Abstract
Peptide therapeutics are of increasing interest due to their biological specificity. We used a simple technique to study the efficacy of inducing peptides into adherent chondrocytes by transiently permeabilizing the membrane with electric pulses (in situ electroporation). Mechano-growth factor (MGF) was selected as a model peptide. FITC-labeled MGF was added to cultures of adherent primary chondrocytes grown on ITO coated glass slides. Cells were subjected to 3–9 pulses of 175–275 V and evaluated by flow cytometry. Under optimal conditions, an electroporation efficiency of close to 50% could be achieved. This technique can be used to study the functional domains of intracellular peptides, peptide inhibition of signal transduction and intracrine-mediated effects of peptides in adherent cells.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Barton ER (2006) The ABCs of IGF-I isoforms: impact on muscle hypertrophy and implications for repair. Appl Physiol Nutr Metab 31:791–797
Brownell HL, Firth KL, Kawauchi K, Delovitch T, Raptis L (1997) A novel technique for the study of Ras activity: electroporation of [α32P]GTP. DNA Cell Biol 16:103–110
Carpenter V, Matthews K, Devlin G, Stuart S, Jensen J, Conaglen J et al (2007) Mechano-growth factor reduces loss of cardiac function in acute myocardial infarction. Heart Lung Circ 17:33–39
Dluzniewska J, Sarnowska A, Beresewicz M, Johnson I, Srai SK, Ramesh B et al (2005) A strong neuroprotective effect of the autonomous C-terminal peptide of IGF-1 Ec (MGF) in brain ischemia. FASEB J 19:1896–1898
Dom Gv, Shaw-Jackson C, Matis C, Bouffioux O, Picard JJ, Prochiantz A et al (2003) Cellular uptake of antennapedia penetratin peptides is a two-step process in which phase transfer precedes a tryptophan-dependent translocation. Nucleic Acids Res 31:556–561
Firth KL, Brownell HL, Raptis L (1997) Improved procedure for electroporation of peptides into adherent cells in situ. Biotechniques 23:644–646
Fujimoto H, Yoshizako S, Kato K, Iwata H (2006) Fabrication of cell-based arrays using micropatterned alkanethiol monolayers for the parallel silencing of specific genes by small interfering RNA. Bioconj Chem 17:1404–1410
Fujimoto H, Kato K, Iwata H (2008) Electroporation microarray for parallel transfer of small interfering RNA into mammalian cells. Anal Bioanal Chem 392:1309–1316
Goldfine ID, Smith GJ, Wong KY, Jones AL (1977) Cellular uptake and nuclear binding of insulin in human cultured lymphocytes: evidence for potential intracellular sites of insulin action. Proc Natl Acad Sci USA 74:1368–1372
Goldspink G (2005) Mechanical signals, IGF-I gene splicing, and muscle adaptation. Physiology 20:232–238
Hill M, Goldspink G (2003) Expression and splicing of the insulin-like growth factor gene in rodent muscle is associated with muscle satellite (stem) cell activation following local tissue damage. J Physiol 549:409–418
Khine M, Ionescu-Zanetti C, Blatz A, Wang L-P, Lee LP (2007) Single-cell electroporation arrays with real-time monitoring and feedback control. Lab Chip 7:457–462
Nelson WJ, Nusse R (2004) Convergence of Wnt, beta-catenin, and cadherin pathways. Science 303:1483–1487
Raptis L, Firth KL (2008) Electrode assemblies used for electroporation of cultured cells. Methods Mol Biol 423:61–76
Swartz M, Eberhart J, Mastick GS, Krull CE (2001) Sparking new frontiers: using in vivo electroporation for genetic manipulations. Dev Biol 233:13–21
Torchilin VP, Rammohan R, Weissig V, Levchenko TS (2001) TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors. Proc Natl Acad Sci USA 98:8786–8791
Wong M, Siegrist M, Wang X, Hunziker E (2001) Development of mechanically stable alginate/chondrocyte constructs: effects of guluronic acid content and matrix synthesis. J Orthop Res 19:493–499
Yamauchi F, Kato K, Iwata H (2004) Spatially and temporally controlled gene transfer by electroporation into adherent cells on plasmid DNA-loaded electrodes. Nucleic Acids Res 32:e187
Yamauchi F, Kato K, Iwata H (2005) Layer-by-layer assembly of poly(ethyleneimine) and plasmid DNA onto transparent indium-tin oxide electrodes for temporally and spatially specific gene transfer. Langmuir 21:8360–8367
Yang SY, Goldspink G (2002) Different roles of the IGF-I Ec peptide (MGF) and mature IGF-I in myoblast proliferation and differentiation. FEBS Lett 522:156–160
Acknowledgments
This work was funded by a grant from the AO Research Foundation and a Marie Heim-Vögtlin grant (PMPDP2_122997) from the Swiss National Science Foundation. We acknowledge the advice and support from Leda Raptis, Kevin Firth and Tom Hole. Dr. Malgorzata Kisielow is acknowledged for helping with the flow cytometry analyses.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Schönenberger, C., Schütz, A., Franco-Obregón, A. et al. Efficient electroporation of peptides into adherent cells: investigation of the role of mechano-growth factor in chondrocyte culture. Biotechnol Lett 33, 883–888 (2011). https://doi.org/10.1007/s10529-010-0508-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-010-0508-1