## 2 Molecular Mechanism of Gene Electrotransfer  
The pores that are shaped after using a transmembrane electric field are large enough to serve as channels for ions and small molecules. This process occurs within a few nanoseconds and is more rapid for the highest field applied. Once the electric field is turned off, pore annihilation begins, and phospholipid head groups move out of the bilayer interior. The head groups separate again into two distinct layers, rapidly removing water. The relocalization of lipid molecules is independent of their initial location and achieved within a few nanoseconds [14]. Gene electrotransfer (GET) is a multistep process and an efficient method for introducing genes into cells in vitro and in vivo. GET involves the following steps: (1) the electropermeabilization of the plasma membrane, (2) the electrophoretic migration of the DNA towards the membrane, (3) DNA/membrane interaction, (4) DNA translocation across the membrane, (5) the intracellular migration of DNA through dense cell cytoplasm, (6) DNA passage through the nuclear envelope, and (7) gene expression (Figure 2). It occurs successfully when both electropermeabilization and viability are optimal, depending on the electrical field strength, pulse number, and duration. The plasma membrane is impervious to hydrophilic molecules, such as nucleic acids. DNA molecules cannot access the cytoplasm because negatively charged plasmid molecules cannot interact with the cell plasma membrane that bears the same charge [46]. The field strength has been observed to play a fundamental role. GET is only detected for electric field values leading to membrane permeabilization (Ec > Ep). Plasmid molecules, negatively charged, migrate when submitted to an electric field. In the case of a \"low\" electric field (i.e., Ec < Ep), the plasmid simply electrophoretically flows along the cell membrane towards the anode [47]. Long pulses (1 ms to 5 ms) and a combination of high- and low-voltage pulses have been suggested in vitro. It has been demonstrated that short HV pulses are crucial for the efficient permeabilization of the cell membrane, enabling transfection, and are sufficient to successfully deliver DNA into cells at optimal plasmid concentrations [48]. Therefore, it is generally difficult to separate the role of HV pulses as being only permeabilizing and LV pulses as being electrophoretic since both effects can be present during HV pulses. Whereas for in vivo applications, where suboptimal plasmid concentration is the limiting factor for efficient transfection, cell viability is better preserved with shorter duration pulses (od 100 us do 500 us) [49; 50; 51]. During the application of the electric field, the plasma membrane is permeabilized. DNA is electrophoretically pushed onto the cell membrane side facing the cathode, which results in DNA-membrane interactions. The involvement of the electrophoretic component leads to an asymmetrical interaction of the DNA with the membrane, which remains together for about ten minutes. After the application of the electric field and resealing of the membrane, DNA is mainly internalized by endocytosis and caveolin/raft-mediated endocytosis. The insertion of DNA into the membrane and the relatively large size of DNA aggregates may exert a sufficiently large curvature on the membrane to be recognized by the endocytic machinery, which would then puncture the membrane and, eventually, the vesicle. DNA uptake by several pathways would likely be based on aggregate size [46]. DNA is internalized by endocytosis with the following approximate contributions: 50% caveolin/raft-mediated endocytosis (caveolin/raft-ME), 25% macropinocytosis, and 25% clathrin-mediated endocytosis (clathrin-ME) [52]. Figure 2: The chain of events that take place while inserting plasmid DNA into the cell to provoke gene expression. Once the plasma membrane is subjected to electropermeabilization, DNA electrophoretically migrates toward the membrane and interacts with it.