title: 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.
authors.affiliation: Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Ono
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Ito
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Tomonaga
authors.initials: T
authors.lastname: Sato
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Yamaguchi
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Taiga
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kawabe
authors.affiliation: 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. Electronic address: kamihira@chem-eng.kyushu-u.ac.jp.
authors.forename: Masamichi
authors.initials: M
authors.lastname: Kamihira
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015687:Cell Hypoxia / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D010100:Oxygen / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D000072417:Protein Domains; D015534:Trans-Activators / Q000235:genetics*; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes / Q000235:genetics
chemical_list: C469991:DDIT4 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D010100:Oxygen
keywords: Gene expression system; Hypoxia; Oxygen-dependent degradation domain; RTP801 promoter; Three-dimensional culture; Transcriptional amplification