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High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay
Published on: June 1, 2014
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[Constructing a p53-fused dual luciferase reporter and verifying its function]
Jing Tan1, Xianzhen Jiang, Leye He
1Department of Urology, Third Xiangya Hospital, Central South University, Changsha 410013, China.
Summary
A novel dual luciferase reporter system was developed to mimic wild-type p53 activities. This p53 reporter effectively identifies modulators of p53 protein levels in high-throughput screening.
Area of Science:
- Molecular Biology
- Cancer Research
- Biotechnology
Background:
- The tumor suppressor protein p53 plays a critical role in cellular responses to stress.
- Developing tools to accurately assess p53 activity is crucial for cancer research and drug discovery.
Purpose of the Study:
- To construct a p53-fused dual luciferase reporter system.
- To validate the reporter's ability to mimic wild-type p53 functions in a high-throughput screening context.
Main Methods:
- Engineered a fusion protein linking full-length p53 to firefly luciferase.
- Inserted the fusion construct upstream of a renilla luciferase reporter gene.
- Utilized PCR to modify open reading frames and added restriction sites for cloning.
- Assessed reporter function by testing MDM2-mediated degradation, nuclear localization, and promoter induction.
Main Results:
- Successfully constructed the p53-fused dual luciferase reporter (p53FL/IRES/RL).
- The expressed p53 fusion protein demonstrated MDM2-mediated degradation.
- The fusion protein exhibited predominantly nuclear localization.
- The reporter successfully induced a p53-responsive promoter.
Conclusions:
- The developed p53-fused dual luciferase reporter accurately reflects wild-type p53 activities.
- This reporter system is suitable for high-throughput screening to identify modulators of p53 protein levels.
