The Journal of Membrane Biology

, Volume 248, Issue 2, pp 273–283 | Cite as

Electrotransfection and Lipofection Show Comparable Efficiency for In Vitro Gene Delivery of Primary Human Myoblasts

  • Tomaz MarsEmail author
  • Marusa Strazisar
  • Katarina Mis
  • Nejc Kotnik
  • Katarina Pegan
  • Jasna Lojk
  • Zoran Grubic
  • Mojca PavlinEmail author


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.


Myoblasts Lipofection Electroporation Gene electrotransfer Gene therapy 



This study was supported by the Slovenian Research Agency (Grant No. J4-4324, research programme P3-0043). We gratefully acknowledge help from Zvonka Frelih, Marko Narobe and Romain Teruel.

Conflict of interest

The authors report no conflicts of interest.


  1. Aihara H, Miyazaki J (1998) Gene transfer into muscle by electroporation in vivo. Nat Biotechnol 16:867–870CrossRefPubMedGoogle Scholar
  2. André FM, Gehl J, Sersa G, Préat V, Hojman P, Eriksen J, Golzio M, Cemazar M, Pavselj N, Rols MP, Miklavcic D, Neumann E, Teissié J, Mir LM (2008) Efficiency of high- and low-voltage pulse combinations for gene electrotransfer in muscle, liver, tumor, and skin. Hum Gene Ther 19:1261–1272CrossRefPubMedGoogle Scholar
  3. André C, Swain WF, Page CP, Macklin MD, Slama J, Hatzis D, Eriksson E (1994) In vivo transfer and expression of a human epidermal growth factor gene accelerates wound repair. Proc Natl Acad Sci USA 91:12188–12192CrossRefGoogle Scholar
  4. Boulaiz H, Marchal JA, Prados J, Melguizo C, Aránega A (2005) Non-viral and viral vectors for gene therapy. Cell Mol Biol 51:3–22PubMedGoogle Scholar
  5. Bregar VB, Lojk J, Suštar V, Veranič P, Pavlin M (2013) Visualization of internalization of functionalized cobalt ferrite nanoparticles and their intracellular fate. Int J Nanomed 8:919–931Google Scholar
  6. Cavazzana-Calvo M, Thrasher A, Mavilio F (2004) The future of gene therapy. Nature 427:779–781CrossRefPubMedGoogle Scholar
  7. Cemazar M, Golzio M, Sersa G, Rols M, Teissie J (2006) Electrically-assisted nucleic acids Delivery to tissues in vivo: where do we stand? Curr Pharm Des 12:3817–3825CrossRefPubMedGoogle Scholar
  8. Cemazar M, Sersa G (2007) Electrotransfer of therapeutic molecules into tissues. Curr Opin Mol Ther 9:554–562PubMedGoogle Scholar
  9. Dalby B, Cates S, Harris A, Ohki EC, Tilkins ML, Price PJ, Ciccarone VC (2004) Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications. Methods 33:95–103CrossRefPubMedGoogle Scholar
  10. Durieux A-C, Bonnefoy R, Manissolle C, Freyssenet D (2002) High-efficiency gene electrotransfer into skeletal muscle: description and physiological applicability of a new pulse generator. Biochem Biophysic Res Comm 296:443–450CrossRefGoogle Scholar
  11. Faurie C, Rebersek M, Golzio M, Kanduser M, Escoffre JM, Pavlin M, Teissie J, Miklavcic D, Rols MP (2010) Electro-mediated gene transfer and expression are controlled by the life-time of DNA/membrane complex formation. J Gene Med 12:117–125CrossRefPubMedGoogle Scholar
  12. Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA 84:7413–7417PubMedCentralCrossRefPubMedGoogle Scholar
  13. Ferber D (2001) Safer and virus-free? Science 294:1638–1642CrossRefPubMedGoogle Scholar
  14. Frandsen SK, Gissel H, Hojman P, Tramm T, Eriksen J, Gehl J (2012) Direct therapeutic applications of calcium electroporation to effectively induce tumor necrosis. Cancer Res 72:1336–1341CrossRefPubMedGoogle Scholar
  15. Gehl J (2003) Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol Scand 177:437–447CrossRefPubMedGoogle Scholar
  16. Golicnik A, Podbregar M, Lainscak M, Anker SD, Grubic Z, Mars T (2012) Atorvastatin modulates constitutive and lipopolysaccharide induced IL-6 secretion in precursors of human skeletal muscle. Afr J Pharm Pharmacol 6:241–247CrossRefGoogle Scholar
  17. Golzio M, Mora MP, Raynaud C, Delteil C, Teissié J, Rols MP (1998) Control by osmotic pressure of voltage-induced permeabilization and gene transfer in mammalian cells. Biophys J 74:3015–3022PubMedCentralCrossRefPubMedGoogle Scholar
  18. Golzio M, Teissie J, Rols M-P (2002) Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci USA 99:1292–1297PubMedCentralCrossRefPubMedGoogle Scholar
