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High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay
Published on: June 1, 2014
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High-throughput functional screening using a homemade dual-glow luciferase assay
Jessica M Baker1, Frederick M Boyce2
1Department of Neurology, Massachusetts General Hospital.
Journal of Visualized Experiments : Jove
|June 26, 2014
Summary
We developed a fast, affordable high-throughput screening method to find genes regulating alpha-synuclein, a key factor in Parkinson's disease. This approach identified 68 new regulators, offering insights into disease mechanisms.
Area of Science:
- Molecular Biology
- Neuroscience
- Genetics
Background:
- Alpha-synuclein is a gene strongly associated with Parkinson's disease.
- Identifying its transcriptional regulators is crucial for understanding disease mechanisms and developing therapies.
Purpose of the Study:
- To develop and validate a rapid, inexpensive, high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein.
- To screen a large library of human genes for their effect on alpha-synuclein transcription.
Main Methods:
- Utilized 293T cells transiently transfected with an arrayed ORF expression library and luciferase reporter plasmids in a 96-well format.
- Employed automated liquid handling for aseptic transfections and a novel, cost-effective reagent to suppress firefly luciferase activity.
- Normalized luciferase activity to an internal control (Renilla luciferase) to assess gene effects on alpha-synuclein transcription.
Main Results:
- Screened 7,670 human genes using the developed protocol.
- Identified 68 novel transcriptional regulators of alpha-synuclein.
- Demonstrated the protocol's adaptability for other genes and high-throughput applications.
Conclusions:
- The presented protocol is a powerful and economical tool for identifying gene regulators in a high-throughput manner.
- This method significantly advances the study of alpha-synuclein and Parkinson's disease by uncovering new regulatory factors.
- The protocol's flexibility allows for broad application in functional genomics and drug discovery.
