Yoshida Y, Hamada H
Department of Molecular Biotherapy Research, Cancer Chemotherapy Center, Cancer Institute, Toshima-ku, Tokyo, Japan.
Biochem Biophys Res Commun. 1997 Jan 13;230(2):426-30. doi: 10.1006/bbrc.1996.5975.
Tetracycline-controllable expression vectors are widely used for inducible expression in mammalian cells. The limitation of this system is the difficulty in expressing high levels of the chimeric transactivator tTA. In this study, we demonstrate a utility of recombinant adenoviruses for the tetracycline-controllable expression system. Unexpectedly, the original tTA transactivator did not show sufficient regulation of the reporter gene expression driven by the tetracycline-responsive promoter (Tet). By adding the nuclear localization signal on the tTA transactivator (NtTA), we achieved tight regulation and high-level induction of the reporter gene expression. The NtTA driven by various promoters demonstrated strict tetracycline controllability at 1 microg/ml of tetracycline and above. The methodology for adenovirus-mediated inducible gene expression has wide applicability. Controllable expression of cytotoxic viral proteins will be applicable for antiviral vaccine productions and pseudotype viral vector generations.
四环素可控表达载体广泛用于哺乳动物细胞的诱导表达。该系统的局限性在于难以高水平表达嵌合反式激活因子tTA。在本研究中，我们证明了重组腺病毒在四环素可控表达系统中的效用。出乎意料的是，原始的tTA反式激活因子对四环素反应性启动子（Tet）驱动的报告基因表达没有充分的调控作用。通过在tTA反式激活因子上添加核定位信号（NtTA），我们实现了对报告基因表达的严格调控和高水平诱导。由各种启动子驱动的NtTA在1微克/毫升及以上的四环素浓度下表现出严格的四环素可控性。腺病毒介导的诱导基因表达方法具有广泛的适用性。细胞毒性病毒蛋白的可控表达将适用于抗病毒疫苗生产和假型病毒载体的产生。
