Kühnel Florian, Fritsch Corinna, Krause Sabine, Mundt Bettina, Wirth Thomas, Paul Yasmin, Malek Nisar Peter, Zender Lars, Manns Michael Peter, Kubicka Stefan
Department of Gastroenterology and Hepatology, Medical School Hannover, Carl Neuberg Str. 1, D-30625 Hannover, Germany.
Nucleic Acids Res. 2004 Feb 13;32(3):e30. doi: 10.1093/nar/gnh034.
The tetracycline system has limitations in liver cells, such as toxic effects and low controllability. We generated different retroviral vectors for controlled gene expression in liver cells, in which the regulatory elements were arranged in different patterns. Only the organization of the tetracycline system in an autoregulatory loop in the sense orientation results in high retroviral titres and in tight regulation of gene expression in highly differentiated hepatoma cells. Because of the toxicity of the transactivator tTA, it was impossible to establish doxycycline-dependent stable HepG2 cell lines. To avoid sequelching-related toxicity in liver cells, we replaced tTA with new non-toxic transactivators. By using tTA2, tTA3 and tTA4, we observed tight doxycycline-dependent gene expression in 23, 49 and 45% of the isolated clones. The tTA4 vector was used to transduce hepatocytes of mice in vivo. Tight doxycycline-controllable gene regulation was also observed in the liver of mice, confirming our hypothesis that retroviral vectors with autoregulatory loops of the tetracycline system facilitate inducible gene expression in the liver in vivo. Our new retroviral vector system allows rapid isolation of controllable clones in a very high yield and should make the tetracycline system more applicable to liver-derived cells and in liver gene therapy in vivo.
四环素系统在肝细胞中存在局限性，如毒性作用和低可控性。我们构建了不同的逆转录病毒载体用于肝细胞中的可控基因表达，其中调控元件以不同模式排列。只有四环素系统以正向自调控环的形式组织时，才能在高度分化的肝癌细胞中产生高逆转录病毒滴度并实现基因表达的严格调控。由于反式激活因子tTA具有毒性，因此无法建立强力霉素依赖性稳定的HepG2细胞系。为避免肝细胞中与封闭相关的毒性，我们用新的无毒反式激活因子取代了tTA。通过使用tTA2、tTA3和tTA4，我们在分离的克隆中分别有23%、49%和45%观察到了强力霉素依赖性的严格基因表达。tTA4载体用于体内转导小鼠肝细胞。在小鼠肝脏中也观察到了强力霉素可控的严格基因调控，证实了我们的假设，即具有四环素系统自调控环的逆转录病毒载体有助于体内肝脏中的诱导型基因表达。我们新的逆转录病毒载体系统能够以非常高的产量快速分离可控克隆，并且应该会使四环素系统更适用于肝脏来源的细胞以及体内肝脏基因治疗。
