title: siRNA delivery into cultured primary human myoblasts--optimization of electroporation parameters and theoretical analysis.
abstract: Introduction of genetic material into muscle tissue has been extensively researched, including isolation and in vitro expansion of primary myoblasts as a potential source of cells for skeletal and heart muscle tissue engineering applications. In this study, we optimized the electroporation protocol for introduction of short interfering ribonucleic acid (siRNA) against messenger RNA for Hypoxia Inducible Factor 1α (HIF-1α) into cultured primary human myoblasts. We established optimal pulsing protocol for siRNA electro transfection, and theoretically analyzed the effect of electric field and pulse duration on silencing efficiency and electrophoretic displacement of siRNA. Silencing of HIF-1α was determined with quantitative polymerase chain reaction and Western Blot. The most efficient silencing (71% knockdown) was achieved with 8 × 2 ms pulses, E = 0.6 kV/cm. Viability was determined immediately, 1 h and 48 h after electroporation. In general, there was a trade-off between efficient silencing and preserved viability. Electric field and pulse duration are crucial parameters for silencing, since both increase membrane permeabilization and electrophoretic transfer of siRNA. Short-term viability showed immediate toxicity of pulses due to membrane damage, while indirect effects on cell proliferation were observed after 48 h. Presented results are important for faster optimization of electroporation parameters for ex vivo electrotransfer of short RNA molecules into primary human myoblasts.
authors.affiliation: Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Jasna
authors.initials: J
authors.lastname: Lojk
authors.affiliation: Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Katarina
authors.initials: K
authors.lastname: Mis
authors.affiliation: Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Sergej
authors.initials: S
authors.lastname: Pirkmajer
authors.affiliation: Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Mojca
authors.initials: M
authors.lastname: Pavlin
mesh_terms: D002470:Cell Survival; D002478:Cells, Cultured; D018274:Electroporation / Q000379:methods*; D055785:Gene Knockdown Techniques; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000172:deficiency / Q000235:genetics; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods*
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D034741:RNA, Small Interfering
keywords: electrophoretic displacement; electrotransfection; mechanisms; silencing; viability