title: Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level.
abstract: BACKGROUND
  Gene electrotransfer is a nonviral method used for DNA delivery into cells. Several steps are involved. One of them is the interaction of DNA with the cell membrane, which is crucial before DNA can enter the cell. We analysed the level of DNA-membrane interaction in relation to electrotransfer efficiency and the importance of the electrophoretic accumulation of DNA at the cell membrane. Systematic comparison of long-duration, short-duration and combinations of electropermeabilizing short (high-voltage; HV) and electrophoretic long (low-voltage; LV) pulses were performed. The effect of Mg(2+) ion concentrations on electrotransfer and their effect on DNase activity were explored.
  
  
  METHODS
  To visualize the DNA-membrane interaction, TOTO-1 labeled DNA was used. Transfection efficiency was assessed with plasmid DNA coding for green fluorescent protein.
  
  
  RESULTS
  Higher relative electrotransfer efficiency was obtained by using longer pulses, whereas shorter pulses preserved cell viability. Short-duration pulses enabled higher (24%) overall transfection yield compared to long-duration pulses (12%), although a higher DNA-membrane interaction was observed. No significant difference in transfection was obtained between different HV-LV pulsing protocols, although the highest DNA-membrane interaction was observed with HV + LV pulses. The formation of the DNA-membrane complex depended on the Mg(2+) concentration, whereas DNase inhibitor did not affect gene expression.
  
  
  CONCLUSIONS
  Gene electrotransfer is a complex phenomenon, where many factors mutually affect the process and the DNA-membrane interaction only comprises the first step. We showed that longer electric pulses are optimal for higher transfection efficiency but reduce viability, whereas shorter pulses enable moderate transfection efficiency and preserve viability. Thus, each application needs a careful choice of pulsing protocol.
authors.affiliation: University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Biocybernetics, Ljubljana, Slovenija.
authors.forename: Saša
authors.initials: S
authors.lastname: Haberl
authors.forename: Maša
authors.initials: M
authors.lastname: Kandušer
authors.forename: Karel
authors.initials: K
authors.lastname: Flisar
authors.forename: Duša
authors.initials: D
authors.lastname: Hodžić
authors.forename: Vladimir Boštjan
authors.initials: VB
authors.lastname: Bregar
authors.forename: Damijan
authors.initials: D
authors.lastname: Miklavčič
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Escoffre
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Rols
authors.forename: Mojca
authors.initials: M
authors.lastname: Pavlin
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D006224:Cricetinae; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D018274:Electroporation; D015870:Gene Expression; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
chemical_list: C086329:TOTO-1 thiazole orange dye; D013844:Thiazoles; D049452:Green Fluorescent Proteins; D004247:DNA; D008274:Magnesium