  19. Gothelf A, Gehl J (2012) What you always needed to know about electroporation based DNA vaccines. Hum Vaccin Immunother 8:1694–1702PubMedCentralCrossRefPubMedGoogle Scholar
  20. Gothelf A, Eriksen J, Hojman P, Gehl J (2010) Duration and level of transgene expression after gene electrotransfer to skin in mice. Gene Ther 17:839–845CrossRefPubMedGoogle Scholar
  21. Haberl S, Kandušer M, Flisar K, Hodžić D, Bregar VB, Miklavčič D, Escoffre JM, Rols MP, Pavlin M (2013) 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. J Gene Med 15:169–181CrossRefPubMedGoogle Scholar
  22. Heller R, Cruz Y, Heller LC, Gilbert RA, Jaroszeski MJ (2010) Electrically mediated delivery of plasmid DNA to the skin, using a multielectrode array. Hum Gene Ther 21:357–362PubMedCentralCrossRefPubMedGoogle Scholar
  23. Hojman P (2010) Basic principles and clinical advancements of muscle electrotransfer. Curr Gene Ther 10:128–138CrossRefPubMedGoogle Scholar
  24. Hojman P, Zibert JR, Gissel H, Eriksen J, Gehl J (2007) Gene expression profiles in skeletal muscle after gene electrotransfer. BMC Mol Biol 8:56PubMedCentralCrossRefPubMedGoogle Scholar
  25. Hunt MA, Currie MJ, Robinson BA, Dachs GU (2010) Optimizing transfection of primary human umbilical vein endothelial cells using commercially available chemical transfection reagents. J Biomol Tech. 21:66–72PubMedCentralPubMedGoogle Scholar
  26. Kanduser M, Miklavcic D, Pavlin M (2009) Mechanisms involved in gene electrotransfer using high- and low-voltage pulses—an in vitro study. Bioelectrochemistry 74:265–271CrossRefPubMedGoogle Scholar
  27. Katalinić M, Miš K, Pirkmajer S, Grubič Z, Kovarik Z, Marš T (2012) The cholinergic and non-cholinergic effects of organophosphates and oximes in cultured human myoblasts. Chem Biol Interact 203:144–148CrossRefPubMedGoogle Scholar
  28. Konieczny P, Swiderski K, Chamberlain JS (2013) Gene and cell-mediated therapies for muscular dystrophy. Muscle Nerve 47:649–663PubMedCentralCrossRefPubMedGoogle Scholar
  29. Kotnik N, Haberl S, Pegan K, Marš T, Grubič Z, Pavlin M (2010) Comparison of electroporation and lipofection for in vitro transfer of plasmid PEGFP-N1 into human myoblasts. Proceedings of the Eighteenth International Electrotechnical and Computer Science Conference ERK 2009, pp 287–290Google Scholar
  30. Lehrman S (1999) Virus treatment questioned after gene therapy death. Nature 401:517–518CrossRefPubMedGoogle Scholar
  31. Li S, Benninger M (2002) Applications of muscle electroporation gene therapy. Curr Gene Ther 2:101–105CrossRefPubMedGoogle Scholar
  32. Marshall DJ, Leiden JM (1998) Recent advances in skeletal-muscle-based gene therapy. Curr Opin Genet Dev 8:360–365CrossRefPubMedGoogle Scholar
  33. Mathiesen I (1999) Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Ther 6:508–514CrossRefPubMedGoogle Scholar
  34. Maurisse R, De Semir D, Emamekhoo H, Bedayat B, Abdolmohammadi A, Parsi H, Gruenert DC (2010) Comparative transfection of DNA into primary and transformed mammalian cells from different lineages. BMC Biotechnol 10:9PubMedCentralCrossRefPubMedGoogle Scholar
  35. Menasché P (2004) Skeletal myoblast transplantation for cardiac repair. Expert Rev Cardiovasc Ther 2:21–28CrossRefPubMedGoogle Scholar
  36. Meregalli M, Farini A, Colleoni F, Cassinelli L, Torrente Y (2012) The role of stem cells in muscular dystrophies. Curr Gene Ther 12:192–205CrossRefPubMedGoogle Scholar
  37. Mir LM, Bureau MF, Rangara R, Schwartz B, Scherman D (1998) Long-term, high level in vivo gene expression after electric-pulse-mediated gene transfer into skeletal muscle. C R Acad Sci III Sci Vie 321:893–899CrossRefGoogle Scholar
  38. Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D, Caillaud JM, Delaere P, Branellec D, Schwartz B, Scherman D (1999) High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci USA 96:4262–4267PubMedCentralCrossRefPubMedGoogle Scholar
  39. Modlich U, Kustikova OS, Schmidt M, Rudolph C, Meyer J, Li Z, Kamino K, von Neuhoff N, Schlegelberger B, Kuehlcke K, Bunting KD, Schmidt S, Deichmann A, von Kalle C, Fehse B, Baum C (2005) Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis. Blood 105:4235–4246CrossRefPubMedGoogle Scholar
