Structural Biochemistry/DNA recombinant techniques/Electroporation

Overview

Electroporation can be used to insert genes into eukaryotic cells such as plants cells and animal cells, as well as prokaryotic cells such as bacterial cells. In electroporation, cereal monocots and dicots can be introduced to foreign DNA by applying intense electric fields.

Procedure

Lets take a plant cell as an example. First, a plant cell will consist of a cell wall and a plasma membrane. Cellulose is used to digest the cell wall, creating a plant cell that is has an exposed membrane. This is known as a protoplast. Second, foreign DNA is added into the cell. High electric fields are used to create a transiently permeable membrane so that large molecules will be able to pass through the membrane. This is caused by transient electric pulses. Thirdly, the cell wall is able to regrow back. In the end, the plant cell is now a viable plant cell with an insert of foreign DNA.

Considerations for Optimization

When in vitro electroporation is performed, there are several factors that must be taken into account.

• Waveform - when the electroporation technique was originally developed, exponential pulses were used. Since then, square waves have been found to be more effective and less damaging to cells. The pulse length and amplitude can be modified to fit the needs of the experimenter.
• Cell size - transmembrane potential is described by the equation ΔV(m) = fE(ext)r cosθ, where V(m) is the transmembrane potential, f is a proportionality constant relating the external field to its impact on the cell, E(ext) is the external applied electric field, r is the cell radius, and θ is the angle of the field with respect to the cell poles. This means that the smaller a cell is, the larger the external field must be to achieve the same electroporetic effect. Also, a more homogenous cell sampling will yield better results of electroporation.
• Resealing - membrane resealing after electroporation is temperature-dependent, with lower temperature correlating to slower resealing times. An incubator often increases the quality of the results. Waiting for 15 minutes after electroporation to disturb cells by pipetting betters results by allowing cells to reseal. The use of surfactant poloxamer 118 increases the rate of resealing for better results as well. Extremely strong pulses can result in irreversible permeabilization.
• Metal electrodes - for longer pulses, the release of aluminum metal from the electrodes in disposable cuvettes is a concern; low-conductivity metal is sometimes recommended.
• Metal in pulsing media - Generally, calcium is to be avoided in the pulsing medium because it disrupts the intracellular levels of this ion. However, calcium or magnesium in media are often recommended for DNA transfer.

Importance

Electroporation is usually used in molecular biology to introduce a substance into a cell. This can be done with a molecular probe loading the substance into the cell, by a piece of coding DNA, or by a drug that is able to change the functions of a cell. Electroporation is highly efficient when trying to introduce foreign genes into tissue culture cells. An example is mammalian cells. This process is used in the production of knockout mice. Electroporation is beneficial because it can be used to treat tumors, as well as cell-based therapy and gene therapy. This is known as transfection, the process that is used to introduce foreign DNA into eukaryotic cells.