GFP-based fluorescence assay for CAG repeat instability in cultured human cells
Beatriz A Santillan1, Christopher Moye2, David Mittelman3
1Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, United States of America; Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.
Plos One
|November 26, 2014
Summary
Scientists developed a GFP-based assay to screen for modifiers of CAG repeat instability in human cells. This tool quantifies repeat changes, aiding research into neurological diseases caused by trinucleotide repeat expansions.
Area of Science:
- Genetics
- Molecular Biology
- Neuroscience
Background:
- Trinucleotide repeats, particularly CAG repeats, are prone to instability and expansion.
- CAG repeat expansions are implicated in numerous human neurological disorders, including Huntington's disease and spinocerebellar ataxias.
- Understanding the mechanisms of CAG repeat instability is crucial for developing therapeutic strategies.
Purpose of the Study:
- To develop and validate a quantitative, scalable assay for screening modifiers of CAG repeat instability in human cells.
- To facilitate the discovery of potential therapeutic targets for trinucleotide repeat expansion disorders.
Main Methods:
- Development of a GFP-based assay utilizing an engineered intronic CAG repeat tract.
- The assay links CAG repeat length to the expression of a reporter gene (GFP), where longer repeats impair GFP function.
- Validation using known inducers of CAG repeat instability, such as transcription and engineered nucleases.
Main Results:
- The GFP-based assay demonstrates a length-dependent inverse correlation between CAG repeat length and GFP fluorescence intensity.
- The assay successfully detected changes in repeat tract length in live cells.
- Validation experiments confirmed the assay's ability to respond to known modifiers of repeat instability.
Conclusions:
- The developed GFP-based assay provides a quantitative and scalable method for studying CAG repeat instability in human cells.
- This assay is a valuable tool for high-throughput screening of chemical and shRNA libraries to identify modifiers and potential therapeutic interventions for neurological diseases associated with CAG repeat expansions.
