Hypoxia-responsive transgene expression system using RTP801 promoter and synthetic transactivator fused with oxygen-dependent degradation domain

Abstract

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.

Keywords: Gene expression system; Hypoxia; Oxygen-dependent degradation domain; RTP801 promoter; Three-dimensional culture; Transcriptional amplification.