondividing cells. The first cassette was used to express the transgene under the control of the Tet-dependent minimal cytomegalovirus promoter. The second cassette was employed to express constitutively the Tet-dependent transactivator rtTA2-M2, which activates the Tet-dependent promoter after binding of doxycycline (Tet-on system). Vectors carrying luciferase and tyrosine hydroxylase as the transgene were constructed, tested in astroglia-rich primary cultures, and injected into the striata of rats. The constructs allowed in vitro and in vivo robust expression of the transgene that could be regulated over two orders of magnitude by the addition and withdrawal of doxycycline. The vector may thus be useful for many applications in gene therapy research, including the development of a therapeutic protocol for the treatment of Parkinson's disease based on the restoration of regulated dopamine production.
通过将四环素（Tet）依赖性调控系统整合到载体骨架中，构建了一种可在脑内实现强力霉素调控转基因表达的慢病毒载体。在中央Flap序列的上下游插入了两个不同的表达盒，该序列可增强对非分裂细胞的转导。第一个表达盒用于在Tet依赖性最小巨细胞病毒启动子的控制下表达转基因。第二个表达盒用于组成型表达Tet依赖性反式激活因子rtTA2-M2，其在强力霉素结合后激活Tet依赖性启动子（Tet-on系统）。构建了携带荧光素酶和酪氨酸羟化酶作为转基因的载体，在富含星形胶质细胞的原代培养物中进行测试，并注射到大鼠纹状体中。这些构建体允许转基因在体外和体内进行强力表达，通过添加和去除强力霉素，表达可在两个数量级上进行调控。因此，该载体可能在基因治疗研究的许多应用中有用，包括基于恢复受调控的多巴胺产生来开发治疗帕金森病的治疗方案。
