Electroporation methods

Combined Chemical and Electroporation Methods of Skin Penetration Enhancement... 

Most of electroporation methods have adopted total population cell treatments. Total population methods yield information about average electroporative behavior of cells. Since cells are heterogenic cell population, it is important to examine if average... 

Buchser, W.J. et al., 96-WeU electroporation method for transfection of mammahan central neurons, BioTechniques, 41, 619, 2006. 

Nakamura H, Funahashi J (2001) Introduction of DNA into chick embryos by in ovo electroporation. Methods 24 43 8. 

Electroporation. When bacteria are exposed to an electric field a number of physical and biochemical changes occur. The bacterial membrane becomes polarized at low electric field. When the membrane potential reaches a critical value of 200—300 mV, areas of reversible local disorganization and transient breakdown occur resulting in a permeable membrane. This results in both molecular influx and efflux. The nature of the membrane disturbance is not clearly understood but bacteria, yeast, and fungi are capable of DNA uptake (see Yeasts). This method, called electroporation, has been used to transform a variety of bacterial and yeast strains that are recalcitrant to other methods (2). Apparatus for electroporation is commercially available, and constant improvements in the design are being made. 

Edwards [105] has extended the macrotransport method, originally developed by Brenner [48] and based upon a generalization of Taylor-Aris dispersion theory, to the analysis of electrokinetic transport in spatially periodic porons media. Edwards and Langer [106] applied this methodology to transdermal dmg delivery by iontophoresis and electroporation. 

Classical gene transfer methods still in use today are diethylamino ethyl (DEAE)-dextran and calcium phosphate precipitation, electroporation, and microinjection. Introduced in 1965, DEAE-dextran transfection is one of the oldest gene transfer techniques [2]. It is based on the interaction of positive charges on the DEAE-dextran molecule with the negatively charged backbone of nucleic acids. The DNA-DEAE-dextran complexes appear to adsorb onto cell surfaces and be taken up by endocytosis. 

Tjelle TE, Rabussay D, Ottensmeier C, Mathiesen I, Kjeken R (2008) Taking electroporation-based delivery of DNA vaccination into humans a generic clinical protocol. Methods Mol Biol 423 497-507... 

The uptake of the plasmid into the bacterial cell is called transformation and in the laboratory can be induced in two ways. In one method the bacteria and DNA are placed in ice-cold CaCl2 (50 mmol l-1). This induces the DNA to stick to the outside of the bacteria. The temperature is then increased to 42 °C and the DNA enters the cell. The second method is by electroporation,... 

The DNA or cDNA library is then introduced into a preparation of bacterial host cells. Usually, the first host selected is a laboratory strain of E. coli which has been grown and pretreated with inorganic salts to make uptake of DNA easier. The ability to take up foreign DNA is called competence, cells which have been specially prepared for the purpose are called competent cells. Other methods to transfer DNA into cells include electroporation (application of an external electric field to permeabUize the cell wall), transfection (where a recombinant bacterial virus is used to transfer the DNA to the target cell) or ballistic methods (by using DNA-coated particle projectiles). The last method has been used to introduce foreign DNA into plant cells and mammalian cells. 

Transfection is the process of introducing DNA or RNA into eukaryotic ceils. The use of transfection is to study the role and regulation of proteins or to understand the mechanisms of a pathway. Transfection can be transient for rapid analysis or stable , mostly for induction of expression. There are various methods of transfection which include electroporation, viral vectors, DEAE-Dextran, calcium phosphate or Lipofectamine. The choice of transfection depends on the cell type used. The most desirable technique is the one which gives high efficiency of nucleic acid transfection with less interference to the cells physiology and high reproducibility. 

Table 11.2. Vector systems used to deliver genes into mammalian cells. To date, the majority of clinical trials undertaken have utilized retroviral vector systems. Non-viral systems have generally been employed least often, although some, e.g. nucleic acid-containing liposomes, may be used more extensively in the future. Some of the methods tested, e.g. calcium phosphate precipitation, electroporation and particle acceleration, are unlikely to be employed to any great extent in gene therapy protocols...

Some investigators have used electrical current (electroporation) to improve DNA (or drug) entry into tumor cells with some preliminary success. Liposomes are attractive vehicles for gene delivery, since they can carry plasmid, antisense, or viral DNA. Compared with viral approaches, however, liposomes remain relatively inefficient at facilitating gene transfer, although their safety profile remains more desirable. Some of the attributes and limitations of the nonviral methods are listed in Table 58.1. 

Agrobacterium-mQdi iQd DNA transfer technique and direct gene transfer into protoplasts on the basis of the electroporation technique have frequently been used for the gene transfer methods in plants. However, these methods were hardly adapted to monocotyledonous plants or to varieties in which regeneration system was not established. These deficiencies found in gene transfer techniques will be largely overcome if pollen could be used as a DNA vector. 

Gel, J. (2003). Electroporation theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol. Scand., Ill, 437-447. 

Nickoloff, J.A. (Ed.) (1995). Methods in Molecular Biology, Vol. 48 Animal Cell Electroporation and Electrofusion Protocols. Human Press, pp. 3-38. 

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