Investigating relationship between transfection and permeabilization by the electric field and/or the Pluronic® L64 in vitro and in vivo.
BACKGROUND
Electrotransfer can be obtained by the successive delivery of a high voltage short duration pulse (HV) inducing membrane destabilization and then a low voltage long duration pulse (LV), allowing DNA electrophoresis (HVLV mode). Pluronic® L64 (L64) (Fluka, Sigma-Aldrich, L'Isle-d'Abeau Chesnes, Saint-Quentin Fallavier, France) has permeabilizing properties and amplifies the expression of DNA. We aimed to determine whether L64 could have an adjuvant effect on transfection by electrotransfer and whether the sequence L64 injection and then application of a LV pulse could induce transfection comparable to that observed with the HVLV mode.


METHODS
In vitro, we used fluorescence-activated cell sorting to evaluate Chinese hamster ovary (CHO) cell transfection by a plasmid coding green fluorescent protein, and permeabilization to propidium iodide. In vivo, the transfection efficiency of mice tibial cranial muscle was evaluated by optical imaging using a plasmid DNA encoding luciferase. For the same animals, permeabilization indices were evaluated by magnetic resonance imaging from the uptake of a T(1) contrast agent.


RESULTS
Using the HVLV mode, transfection efficiency was low in vitro on CHO cells but high for muscles in vivo. Pre-treatment by L64 increased the transfection efficiency of electrotransfer for CHO cells but not for muscle. In mice muscles, the L64 amplified the expression of DNA. Nevertheless, neither transgene expression, nor permeability indices were further amplified by subsequent delivery of one LV pulse.


CONCLUSIONS
A major finding of the present study is that the nature of the membrane modification induced by electric pulses is not comparable to that mediated by L64. The electrophoretic LV pulse does not induce additive effects to that of L64 for transfection improvement.
Unité de Pharmacologie chimique et Génétique et d'Imagerie, CNRS UMR 8151, Paris, France; INSERM U 1022, Paris, France. michel-francis.bureau@parisdescartes.fr
Michel F
MF
Bureau
Luc
L
Wasungu
Lauriane
L
Jugé
Daniel
D
Scherman
Marie-Pierre
MP
Rols
Nathalie
N
Mignet
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004586:Electrophoresis / Q000379:methods; D018274:Electroporation / Q000379:methods*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D008156:Luciferases; D008279:Magnetic Resonance Imaging; D051379:Mice; D008807:Mice, Inbred BALB C; D018482:Muscle, Skeletal / Q000378:metabolism*; D010539:Permeability; D010957:Plasmids / Q000235:genetics; D020442:Poloxamer / Q000737:chemistry / Q000378:metabolism*; D018709:Statistics, Nonparametric; D014162:Transfection / Q000379:methods*
D020442:Poloxamer; D049452:Green Fluorescent Proteins; C514452:pluronic L64; D008156:Luciferases