Abstract
The improvement of gene therapy protocols is intimately related to the establishment of efficient gene transfer methods. Electroporation has been increasingly employed in in vitro and in vivo protocols, and much attention has been given to increasing its transfection potential. The method is based on the application of an electric field of short duration and high voltage to the cells, forming reversible pores through which molecules can enter the cell. In this work, we describe the optimization of a protocol for the electroporation of K562 cells involving the combination of electric field, resistance and capacitance values. Using RPMI 1640 as pulsing buffer and 30 μg of pEGFP-N1 plasmid, 875 V cm−1, 500 μF and infinite resistance, we achieved transfection rates of 82.41 ± 3.03%, with 62.89 ± 2.93% cell viability, values higher than those reported in the literature. Analyzing cell cycle after electroporation, with three different electric field conditions, we observed that in a heterogeneous population of cells, viability of G1 cells is less affected by electroporation than that of cells in late S and G2/M phases. We also observed that efficiency of electroporation can be improved using the DNAse inhibitor Zn, immediately after the pulse. These results can represent a significant improvement of current methods of electroporation of animal and plant cells.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Blomberg P, Eskandarpour M, Xia S, Sylvén C, Islam KB (2002). Electroporation in combination with a plasmid vector containing SV40 enhancer elements results in increased and persistent gene expression in mouse muscle. Biochem Biophys Res Commun 298:505–510
Bureau MF, Naimi S, Torero Ibad R, Seguin J, Georger C, Arnould E, Maton L, Blanche F, Delaere P, Scherman D (2004). Intramuscular plasmid DNA electrotransfer: biodistribution and degradation. Biochim Biophys Acta 1676:138–148
Dean DA (1997) Import of plasmid DNA into the nucleus is sequence specific. Exp Cell Res 230:293–302
Dean DA, Dean BS, Muller S, Smith LC (1999) Sequence requirements for plasmid nuclear import. Exp Cell Res 253:713–722
Gehl J (2003) Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol Scand 177:437–447
Goldstein S, Fordis CM, Howard BH (1989) Enhanced transfection efficiency and improved cell survival after electroporation of G2/M-synchronized cells and treatment with sodium butyrate. Nucleic Acids Res 17:3959–3971
Golzio M, Mora MP, Raynaud C, Delteil C, Teissie J, Rols MP (1998) Control by osmotic pressure of voltage-induced permeabilization and gene transfer in mammalian cells. Biophys J 74:3015–3022
Golzio M, Teissie J, Rols MP (2002) Cell synchronization effect on mammalian cell permeabilization and gene delivery by electric field. Biochim Biophys Acta 1563:23–28
Haynes J Jr, Baliga BS, Obiako B, Ofori-Acquah S, Pace B (2004) Zileuton induces hemoglobin F synthesis in erythroid progenitors: role of the L-arginine-nitric oxide signaling pathway. Blood 103:3945–3950
Koeffler HP, Golde DW (1980) Human myeloid leukemia cell lines: a review. Blood 56:344–350
Kunz BA, Kohalmi SE (1991). Modulation of mutagenesis by deoxyribonucleotide levels. Annu Rev Genet 25:339–359
Lechardeur D, Sohn KJ, Haardt M, Joshi PB, Monck M, Graham RW, Beatty B, Squire J, O’Brodovich H, Lukacs GL (1999) Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Ther 6:482–497
Li LH, Ross P, Hui SW (1999) Improving electrotransfection efficiency by post-pulse centrifugation. Gene Ther 6:364–372
Lozzio CB, Lozzio BB (1975) Human chronic myelogenous leukemia cell-line with positive philadelphia chromosome. Blood 45:321–334
Mastrangeli A, O’Connell B, Aladib W, Fox PC, Baum BJ, Crystal RG (1994) Direct in vivo adenovirus-mediated gene transfer to salivary glands. Am J Physiol 266:1146–1155
Neumann E, Kakorin S, Tsoneva I, Nikolova B, Tomov T (1996) Calcium-mediated DNA adsorption to yeast cells and kinetics of cell transformation by electroporation. Biophys J 71:868–877
Niedzinski EJ, Chen YJ, Olson DC, Parker EA, Park H, Udove JA, Scollay R, McMahon BM, Bennett MJ (2003) Enhanced systemic transgene expression after nonviral salivary gland transfection using a novel endonuclease inhibitor/DNA formulation. Gene Ther 10:2133–2138
Overton WR, McCoy JP Jr (1994) Reversing the effect of formalin on the binding of propidium iodide to DNA. Cytometry 16:351–356
Park JI, Choi HS, Jeong JS, Han JY, Kim IH (2001) Involvement of p38 kinase in hydroxyurea-induced differentiation of K562. Cell Growth Differ 12:481–486
Pucihar G, Kotnik T, Kanduser M, Miklavcic D (2001) The influence of medium conductivity on electropermeabilization and survival of cells in vitro. Bioelectrochemistry 54:107–115
Rodrigue CM, Arous N, Bachir D, Smith-Ravin J, Romeo PH, Galacteros F, Garel MC (2001) Resveratrol, a natural dietary phytoalexin, possesses similar properties to hydroxyurea towards erythroid differentiation. Br J Haematol 113:500–507
Schakowski F, Buttgereit P, Mazur M, Marten A, Schottker B, Gorschluter M, Schmidt-Wolf IGH (2004) Novel non-viral method for transfection of primary leukemia cells and cell lines. Genet Vaccines Ther 2:1
Takahashi M, Furukawa T, Nikkuni K, Aoki A, Nomoto N, Koike T, Moriyama Y, Shinada S, Shibata A (1991) Efficient introduction of a gene into hematopoietic cells in S-phase by electroporation. Exp Hematol 19:343–346
Teixeira LAK, Fricke CH, Bonorino CB, Bogo MR, Nardi NB (2001) An efficient gene transfer system for hematopoietic cell line using transient and stable vectors. J Biotechnol 88:159–165
Van Tendeloo VF, Ponsaerts P, Lardon F, Nijs G, Lenjou M, Van Broeckhoven C, Van Bockstaele DR, Berneman ZN (2001) Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood 98:49–56
Acknowledgments
The authors would like to thank Dr. Leonardo A. Karan Teixeira and Dr. Guido Lenz for helpful discussion and Dr. Arnaldo Zaha, Dr. Henrique B. Ferreira and their staff for gently sharing with us the Gene Pulser Transfection Apparatus. This research was funded by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and Instituto Do Milênio-CNPq/Rede De Terapia Gênica, Brazil.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Delgado-Cañedo, A., Santos, D.G.d., Chies, J.A.B. et al. Optimization of an electroporation protocol using the K562 cell line as a model: role of cell cycle phase and cytoplasmic DNAses. Cytotechnology 51, 141–148 (2006). https://doi.org/10.1007/s10616-006-9028-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-006-9028-1