  40. Muramatsu T, Arakawa S, Fukazawa K, Fujiwara Y, Yoshida T, Sasaki R, Masuda S, Park HM (2001) In vivo gene electroporation in skeletal muscle with special reference to the duration of gene expression. Int J Mol Med 7:37–42PubMedGoogle Scholar
  41. Negroni E, Vallese D, Vilquin JT, Butler-Browne G, Mouly V, Trollet C (2011) Current advances in cell therapy strategies for muscular dystrophies. Expert Opin Biol Ther 11:157–176CrossRefPubMedGoogle Scholar
  42. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982) Gene transfer into mouse lymphoma cells by electroporation in high electric fields. EMBO J 1:841–845PubMedCentralPubMedGoogle Scholar
  43. Nguyen LT, Atobe K, Barichello JM, Ishida T, Kiwada H (2007) Complex formation with plasmid DNA increases the cytotoxicity of cationic liposomes. Biol Pharm Bull 30:751–757CrossRefPubMedGoogle Scholar
  44. Parker AL, Newman C, Briggs S, Seymour L, Sheridan PJ (2003) Nonviral gene delivery: techniques and implications for molecular medicine. Expert Rev Mol Med 5:1–15CrossRefPubMedGoogle Scholar
  45. Pavlin M, Miklavcic D (2003) Effective conductivity of a suspension of permeabilized cells: a theoretical analysis. Biophys J 85:719–729PubMedCentralCrossRefPubMedGoogle Scholar
  46. Pavlin M, Flisar K, Kanduser M (2010) The role of electrophoresis in gene electrotransfer. J Membr Biol 236:75–79CrossRefPubMedGoogle Scholar
  47. Pavlin M, Pucihar G, Kandušer M (2012) The role of electrically stimulated endocytosis in gene electrotransfer. Bioelectrochemistry 83:38–45CrossRefPubMedGoogle Scholar
  48. Pavšelj N, Préat V (2005) DNA electrotransfer into the skin using a combination of one high- and one low-voltage pulse. J Controlled Release 106:407–415CrossRefGoogle Scholar
  49. Prud’homme GJ, Glinka Y, Khan AS, Draghia-Akli R (2006) Electroporation-enhanced nonviral gene transfer for the prevention or treatment of immunological, endocrine and neoplastic diseases. Curr Gene Ther 6:243–273CrossRefPubMedGoogle Scholar
  50. Reed SD, Li S (2009) Electroporation advances in large animals. Curr Gene Ther 9:316–326PubMedCentralCrossRefPubMedGoogle Scholar
  51. Rols M-P, Teissié J (1998) Electropermeabilization of mammalian cells to macromolecules: control by pulse duration. Biophysical J 75:1415–1423CrossRefGoogle Scholar
  52. Skuk D, Goulet M, Tremblay JP (2013) Electroporation as methods to induce myofiber regeneration and increase the engraftment of myogenic cells in skeletal muscles of primates. J Neuropathol Exp Neurol 72:723–734CrossRefPubMedGoogle Scholar
  53. Shirota H, Petrenko L, Hong C, Klinman DM (2007) Potential of transfected muscle cells to contribute to DNA vaccine immunogenicity. J Immunol 179:329–336CrossRefPubMedGoogle Scholar
  54. Tsong TY (1991) Electroporation of cell membranes. Biophys J 60:297–306PubMedCentralCrossRefPubMedGoogle Scholar
  55. van den Hoff MJ, Moorman AF, Lamers WH (1992) Electroporation in ‘intracellular’ buffer increases cell survival. Nucleic Acids Res 20:2902PubMedCentralCrossRefPubMedGoogle Scholar
  56. Vernier PT, Levine ZA, Wu YH, Joubert V, Ziegler MJ, Mir LM, Tieleman DP (2009) Electroporating fields target oxidatively damaged areas in the cell membrane. PLoS One 4:e7966PubMedCentralCrossRefPubMedGoogle Scholar
  57. Wells DJ (2004) Gene therapy progress and prospects: electroporation and other physical methods. Gene Ther 11:1363–1369CrossRefPubMedGoogle Scholar
  58. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL (1990) Direct gene transfer into mouse muscle in vivo. Science 247:1465–1468CrossRefPubMedGoogle Scholar
  59. Zupanič A, Čorović S, Miklavčič D, Pavlin M (2010) Numerical optimization of gene electrotransfer into muscle tissue. Biomed Eng Online 9:66PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Tomaz Mars
    • 1
    Email author
  • Marusa Strazisar
    • 2
  • Katarina Mis
    • 1
  • Nejc Kotnik
    • 1
  • Katarina Pegan
    • 1
  • Jasna Lojk
    • 2
  • Zoran Grubic
    • 1
  • Mojca Pavlin
    • 2
    Email author
  1. 1.Institute of Pathophysiology, Faculty of MedicineUniversity of LjubljanaLjubljanaSlovenia
  2. 2.Faculty of Electrical EngineeringUniversity of LjubljanaLjubljanaSlovenia

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