Electroporation is a microbiology technique where an electrical field is applied to cells to increase the permeability of the cell membrane. It allows chemicals or DNA to be instigated into the cell. This technique is also known as electropermeabilization. In microbiology, the process of electrotransfer is often used to transform yeast, bacteria, or plant protoplasts by introducing new coding DNA. If plasmids and bacteria plasmids are mixed together, the plasmids can be sending into the bacteria after electropermeabilization, though depending on what is being transferred Cell Squeeze or cell-penetrating peptides could also be used. Electropermeabilization runs by passing thousands of volts over a distance of one to two millimeters of suspended cells in an electropermeabilization cuvette. After that, the cells have to be handled properly until they have had a chance to split, producing new cells that contain reproduced plasmids. This process is around ten times more effective than chemical transformation. Electrotransfer is the use of high-voltage electric shocks, which is introduced DNA into cells. This can be used with most cell types, transient gene expression and yields a high frequency of both stable transformation and, because it requires closer steps, so it can be easier than alternate techniques. This unit describes electropermeabilization of mammalian cells including ES cells for the production of knock in, knockout transgenic mice. It describes rules for using electroporation in vivo to perform gene therapy, cancer therapy and DNA vaccination.

Basic protocols of Electroporation:

ELECTROPORATION INTO MAMMALIAN CELLS:

1. Transfer 0.5-ml aliquots of the cell suspension into desired number of electrotransfercuvettes set on ice.

2. Resuspend cell pellet in half its original volume of ice-cold electrotransfer buffer.

3. Harvest cells by centrifuging 5 min at 640 × g (1500 rpm in a JS-4.2 rotor), 4°C.

4. Harvest cells by centrifuging 5 min.

5. Grow cells to be transfected to late-log phase in complete medium. Each permanent transfection will usually require 5 × 106 cells to yield a reasonable number of transfectants. Each transient expression may require 1–4 × 107 cells, depending on the promoter.

ELECTROPORATION INTO MUSCLE OR SKIN:

1. Inject DNA into tissue. A standard injection volume is 50 μl, although volumes between 10–100 μl have been used. For muscle, concentration of DNA should be between 0.5–1.0 μg/μl and for skin the concentration should be between 1.0–2.0 μg/μl.

2. Anesthetize the animals. Using an induction chamber, animals can be anesthetized in 2–4% isoflurane in oxygen. Once animal is anesthetized, they are fitted with an appropriate mask and kept under general anesthesia (2–3% isoflurane in oxygen) for the entire procedure.

3. Remove hair from area (skin or skin above muscle) to be transfected. This can be done with an electric razor, disposable razor or hair removal product.

4. Placement of electrodes. Electrodes are placed around the injection site.

ELECTROPORATION INTO PLANT PROTOPLASTS:

1. Rinse screen with 4 ml plant electroporation buffer. Combine protoplasts in a sterile 15-ml conical centrifuge tube.

2. Remove debris by filtration through an 80-μm-mesh nylon screen.

3. Centrifuge 5 min at 300 × g (1000 rpm in a JS-4.2 rotor). Discard supernatant, add 5 ml plant electrotransfer buffer, and repeat wash step. Resuspend in plant electrotransfer buffer at 1.5–2 × 106protoplasts/ml.

4. Obtain protoplasts from carefully sliced 5-mm strips of sterile plant material by incubating in 8 ml protoplast solution for 3 to 6 hr at 30°C on a rotary shaker.

Consideration:

1. Temperature

2. Cell size

3. Composition of electrodes

The advantages of this method:

1. Simplicity :  Just one kit for all cell types.

2. Efficiency : A high percentage of cells are transfected without jeopardizing viability

3. Versatility : Electrotransferwith the Neon® system is effective with mammalian cell types.

4. Flexibility : A range of cell numbers can be used: from 104 to 106 cells.

5. Novel : The Neon system employs a unique electrotransferchamber, as easy to use as a pipette and with a circular shape that provides advantages over cuvette-based systems.