Identification and Screening Solutions for Antioxidant Gene Transcription Activators
One mechanism by which humans protect themselves from oxidative stress is through endogenous stress responses leading to transcriptional activation of antioxidant response elements (ARE). Increasing levels of antioxidant enzymes without causing oxidative stress can potentially resist degeneration and may be therapeutically useful.
Creative Bioarray can identify ARE activators on a genomic scale by screening expression cDNA libraries in high-throughput reporter gene analysis and testing the ability of cDNAs to upregulate endogenous AREs at the transcriptional and protein levels to regulate gene expression and their ability to protect cells from oxidative damage.
Identification of ARE Activators Gene
In traditional small-scale or one-gene at a time studies, it is not possible to rapidly identify positive ARE regulators. So we screened identified ARE activators by a high-throughput genome-wide cDNA library, using another reporter gene that was unresponsive to ARE activators, to find genes involved in protection against oxidative stress.
High-Throughput Genome-Wide Screening
Approximately 15,000 expression cDNAs, standardized and arranged in 384-well plates, are transfected into cells along with the ARE-luciferase reporter construct. After 48 hours of incubation, luciferase activity was assessed by measuring the luminescence output of each well.
Hit Confirmation
ARE Activators candidate genes obtained from high-throughput screening were transfected into cells, which were treated with an excess of antioxidant, N-acetylcysteine (1 mM final concentration) or solvent (DMEM). Luciferase activity was analyzed by luminescence.
Validation of ARE Activators Gene Candidates
Verify whether candidate ARE-activated genes can induce endogenous ARE-regulated genes at the transcriptional level (real-time PCR)
- Overexpression of candidate cDNAs in cells
- Real-time fluorescence quantitative PCR assay
Verify whether candidate ARE activation genes can induce endogenous ARE regulatory genes at the protein level (immunoblot analysis)
- Overexpression of candidate cDNAs in cells
- Protein blotting
Protection Assay of ARE Activators Gene Candidates
- Overexpression of candidate cDNAs in cells
- Oxidative stimulation
- Cell viability assay
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