Generation of Cell Models with Stable Expression of a Caspase-3 Activity Fluorescent Sensor

Area of Science:

• Biotechnology and Biomedical Research
• Cell Biology and Molecular Medicine

Background:

• Apoptosis, or programmed cell death, is crucial in development and disease.
• Studying apoptosis in specific cell types like mesenchymal stem cells (MSCs) and cancer stem cells (CSCs) requires precise monitoring tools.
• Existing methods for apoptosis detection can be limited in real-time monitoring within live cell populations.

Purpose of the Study:

• To develop and validate a lentiviral vector for delivering the ZipGFP-Casp3 biosensor into various cell cultures.
• To establish reliable cell models for studying apoptosis in MSCs and colorectal CSCs.
• To assess the sensitivity of these engineered cell models to known apoptosis-inducing agents.

Main Methods:

• Construction of a lentiviral vector for the fluorogenic protease biosensor ZipGFP-Casp3.
• Transduction of mesenchymal stem cells (liver and placenta), dermal fibroblasts, and colorectal carcinoma cell lines (HT-29, Caco2) with CD133 knockout and control clones.
• Cell sorting, monitoring of GFP fluorescence for apoptosis, and treatment with apoptosis inducers (staurosporine, TRAIL).

Main Results:

• Successful transduction of target cell cultures with the ZipGFP-Casp3 sensor.
• Observation of stable GFP fluorescence in cells undergoing spontaneous apoptosis.
• Demonstrated sensitivity of engineered MSCs and HT-29 clones to staurosporine and TRAIL, confirming apoptosis induction.

Conclusions:

• The developed lentiviral vector and engineered cell cultures provide a robust platform for studying apoptosis.
• These models are suitable for investigating apoptosis mechanisms in both mesenchymal stem cells and cancer stem cell populations.
• The ZipGFP-Casp3 biosensor facilitates real-time, quantitative analysis of apoptosis in complex cellular contexts.