Hypoxia-Sensitive Reporter System for High-Throughput Screening

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Author(s)

    • Tsujita Tadayuki
    • Department of Medical Biochemistry, Tohoku University Graduate School of Medicine|Department of Molecular Medicine and Therapy, Tohoku University Graduate School of Medicine
    • Kawaguchi Shin-ichi
    • Department of Molecular Medicine and Therapy, Tohoku University Graduate School of Medicine
    • Dan Takashi
    • Department of Molecular Medicine and Therapy, Tohoku University Graduate School of Medicine
    • Baird Liam
    • Department of Medical Biochemistry, Tohoku University Graduate School of Medicine
    • Miyata Toshio
    • Department of Molecular Medicine and Therapy, Tohoku University Graduate School of Medicine
    • Yamamoto Masayuki
    • Department of Medical Biochemistry, Tohoku University Graduate School of Medicine

Abstract

The induction of anti-hypoxic stress enzymes and proteins has the potential to be a potent therapeutic strategy to prevent the progression of ischemic heart, kidney or brain diseases. To realize this idea, small chemical compounds, which mimic hypoxic conditions by activating the PHD-HIF-α system, have been developed. However, to date, none of these compounds were identified by monitoring the transcriptional activation of hypoxia-inducible factors (HIFs). Thus, to facilitate the discovery of potent inducers of HIF-α, we have developed an effective high-throughput screening (HTS) system to directly monitor the output of HIF-α transcription. We generated a HIF-α-dependent reporter system that responds to hypoxic stimuli in a concentration- and time-dependent manner. This system was developed through multiple optimization steps, resulting in the generation of a construct that consists of the secretion-type luciferase gene (<i>Metridia</i> luciferase, MLuc) under the transcriptional regulation of an enhancer containing 7 copies of 40-bp hypoxia responsive element (HRE) upstream of a mini-TATA promoter. This construct was stably integrated into the human neuroblastoma cell line, SK-N-BE(2)c, to generate a reporter system, named SKN:HRE-MLuc. To improve this system and to increase its suitability for the HTS platform, we incorporated the next generation luciferase, Nano luciferase (NLuc), whose longer half-life provides us with flexibility for the use of this reporter. We thus generated a stably transformed clone with NLuc, named SKN:HRE-NLuc, and found that it showed significantly improved reporter activity compared to SKN:HRE-MLuc. In this study, we have successfully developed the SKN:HRE-NLuc screening system as an efficient platform for future HTS.

Journal

  • The Tohoku Journal of Experimental Medicine

    The Tohoku Journal of Experimental Medicine 235(2), 151-159, 2015

    Tohoku University Medical Press

Codes

  • NII Article ID (NAID)
    130004822440
  • Text Lang
    ENG
  • ISSN
    0040-8727
  • Data Source
    J-STAGE 
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