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Direct visualization at the single-cell level of electrically mediated gene delivery
Muriel Golzio1, Justin Teissie, Marie-Pierre Rols
1Institut de Pharmacologie et de Biologie Structurale Centre National de la Recherche Scientifique/Unité Mixte de Recherche-5089, 205, Route de Narbonne, 31077 Toulouse Cedex, France.
Summary
Electropermeabilization facilitates gene transfer by creating localized plasmid aggregates on the cell membrane. These aggregates form after specific electrical pulses and are crucial for subsequent DNA uptake and intracellular transport.
Area of Science:
- Biotechnology
- Cell Biology
- Molecular Biology
Background:
- Electropermeabilization is a key nonviral method for gene transfer in gene therapy.
- The fundamental mechanisms of DNA transfer via electropermeabilization remain largely uncharacterized.
- Understanding these processes is vital for advancing gene therapy applications.
Purpose of the Study:
- To investigate the single-cell dynamics of DNA transfer during electropermeabilization.
- To elucidate the spatiotemporal interactions between plasmids and cell membranes post-electroporation.
- To identify critical parameters influencing DNA uptake and intracellular trafficking.
Main Methods:
- Digitized fluorescence microscopy was employed for real-time, single-cell analysis.
- Propidium iodide (PI) penetration was used to assay membrane permeabilization.
- Fluorescently labeled plasmids were tracked to observe their interaction with cell membranes and intracellular localization.
Main Results:
- Permeabilization occurs at electrode-facing membrane regions; plasmid interaction is limited to the cathode-facing side.
- Localized plasmid-membrane aggregates form after electrical pulses exceeding a critical duration.
- These aggregates are stable, protected from external dyes after 10 minutes, and translocate to the cytoplasm and nucleus over 24 hours.
Conclusions:
- Electropermeabilization induces specific, localized plasmid-membrane interactions crucial for gene transfer.
- The duration of electrical pulses significantly influences aggregate formation and subsequent DNA trafficking.
- These findings provide fundamental insights into the mechanism of electropermeabilization-mediated gene delivery.