title: Electrotransfection and lipofection show comparable efficiency for in vitro gene delivery of primary human myoblasts.
abstract: Transfection of primary human myoblasts offers the possibility to study mechanisms that are important for muscle regeneration and gene therapy of muscle disease. Cultured human myoblasts were selected here because muscle cells still proliferate at this developmental stage, which might have several advantages in gene therapy. Gene therapy is one of the most sought-after tools in modern medicine. Its progress is, however, limited due to the lack of suitable gene transfer techniques. To obtain better insight into the transfection potential of the presently used techniques, two non-viral transfection methods--lipofection and electroporation--were compared. The parameters that can influence transfection efficiency and cell viability were systematically approached and compared. Cultured myoblasts were transfected with the pEGFP-N1 plasmid either using Lipofectamine 2000 or with electroporation. Various combinations for the preparation of the lipoplexes and the electroporation media, and for the pulsing protocols, were tested and compared. Transfection efficiency and cell viability were inversely proportional for both approaches. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for lipofection, while for electroporation, RPMI medium and a pulsing protocol using eight pulses of 2 ms at E = 0.8 kV/cm proved to be the optimal combination. The transfection efficiencies for the optimal lipofection and optimal electrotransfection protocols were similar (32 vs. 32.5%, respectively). Both of these methods are effective for transfection of primary human myoblasts; however, electroporation might be advantageous for in vivo application to skeletal muscle.
authors.affiliation: Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska cesta 4, 1000, Ljubljana, Slovenia, tomaz.mars@mf.uni-lj.si.
authors.forename: Tomaz
authors.initials: T
authors.lastname: Mars
authors.forename: Marusa
authors.initials: M
authors.lastname: Strazisar
authors.forename: Katarina
authors.initials: K
authors.lastname: Mis
authors.forename: Nejc
authors.initials: N
authors.lastname: Kotnik
authors.forename: Katarina
authors.initials: K
authors.lastname: Pegan
authors.forename: Jasna
authors.initials: J
authors.lastname: Lojk
authors.forename: Zoran
authors.initials: Z
authors.lastname: Grubic
authors.forename: Mojca
authors.initials: M
authors.lastname: Pavlin
mesh_terms: D000293:Adolescent; D000328:Adult; D002470:Cell Survival; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D018274:Electroporation / Q000379:methods; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006801:Humans; D007223:Infant; D008055:Lipids; D032446:Myoblasts / Q000378:metabolism*; D061251:Primary Cell Culture; D014162:Transfection / Q000379:methods; D055815:Young Adult
chemical_list: D008055:Lipids; C086724:Lipofectamine