Electroporation

Sachin Mehta Reno Nevada

1. Electroporation

2. What is Electroporation!? A method used to apply an electric current across a cell membrane resulting in temporary “ pore” formation enabling the uptake of exogenous molecules found in the medium to either the cytoplasm or into the nucleus, thereby transfecting or transforming the cell.

3. What is electroporation cont … 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.

4. Electroporation Cell Process

5. How can electroporation be applied?   integral tool in BioTech industry. Tranformation of bacteria, yeast ,and plant protoplasts   tissues in vivo, for in utero applications as well as in ovo transfection. tumor treatment, gene therapy, and cell-based therapy Electroporator with square wave and exponential decay waveforms for in vitro

6. 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.

7. Inovio’s Electroporation Process

8. No electroporation vs. With electroporation Green Light displays the proteins created by the DNA vaccines delivered using electroporation

9. Considerations Cell Size Temperature Composition of Electrodes

10. Optimizing Process Use 8 pulses of 100 microseconds Increasing voltage slowly because cell death can occur

11. 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.

12. 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 

13. Applications to Medical Field Chemotherapy Gene Therapy Transdermal Drug Delivery

14. Electrochemotherap y Specialized drug delivery to affected site in organism First study in 1991 Bleomycin Cisplatin http://www.jove.com/video/1038/electrochemotherapy-of-tumours