Electroporation applications

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. 

Electroporated cells can be used to transfer DNA in bacterial, plant, and mammalian cells. This method offers rapid and efficient incorporation of plasmid and DNA in cells [49]. The in vivo electroporation has been shown to yield enhanced plasmid delivery to a wide range of tissues including muscle, skin, liver, lung, artery, kidney, retina, cornea, spinal cord, brain, synovium, and tumors. The precise mechanisms involved in electroporation applications in vivo are uncertain and require further studies, but appear to involve both electropore formation and an electrophoretic movement of the plasmid DNA. 

Ohtani, K., Nakamura, M., Saito, S. Nagata, K., Sugamura, K., and Hinuma, Y. (1989) Electroporation Application to human lymphoid cell lines for stable introduction of a transactivator gene of human T-cell leukemia virus type I. Nucleic Acids Res. 17, 1589-1604. 

Electroporation (a temporary application of direct current, which disturbs the skin surface and allows penetration of the drug molecules)... 

The simplest nonviral gene transfer system in use for gene therapy is the injection of naked plasmid DNA (pDNA) into local tissues or the systemic circulation (88, 100). Naked DNA systems are composed of a bacterial plasmid that contains the cDNA of a reporter or therapeutic gene under the transcriptional control of various regulatory elements (101, 102). In recent years, work in several laboratories has shown that naked plasmid DNA (pDNA) can be delivered efficiently to cells in vivo either via electroporation, or by intravascular delivery, and has great prospects for basic research and gene therapy (101). Efficient transfection levels have also been obtained on direct application of naked DNA to the liver (103, 104), solid tumours (105), the epidermis (106), and hair follicles (106). 

In one of the first studies of in vivo delivery of pDNA using electroporation, Titomirov etal. (1991) demonstrated that the skin of newborn mice could take up and express a reporter gene after pDNA injection followed by the application of electric pulses. In a later study, Heller et al. (1996) injected the liver of rats with pDNA encoding / -gal followed by the administration of electric pulses. In the rat study, long-term expression of / -gal occurred after pDNA electroporation (up to day 21 post pDNA injection) and 30-35% of the hepatocytes were transfected. 

Figure 18.4 Pulse generator and applicator electrode for in vivo electroporation. The applicator electrode shown here consists of six needles and is used for intra-tumoral and intra-muscular applications in large animals. Two needle applicator electrodes for use in rodent muscles are commercially available.

Potter, H. (1988) Electroporation in biology methods, applications, and instrumentation. Anal. Biochem., 174, 361-373. 

Transdermal iontophoresis involves the application of an electric field across the skin to facilitate (primarily) ionic transport across the membrane. Iontophoresis, it is important to point out, is differentiated from electroporation [14], another electrical approach to enhance transdermal transport, by the low fields employed. Whereas iontophoresis has achieved commercialization, there is (to our knowledge) no active development in progress of a transdermal delivery system employing electroporation. 

It is noteworthy that some therapeutic applications, such as transcutaneous electrical neural stimulation, involve application to the skin of electric pulses of up to hundreds of volts [5], However, a safety limitation is the major concern associated with the use of electroporation, even though several reports indicated that the damage to the skin was mild and reversible [16,23]. The only skin alteration seen with electroporation was slight erythema that decreased within a few hours [34]. Patients submitted to electrochemotherapy seemed to tolerate well the application of 10,000 V/cm for 100 p,s square-wave pulses [35]. However, to avoid pain during electroporation, milder conditions such as lower voltage, shorter pulses, or improved electrode design could be used [36]. 

During the first passive stage, both permeants penetration slowly built up. Upon application of all three electrical protocols, the apparent fluxes for both molecules were always significantly enhanced in relation to their respective steady-state passive values (P < 0.05). During the second passive period, penetration rates considerably reduced, though the apparent fluxes were always significantly greater after electroporation or combined treatment than with iontophoresis alone (P < 0.05). 

As the mechanisms of penetration and acceleration during iontophoresis or electroporation are different, the application of a constant direct current after pulsing may raise penetration even further in comparison with either method alone. Table 15.2 shows that for L-glutamic... 

Application of an electric field to lipid bilayers such as those found in cellular membranes causes short-term depolarization of the membrane and formation of pores and other structural changes [17]. These so-called electropores allow the uptake of hydrophilic macromolecules such as plasmid DNA, siRNA, or proteins that are otherwise unable to diffuse passively through this highly regulated barrier. The use of high-voltage electrical pulses to permeabilize cell membranes was first described as a tool to deliver DNA into mammalian cells in 1982 (Wong and Neumann 1982 Neumann et al. 1982). In cuvette-based methods, cells are... 

Transfection efficacy of naked DNA can be increased by physical methods such as electroporation and sonication. Electroporation employs electric pulses to punch holes in the cell membrane, usually smaller than 10 nm but larger than oligonucleotides. With the use of electroporation, DNA was delivered into the cytosol of cells by diffusion. Since its introduction in 1982, in vivo transfection has been achieved in skeletal muscle, fiver, skin, tumors, testis, and the kidney. Tsujie et al. (2001) developed a method to target glomeruli using electroporation in vivo wherein injection of plasmid DNA via the renal artery was followed by application of electric fields. The kidney was electroporated by sandwiching the organ... 

Cell lysis under a high electric field is referred to as electroporation [6], Under these conditions, the cell membrane experiences dramatic changes in permeability to macromolecules. The main applications of the electroporation include the electrotransformation of cells and the electroporative gene transfer by the uptake of foreign DNA or RNA (in plants, animals, bacteria, and yeast). The electric field generates permeable microspores at the cell membrane, so that the nucleic acid can be introduced by electroosmosis or diffusion. 

Cell lysis on a chip has been carried out by several different approaches. Detergents have been used to lyse cells on a chip. Thermal lysing on a chip has been carried out by placing the cell in the sample reservoir and then raising the temperature of the chip [50], A practical approach for microchip applications is lysis by electroporation. Since fluids are moved around on chips by the application of an electric field, its use in cell lysis is an obvious choice. 

---