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Gene electrotransfer
Gene electrotransfer is a versatile biotechnology technique that enables the transfer of genetic material into prokaryotic or eukaryotic cells. It is based on a physical method named electroporation, where a transient increase in the permeability of cell membrane is achieved when submitted to short and intense electric pulses, thus enabling the transport of large molecules (naked plasmid DNA, antisense oligonucleotides, siRNA) into cells that otherwise cannot permeate through the cell membrane. Gene electrotransfer was first described in the 1980s〔Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982). Gene transfer into mouse lyoma cells by electroporation in high electric fields, EMBO J 7:841-845〕 and since then due to its ease of application and efficiency become a routine method for introducing foreign genes into bacterial,〔Drury L (1996). Transformation of bacteria by electroporation, Methods Mol Biol 58: 249-256〕 yeast,〔Simon JR (1993). Transformation of intact yeast cells by electroporation, Methods Enzymol 217:478-483〕 plant,〔Terzaghi WB, Cashmore AR (1997). Plant cell transfection by electroporation, Methods Mol Biol 62:453-462〕 and animal cells 〔Kanduser M, Miklavcic D, Pavlin M (2009). Mechanisms involved in gene electrotransfer using high- and low-voltage pulses -An in vitro study, Bioelectrochemistry 74:265-271〕 in vitro and into different tissues, including muscle,〔Aihara H, Miyazaki J (1998). Gene transfer into muscle by electroporation in vivo, Nat Biotechnol 16:867-870〕 tumors,〔Cemazar M, Sersa G, Wilson J, Tozer GM, Hart SL, Grosel A, Dachs GU (2002). Effective gene transfer to solid tumors using different nonviral gene delivery techniques: Electroporation, liposomes, and integrin-targeted vector, Cancer Gene Ther 9:399-406〕 liver,〔Heller R, Jaroszeski M, Atkin A, Moradpour D, Gilbert R, Wands J, Nicolau C (1996). In vivo gene electroinjection and expression in rat liver, FEBS Lett 389:225-228〕 and skin〔Pavselj N, Preat V (2005). DNA electrotransfer into the skin using a combination of one high- and one low-voltage pulse, J Control. Release 106:407-415〕 in vivo.
==Physical principle==
Application of electric pulses of sufficient strength to the cell causes an increase in the trans-membrane potential difference, which provokes the membrane destabilization. Cell membrane permeability is increased and otherwise nonpermeant molecules enter the cell.〔Kotnik T, Miklavcic D (2000). Analytical description of transmembrane voltage induced by electric fields on spheroidal cells, Biophys J 79:670-679〕
Although the mechanisms of gene electrotransfer are not yet fully understood, it was shown that the introduction of DNA only occurs in the part of the membrane facing the cathode and that several steps are needed for successful transfection: electrophoretic migration of DNA towards the cell, DNA insertion into the membrane, translocation across the membrane, migration of DNA towards the nucleus, transfer of DNA across the nuclear envelope and finally gene expression.〔Satkauskas S, Bureau MF, Puc M, Mahfoudi A, Scherman D, Miklavcic D, Mir LM (2002). Mechanisms of in vivo DNA electrotransfer: respective contributions of cell electropermeabilization and DNA electrophoresis, Mol Ther 5:133-140〕 There are a number of factors that can influence the efficiency of gene electrotransfer, such as: temperature, parameters of electric pulses, DNA concentration, electroporation buffer used, cell size and the ability of cells to express transfected genes.〔Gehl J (2003). Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research, Acta Physiol Scand 177:437-447〕 In in vivo gene electrotransfer also DNA diffusion through extracellular matrix, properties of tissue and overall tissue conductivity are crucial.〔Miklavcic D, Beravs K, Semrov D, Miklavcic D, Cemazar M, Demsar F, Sersa G (1998). The importance of electric field distribution for effective in vivo electroporation of tissues, Biophys J 74:2152–2158〕
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