Electroporation

Theory

• A method used to apply an electric current across a cell membrane resulting in temporary “pore” formation enabling the cell membrane to be semi-permeable—allowing things to enter the cell for a time being.

Theory, contd.

• Electroporation is a process that is used to introduce foreign genes into a host cell.

• Electric shocks are used as a mechanism for introducing new DNA into a host cell by creating new pores in the plasma membrane of the host cell.

• The new DNA enters the host cell through the new pores and is incorporated into the genome of the new cell.

Electroporation Cell Process

http://www.youtube.com/watch?v=TPPudsNVcqE

Applications

• Transformation of bacteria, yeasts, etc.

• Can be used in vivo or • In vitro• Chemotherapy • Gene Therapy• Diagnostics

Example: Inovio’s Electroporation Technology

Electroporation uses controlled, millisecond electrical pulses to create temporary pores in the cell membrane and allow dramatic cellular uptake of a synthetic DNA vaccine previously injected into muscle or skin.• Technology allows to safely and effectively

deliver DNA-based vaccines. • Numerous human studies demonstrated best in

class immune responses from DNA vaccines.

No electroporation vs. With electroporation

Green Light displays the proteins created by the DNA vaccines delivered using electroporation

Considerations

• Cell Size • Temperature • Composition of Electrodes

Optimizing Process

• Use 8 pulses of 100 microseconds at 0.2 V. • Increasing voltage slowly because

cell death can occur quickly.

Advantages vs. Disadvantages

Advantages:• Versatility: Electroporation is effective

with nearly all cell and species types .• Efficiency: A large majority of cells take in

the target DNA or molecule. In a study on electro transformation of E. coli, for example, 80% of the cells received the foreign DNA .

• Small Scale: The amount of DNA required is smaller than for other methods

• In vivo: The procedure may be performed with intact tissue . A paper published in Developmental Biology showed the successful transfer of a DNA construct with a fluorescent reporter gene into intact mouse brain tissue.

•

• Image of in vivo electroporation in a mouse brain. The mouse brains (telencephalons) in these images are expressing reporter genes (EYFP) introduced in gene constructs by electroporation.

Disadvantages

• Cell Damage: If the pulses are of the wrong length or intensity, some pores may become too large or fail to close after membrane discharge causing cell damage or rupture

• Nonspecific Transport: The transport of material into and out of the cell during the time of electro-permeability is relatively nonspecific. This may result in an ion imbalance that could later lead to improper cell function and cell death

Electrochemotherapy

• Specialized drug delivery to affected site in organism

• First study in 1991 • Bleomycin• Cisplatin

http://www.jove.com/video/1038/electrochemotherapy-of-tumours