Apoptosis induced by DNA uptake limits transfection efficiency.

Area of Science:

• Cell Biology
• Molecular Biology
• Biotechnology

Background:

• Electrotransfection is a key method for introducing genetic material into cells.
• Certain lymphoid cells, like L1210 subclones and NFS-70 pro-B cells, exhibit low transfection efficiency.
• Identifying factors limiting transfection in refractory cells is crucial for improving gene delivery.

Purpose of the Study:

• To investigate the reasons behind low electrotransfection efficiency in specific lymphoid cells.
• To determine the role of apoptosis in limiting transfection success.
• To explore strategies for enhancing cell viability and transgene expression.

Main Methods:

• Electrotransfection of L1210 subclones and NFS-70 pro-B cells with reporter gene plasmids.
• Assessment of cell viability using propidium iodide/acrindine orange staining and DNA laddering.
• Analysis of apoptosis via caspase activity and inhibition.
• Comparison of transfection methods including lipid-based delivery.

Main Results:

• Electrotransfection of refractory lymphoid cells with DNA induced apoptosis, correlating with decreased cell viability and transgene expression.
• Apoptosis was dependent on the presence of DNA during electroporation but not DNA conformation or transgene expression.
• Addition of dextran sulfate or caspase inhibitors (Boc-ASP-FMK) significantly reduced apoptosis and enhanced cell viability and transfection efficiency.
• Lipid-based transfection also induced apoptosis, albeit at lower levels.

Conclusions:

• Apoptosis is a major limiting factor for electrotransfection efficiency in refractory lymphoid cells.
• Caspase inhibition is a viable strategy to improve cell survival and boost transgene expression after electrotransfection.
• Understanding and mitigating apoptosis is essential for optimizing gene delivery into challenging cell types.