title: Inhibitor of endocytosis impairs gene electrotransfer to mouse muscle in vivo.
abstract: Application of electric pulses (electroporation/electropermeabilization) is an effective method for gene transfer (i.e. gene electrotransfer (GET)) in vitro and in vivo. Currently, the mechanisms by which the DNA enters the cell are not yet fully understood. Experimental evidence is building up that endocytosis is the main mechanism by which the DNA, which is later expressed, enters the cell. Therefore the aim of our study was to elucidate whether inhibitors of endocytosis, methyl-β-cyclodextrin (MβCD), Concanavalin A (ConA) and Dynasore, can impair the transfection efficacy of GET in vitro in B16F1 murine melanoma and in vivo in m. tibialis cranialis in mice. We show that MβCD--general inhibitor of endocytosis--can almost prevent GET of EGFP-N1 plasmid in vitro, that ConA--inhibitor of clathrin mediated endocytosis--also abrogates GET but to a lesser extent, and when using Dynasore--reversible inhibitor of dynamin--there is no effect on GET efficacy, if endocytosis is blocked for only 5 min after GET. Moreover, MβCD also reduced GET efficacy in vivo in m. tibialis cranialis and this effect was long lasting. The results of this study show that endocytosis is probably the main mechanism of entrance of DNA after GET in vitro and also in vivo.
authors.affiliation: Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia.
authors.forename: Bostjan
authors.initials: B
authors.lastname: Markelc
authors.affiliation: Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia.
authors.forename: Eva
authors.initials: E
authors.lastname: Skvarca
authors.affiliation: Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia.
authors.forename: Tanja
authors.initials: T
authors.lastname: Dolinsek
authors.affiliation: Department of Cytopathology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia.
authors.forename: Veronika Prevodnik
authors.initials: VP
authors.lastname: Kloboves
authors.affiliation: Department of Natural and Medical Subjects, Faculty of Health Sciences, University of Primorska, Polje 42, SI-6310 Izola, Slovenia.
authors.forename: Andrej
authors.initials: A
authors.lastname: Coer
authors.affiliation: Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia. Electronic address: gsersa@onko-i.si.
authors.forename: Gregor
authors.initials: G
authors.lastname: Sersa
authors.affiliation: Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia; Department of Natural and Medical Subjects, Faculty of Health Sciences, University of Primorska, Polje 42, SI-6310 Izola, Slovenia. Electronic address: mcemazar@onko-i.si.
authors.forename: Maja
authors.initials: M
authors.lastname: Cemazar
mesh_terms: D000818:Animals; D003208:Concanavalin A / Q000494:pharmacology; D004247:DNA / Q000008:administration & dosage; D018274:Electroporation / Q000379:methods; D004705:Endocytosis / Q000187:drug effects* / Q000235:genetics; D005260:Female; D018014:Gene Transfer Techniques; D006835:Hydrazones / Q000494:pharmacology; D008545:Melanoma / Q000188:drug therapy / Q000235:genetics; D008810:Mice, Inbred C57BL; D009132:Muscles / Q000187:drug effects* / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D006835:Hydrazones; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D003208:Concanavalin A; D004247:DNA
keywords: Electropermeabilization; Endocytosis; Gene electrotransfer; Muscle; Plasmid DNA