Yao Feng, Theopold Christoph, Hoeller Daniela, Bleiziffer Oliver, Lu Zheming
Laboratory of Tissue Repair and Gene Transfer, Department of Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Mol Ther. 2006 Jun;13(6):1133-41. doi: 10.1016/j.ymthe.2006.01.009. Epub 2006 Mar 29.
Employing the tetracycline repressor tetR and the wild-type hCMV major immediate-early promoter, we have developed a highly sensitive tetracycline-inducible transcription switch in mammalian cells (T-REx; Invitrogen, Carlsbad, CA, USA). In view of the previous difficulty in achieving regulatable gene expression in recombinant HSV vector systems, we constructed a T-REx-encoding replication-defective HSV-1 recombinant, QR9TO-lacZ, that encodes two copies of the tetR gene controlled by the HSV-1 immediate-early ICP0 promoter and a reporter, the LacZ gene, under the control of the tetO-bearing hCMV major immediate-early promoter. Infection of cells, such as Vero, PC12, and NGF-differentiated PC12 cells, with QR9TO-lacZ led to 300- to 1000-fold tetracycline-regulated gene expression. Moreover, the expression of the LacZ gene by QR9TO-lacZ can be finely controlled by tetracycline in a dose-dependent fashion. Efficiently regulated gene expression can also be achieved in vivo following intracerebral and footpad inoculations in mice. The demonstrated capability of T-REx for achieving high levels of sensitively regulated gene expression in the context of the HSV-1 genome will significantly expand the utility of HSV-based vector systems for studying gene function in the nervous system and delivering regulated gene expression in therapeutic applications, particularly in the treatment of CNS diseases.
利用四环素阻遏物tetR和野生型人巨细胞病毒主要立即早期启动子，我们在哺乳动物细胞中开发了一种高度敏感的四环素诱导转录开关（T-REx；美国加利福尼亚州卡尔斯巴德市英杰公司）。鉴于先前在重组单纯疱疹病毒载体系统中实现可调控基因表达存在困难，我们构建了一个编码T-REx的复制缺陷型单纯疱疹病毒1型重组体QR9TO-lacZ，它编码由单纯疱疹病毒1型立即早期ICP0启动子控制的两个tetR基因拷贝，以及一个由带有tetO的人巨细胞病毒主要立即早期启动子控制的报告基因LacZ基因。用QR9TO-lacZ感染细胞，如Vero细胞、PC12细胞和经神经生长因子分化的PC12细胞，可导致四环素调控的基因表达增加300至1000倍。此外，QR9TO-lacZ对LacZ基因的表达可通过四环素以剂量依赖方式进行精细调控。在小鼠脑内和足垫接种后，体内也能实现高效调控的基因表达。T-REx在单纯疱疹病毒1型基因组背景下实现高水平敏感调控基因表达的能力，将显著扩展基于单纯疱疹病毒的载体系统在研究神经系统基因功能以及在治疗应用（特别是中枢神经系统疾病治疗）中递送调控基因表达方面的用途。
