Ono Akihiko, Ito Akira, Sato Tomonaga, Yamaguchi Masaki, Suzuki Taiga, Kawabe Yoshinori, Kamihira Masamichi
Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
J Biosci Bioeng. 2017 Jul;124(1):115-124. doi: 10.1016/j.jbiosc.2017.02.012. Epub 2017 Mar 9.
Precise control of gene expression using an artificial gene circuit is a major challenge in the application of synthetic biology. Here, we designed a hypoxia-responsive transgene expression system by combining a hypoxia-inducible RTP801 promoter and a tetracycline-responsive transactivator fused with an oxygen-dependent degradation domain (TA-ODD). The reporter gene expression was highly induced by hypoxia when a transactivator-expression plasmid, pRTP801/TA-ODD, harboring a TA-ODD gene driven by the RTP801 promoter, was cotransfected with a reporter plasmid, pTRE/EGFP, harboring an EGFP gene controlled under the transactivator-responsive promoter. A stable cell line into which the expression units RTP801/TA-ODD and TRE/EGFP had been introduced responded to hypoxia and expressed the reporter gene in an oxygen-concentration-dependent manner. Moreover, the cells demonstrated potential as sensors to detect hypoxic conditions in a three-dimensional tissue culture in vitro. These results indicate that the hypoxia-responsive transgene expression system is useful for constructing cell-based hypoxia detection systems.
利用人工基因回路精确控制基因表达是合成生物学应用中的一项重大挑战。在此，我们通过将缺氧诱导的RTP801启动子与融合了氧依赖性降解结构域的四环素反应性反式激活因子（TA-ODD）相结合，设计了一种缺氧反应性转基因表达系统。当携带由RTP801启动子驱动的TA-ODD基因的反式激活因子表达质粒pRTP801/TA-ODD与携带受反式激活因子反应性启动子控制的EGFP基因的报告质粒pTRE/EGFP共转染时，报告基因的表达在缺氧条件下被高度诱导。导入了表达单元RTP801/TA-ODD和TRE/EGFP的稳定细胞系对缺氧有反应，并以氧浓度依赖性方式表达报告基因。此外，这些细胞在体外三维组织培养中显示出作为检测缺氧条件传感器的潜力。这些结果表明，缺氧反应性转基因表达系统可用于构建基于细胞的缺氧检测系统。
