New insights into the mechanisms of gene electrotransfer--experimental and theoretical analysis

Scientific Reports
Mojca Pavlin, Maša Kandušer

Abstract

Gene electrotransfer is a promising non-viral method of gene delivery. In our in vitro study we addressed open questions about this multistep process: how electropermeabilization is related to electrotransfer efficiency; the role of DNA electrophoresis for contact and transfer across the membrane; visualization and theoretical analysis of DNA-membrane interaction and its relation to final transfection efficiency; and the differences between plated and suspended cells. Combinations of high-voltage and low-voltage pulses were used. We obtained that electrophoresis is required for the insertion of DNA into the permeabilized membrane. The inserted DNA is slowly transferred into the cytosol, and nuclear entry is a limiting factor for optimal transfection. The quantification and theoretical analysis of the crucial parameters reveals that DNA-membrane interaction (NDNA) increases with higher DNA concentration or with the addition of electrophoretic LV pulses while transfection efficiency reaches saturation. We explain the differences between the transfection of cell suspensions and plated cells due to the more homogeneous size, shape and movement of suspended cells. Our results suggest that DNA is either translocated through the stabl...Continue Reading

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Citations

Aug 16, 2017·Scientific Reports·Maša KandušerIgor Škrjanc
Jun 18, 2017·Journal of Molecular Neuroscience : MN·Sonja Prpar MihevcTomaz Mars
Aug 23, 2019·International Journal of Molecular Sciences·Sonam ChopraSaulius Satkauskas
Nov 8, 2020·Journal of Controlled Release : Official Journal of the Controlled Release Society·Bastien DuckertMaarten Fauvart
Jan 29, 2017·Molecular Therapy : the Journal of the American Society of Gene Therapy·Mao MaoFan Yuan
Aug 23, 2021·Biomedical Engineering Online·Saša Haberl Meglič, Mojca Pavlin

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Methods Mentioned

BETA
transfection
electrophoresis
flow cytometry
fluorescence microscopy

Software Mentioned

MetaMorph
FlowJo ( Tree Star )

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