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Updated: Feb 3, 2026
A Reporter Based Cellular Assay for Monitoring Splicing Efficiency
Published on: September 15, 2021
Development of a fluorescence-based cellular apoptosis reporter
Lucy A Balderstone1, John C Dawson, Arkadiusz Welman
1Cancer Research UK Edinburgh Centre, Institute of Genetics & Molecular Medicine, University of Edinburgh, Crewe Road South, Edinburgh, EH4 2XR, United Kingdom.
Abstract:
Evasion of apoptosis is a hallmark of human cancer, and a desired endpoint of many anticancer agents is the induction of cell death. With the heterogeneity of cancer becoming increasingly apparent, to understand drug mechanisms of action and identify combination therapies in cell populations, the development of tools to assess drug effects at the single cell level is a necessity for future preclinical drug development. Herein we describe the development of pCasFSwitch, a genetically encoded reporter construct designed to identify cells undergoing caspase-3 mediated apoptosis, by a translocation of a GFP signal from the cell membrane into the nucleus. Anticipated cellular distribution was demonstrated by use of confocal microscopy and cleavage by caspase-3 was shown to be required for the translocation of the GFP signal seen in apoptotic cells. Quantification of apoptosis using the construct revealed similar levels to that obtained with a commercially available apoptosis imaging agent (22.6% versus 20.3%). Moreover, we demonstrated its capacity for use in a high-throughput setting making it a powerful tool for drug development pipelines.
Insights
Researchers developed pCasFSwitch, a novel reporter for tracking apoptosis (programmed cell death) at the single-cell level. This tool aids in understanding anticancer drug mechanisms and identifying new combination therapies.
Area of Science:
- Biotechnology
- Molecular Biology
- Cancer Research
Background:
- Apoptosis evasion is a key feature of human cancers.
- Understanding drug mechanisms and combination therapies requires single-cell analysis.
- Existing methods for apoptosis detection may lack precision or throughput.
Purpose of the Study:
- To develop a genetically encoded reporter for identifying cells undergoing caspase-3 mediated apoptosis.
- To validate the reporter's functionality and accuracy in detecting apoptosis.
- To assess the reporter's utility in high-throughput drug screening.
Main Methods:
- Development of the pCasFSwitch reporter construct.
- Confocal microscopy to visualize GFP signal translocation.
- Caspase-3 cleavage assays to confirm mechanism of action.
- Comparison with a commercial apoptosis imaging agent.
- High-throughput screening assays.
Main Results:
- pCasFSwitch demonstrated correct cellular distribution and GFP signal translocation upon caspase-3 activation.
- Caspase-3 cleavage was confirmed as essential for the observed reporter activity.
- Apoptosis quantification by pCasFSwitch showed comparable results to a commercial agent (22.6% vs. 20.3%).
- The reporter proved effective in a high-throughput setting.
Conclusions:
- pCasFSwitch is a reliable tool for detecting caspase-3 mediated apoptosis at the single-cell level.
- The reporter facilitates the study of drug-induced cell death in cancer research.
- Its high-throughput capability makes it valuable for preclinical drug development pipelines.
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