Electroporation for Gene Delivery
An Overview
Electroporation-the employ of high-voltage electric shocks to introduce DNA into cells-can be used in most cell types, yielding a high frequency of both stable transformation and transient gene expression and. Because it requires fewer steps, it can be easier than alternate techniques.
Electroporation as a microbiological technique, it has now been shown to be effective at delivering plasmid DNA in vivo to a variety of tissue types. This method makes use of the fact that the cell membrane acts as an electrical capacitor that is unable to pass current (except through ion channels) for increasing the permeability of the cell membrane, allowing chemicals, drugs, or DNA to be introduced into the cell.
Figure 1. Electroporation. (Jinturkar, 2011)
How This Method Works?
This technique involves the delivery of a voltage (current) across the surfaces of cells, which will result in the spontaneous creation of pores in the plasma membranes. While the voltage is removed, the pores will close. At the same time, the voltage is applied and the pores open, current will travel through the cell. After that, recalling that DNA has a net negative charge, it should be clear that DNA will migrate in the same direction as electron flow in the applied current. Obviously, when the current is removed, the pores will spontaneously close, trapping some of the DNA within the cells. Membrane-associated DNA aggregates also form in the presence of the voltage, and these aggregates have been seen to remain at the membrane level for up to several minutes after the administration of the current. It is possible that these aggregates enter cells through endocytosis.
Figure 2. Electroporation process. (Mehta, 2015)
Basic Protocol
- Electroporation into Mammalian Cells
For mammalian cells, the process uses at least 200 V/cm for plated cells and to 400V/cm for cells in suspension, with pulses lasting a minimum of 0.5 ms. Multiple pulses are usually delivered. Moreover, it is possible that a small amount of cellular contents escapes during the procedure, especially ions and small proteins, if the voltage is sufficient and the time of delivery is long enough. Electroporation is very stressful for the cell. Although it can be employed on most cell types, it is typically reserved for bacteria because of their high numbers and short cell cycles (meaning they are expendable and have short recovery times).
- Electroporation into Muscle or Skin
Electroporation has been used successfully to deliver plasmid DNA to a variety of tissues in vivo. Because of its physical nature, electroporation can be applied to practically any cell or tissue. Plasmid DNA in the appropriate diluent is injected into the tissue. Electrodes are then placed around the injection site and the cells within the tissue are subjected to a high-voltage electrical pulse of defined magnitude and length. The animals are then allowed to recover and the tissue is evaluated at specified time points following delivery. Factors that can be varied to optimize electroporation effectiveness are pulse width, number, amplitude and electrode configuration.
Advantages of Electroporation
The process of electroporation is applied to multiple cell types with highly efficient, reproducible, and appropriate transient or stable transfections, as required. The strength of the electric field and the duration of cell exposure are varied for optimizing the transfection efficiency for every cell type. The variables influencing the success of electroporation include state of cell growth at the time of electroporation, type of cell to be transfected, amount of transfecting DNA, size of the capacitor used to store the charge, voltage of the electrical charge delivered to the cells, and duration of electric field application.
References
- Jinturkar, K.A.; et al. (2011). Gene Delivery Using Physical Methods-3. In Challenges in Delivery of Therapeutic Genomics and Proteomics. 83-126.
- Mehta, S. (2015). Electroporation.
