Hypoxia-sensitive reporter system for high-throughput screening

Tadayuki Tsujita, Shin Ichi Kawaguchi, Takashi Dan, Liam Baird, Toshio Miyata, Masayuki Yamamoto

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

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 (Metridia 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.

Original languageEnglish
Pages (from-to)151-159
Number of pages9
JournalTohoku Journal of Experimental Medicine
Volume235
Issue number2
DOIs
Publication statusPublished - 2015 Feb 25

Keywords

  • Chemical screening
  • High-throughput screening
  • Hypoxia responsive element
  • Hypoxia stress
  • Reporter cell system

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Fingerprint Dive into the research topics of 'Hypoxia-sensitive reporter system for high-throughput screening'. Together they form a unique fingerprint.

Cite this