Hypoxia-sensitive reporter system for high-throughput screening.
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.
Department of Medical Biochemistry, Sendai, Miyagi, Japan; Department of Molecular Medicine and Therapy, Tohoku University Graduate School of Medicine, tada@med.tohoku.ac.jp
Tadayuki
T
Tsujita
Shin-ichi
S
Kawaguchi
Takashi
T
Dan
Liam
L
Baird
Toshio
T
Miyata
Masayuki
M
Yamamoto
D000818:Animals; D001483:Base Sequence; D015687:Cell Hypoxia / Q000235:genetics; D002460:Cell Line; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D020218:Response Elements / Q000235:genetics; D013312:Stress, Physiological / Q000235:genetics
D008156:Luciferases