Optimization of an electroporation protocol using the K562 cell line as a model: role of cell cycle phase and cytoplasmic DNAses.
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 mug of pEGFP-N1 plasmid, 875 V cm(-1), 500 muF 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 G(1) cells is less affected by electroporation than that of cells in late S and G(2)/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.
Delgado-Cañedo
Andrés
A
Molecular and Cellular Cardiology Laboratory, IC/FUC, Av. Princesa Isabel 370-3 degrees andar, CEP: 90620-00, Porto Alegre, RS, Brazil, andres.lcmc@cardiologia.org.br.
Santos
Daniel Garcia Dos
DG
Chies
José Artur Bogo
JA
Kvitko
Kátia
K
Nardi
Nance Beyer
NB