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Friday, May 13, 2011

Electroporation: Applications and Protocol


Molecular biology / Genetic Engineering works require a foreign gene or protein material to be inserted into a host cell. Since the plasma membrane of the cell is selectively permeable , any polar molecules, including DNA and protein, are unable to freely pass through the membrane.Many methods have been developed to surpass this barrier and allow the insertion of DNA and other molecules into the cells to be studied. These other methods include microprecipitates, microinjection, liposomes, and biological vectors, etc.One such method is electroporation.
Electroporation is technique in which cell plama membrane's permeability is increased by applying electric field which induces the formation of small pores in the membrane through which pieces of foreign materials (usually DNA, Proteins or Dyes) can be taken in to the cell.A quick voltage shock may disrupt areas of the membrane temporarily, allowing polar molecules to pass, but then the membrane may reseal quickly and leave the cell intact.

Procedure

An Image of an Electroporator

The procedure involves placing the cell suspension and the foreign material (eg: Plamsid) to be transferred into plastic or glass cuvette.The cuvettes used here are specially meant for electroporation, they have aluminium electrodes at the sides. The voltage and capacitance is set and the cuvette inserted into the electroporator. Once after electroporation, few ml medium is added , and is incubated at the the culture's (Bacteria, yeast, etc) optimal temperature for an hour or more to allow recovery of the cells.The transformed cells can be screened out using selectable markers in the plasmid (usaually antibiotic resisitance genes are used).
Advantages and Disadvantages of Electroporation
Electroporation has the following advantages and 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 electrotransformation of E. coli, for example, 80% of the cells received the foreign DNA.
  • Small Scale: The amount of DNA required is smaller as compared with other methods.
Disadvantages:
  • Cell Damage: If the electric pulses are given for long time or with more 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 electropermeability is relatively nonspecific. This may result in an ion imbalance that could later lead to improper cell function and cell death.
Applications
As previously mentioned, electroporation is widely used in many areas of molecular biology research and in the medical field. Some applications of electroporation include:
DNA Transfection or Transformation: This is likely the most widespread use of electroporation. Specific genes can be cloned into a plasmid and then this plasmid introduced into host cells in order to investigate gene and protein structure and function.if the host cell used is microbial then it is called Transformation and if the host cell is mammalian then it is called as Transfection.
Direct Transfer of Plasmids Between Cells: Bacterial cells already containing a plasmid may be incubated with another strain that does not contain plasmids but that has some other desireable feature. The voltage of electroporation will create pores, allowing some plasmids to exit one cell and enter another. The desired cells may then be selected by antibiotic resistance or another similar method. This type of transfer may also be performed between species. Thus, large numbers of plasmids may be grown in rapidly multiplying bacterial colonies and then transferred to yeast cells by electroporation for study .
Induced Cell Fusion: The disruption of the membrane that occurs with the quick pulse of electricity in the electroporation procedure has also been shown to induce fusion of cells..
Trans-dermal Drug Delivery: Just as electroporation causes temporary pores to form in plasma membranes, studies suggest that similar pores form in lipid bi-layers of the stratum corneum- the outermost dead layer of skin. These pores could allow drugs to pass through to the skin to a target tissue. This method of drug delivery would be more pleasant than injection for the patient (not requiring a needle) and could avoid the problems of improper absorption or degradation of oral medication in the digestive system.
Cancer Tumor Electrochemotherapy: Scientists are investigating the potential of electroporation to increase the effectiveness of chemotherapy. As in electroporation for DNA transfection, the applied electrical pulse would disrupt the membrane of the tumor cell and increase the amount of drug delivered to the site. Some studies have suggested that increased tumor reduction is seen when this method is applied to cancerous cells in animal model systems.
Gene Therapy: Much like drug delivery, electroporation techniques can allow vectors containing important genes to be transported across the skin and into the target tissue. Once incorporated into the cells of the body, the protein produced from this gene could replace a defective one and thus treat genetic disorder.

Yeast Transformation Electroporation


  1. Grow 50 ml to OD 1.3-1.5. 
  2. Spin cold at 5000 rpm. 
  3. Wash twice in 25 ml cold dH2O. 
  4. Wash in 1 ml 1 M sorbitol. 
  5. Resuspend in 50 µl 1 M sorbitol. 
  6. Take 40 µl aliquot and add <5 µl desalted DNA. 
  7. Incubate on ice 5 min+. 
  8. Electroporate in 0.2 cm cuvette at 1.5 kV (for 5 milli second). 
  9. Immediately add 1 ml cold 1 M sorbitol. 
  10. Plate on selective 1 M sorbitol plate or outgrow in 1 M sorbitol YPD broth first (need 2X media and 2 M sorbitol).
References:
http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/McCord/electroporation.htm
http://en.wikipedia.org/wiki/Electroporation
www.aragonlab.com/protocols/Yeast-Transformation-Electroporation.doc