Electroporation materials

To address biological processes at the single cell level in a live organism, electroporation techniques have been developed [5]. They involve electroporation by injection of various molecules (e.g., DNA, RNA or morpholinos) present in a micropipette brought in the close vicinity of the targeted cell (see Fig. 16.1). The technique is invasive, and the amount of electroporated material is unknown. Moreover, because the whole tissue is displaced by the inserted micropipette, the success rate in targeting a specific cell in a live embryo is very low. It would be desirable if a noninvasive technique could be devised to control the rapid activation of a known concentration of biomolecules in a specific cell of a live organism. 

Transfection by Electroporation. Materials 15 mg of plasmid DNA, 0.4 cm Electroporation cuvettes (BioRad, Hercules, CA, 165-2088), Electro-porator (BioRad Gene Pulser II) and Electroporation media (EM) (20% FBS/SF RPMI), 6-well tissue culture plates, SF RPMI, and complete RPMI. 

The first model of membrane electroporation was suggested by Crowley [1]. In Crowley s model the membrane is viewed as the isotropic elastic material. The necessary background for understanding its voltage-induced instability was discussed in Section II. Crowley s approximation for the elasticity energy term in Eq. (7) is... 

Several enveloped viruses, and some physical gene transfer techniques such as electroporation, deliver the nucleic acid into the cell by direct crossing of the cell membrane. Lipid-based, enveloped systems can do this by a physiological, selfsealing membrane fusion process, avoiding physical damage of the cell membrane. For cationic lipid-mediated delivery of siRNA, most material is taken up by endo-cytotic processes. Recently, direct transfer into the cytosol has been demonstrated to be the bioactive delivery principle for certain siRNA lipid formulations [151]. 

Other systems like electroporation have no lipids that might help in membrane sealing or fusion for direct transfer of the nucleic acid across membranes they have to generate transient pores, a process where efficiency is usually directly correlated with membrane destruction and cytotoxicity. Alternatively, like for the majority of polymer-based polyplexes, cellular uptake proceeds by clathrin- or caveolin-dependent and related endocytic pathways [152-156]. The polyplexes end up inside endosomes, and the membrane disruption happens in intracellular vesicles. It is noteworthy that several observed uptake processes may not be functional in delivery of bioactive material. Subsequent intracellular obstacles may render a specific pathway into a dead end [151, 154, 156]. With time, endosomal vesicles become slightly acidic (pH 5-6) and finally fuse with and mature into lysosomes. Therefore, polyplexes have to escape into the cytosol to avoid the nucleic acid-degrading lysosomal environment, and to deliver the therapeutic nucleic acid to the active site. Either the carrier polymer or a conjugated endosomolytic domain has to mediate this process [157], which involves local lipid membrane perturbation. Such a lipid membrane interaction could be a toxic event if occurring at the cell surface or mitochondrial membrane. Thus, polymers that show an endosome-specific membrane activity are favorable. 

This is a procedure increasingly used to introduce DNA into cells and various methods are available. I have experience with the calcium phosphate technique which is described below but other techniques are described by Gorman (1985) and Spandidos and Wilkie (1984). For example, Bethesda Research Ltd. supply liposomes which will mediate the uptake of nucleic acids at high efficiency (Feigner and Ringold, 1989) and with practice and the appropriate apparatus material may be injected directly into cells (Ansorge and Pepperkok, 1988). Electroporation is particularly useful for introducing DNA into plant spheroplasts. 

Electroporation Creation, by means of an electrical current, of transient pores in the plasmalemma usually for the purpose of introducing exogenous material, especially DNA, from the medium. 

In this sechon some applicahon aspects of giant vesicle electroporation are considered. In parhcular, it will be demonstrated that creahng macropores in GUVs and observing their closing dynamics can be successfully apphed to the evaluation of material properties of membranes. While in Section 7.4.2 we saw that such experiments can be used to characterize membrane stability in terms of the crihcal porahon potenhal f c, here we will find out how one can also evaluate the edge tension of porated membranes. In addition, another apphcation based on electro-porahon, namely vesicle electrofusion, is introduced whereby the use of GUVs as microreactors suitable for the synthesis of nanoparhcles is demonstrated. 

In some circumstances the introduction of a cloning vector into a host cell is a trivial process. For example, phage vectors are designed so they introduce recombinant DNA in an infective process called transfection, and some bacteria take up plasmids unaided. However, most host cells must be induced to take up foreign DNA. Several methods are used. In some prokaryotic and eukaryotic cells, the addition of Ca2+ to the medium promotes uptake. In others, a process called electroporation, in which cells are treated with an electric current, is used. One of the most effective methods for transforming animal and plant cells is the direct microinjection of genetic material. Transgenic animals, for example, are created by the microinjection of recombinant DNA into fertilized ova. 

Fig. 2 (a) Microelectroporation device for cell lysis, (b) Device at various steps of the fabrication process after metallization and electrode-mold formation (left) and after electroplating right). (c) Dielectrophoresis (DEP) effect observed in the flow channels top). Saw-tooth microelectrodes acting as a DEP device for focusing intracellular materials after electroporation bottom). Reproduced from [23] with permission... 

Example 2.11.1 Electroporation Allows Genetic Material to Penetrate the Cell... 

Electroporation can also be used to deliver drugs across the skin and genetic material to target cells, as in gene therapy. 

Esser, A.T., Smith, K.C., Gowiishankar, T.R., Weaver, J.C., 2007. Towards solid tumor treatment hy irreversible electroporation intrinsic redistribution of fields and currents in tissue. Technol. Cancer Res. Treat 6, 261—274. Etter, H.S., Pudenz, R.H., Gersh, I., 1947. The effects of diathermy on tissues contiguous to implanted surgical materials. Arch. Phys. Med. Rehab. 28, 333—344. 

Figure 4 shows a series of images illustrating the release of intercellular materials in the microfluidic devices of Fig. 3. In this figure, the intensified electric field in the electroporative area (section L2 of Fig. 3) is in the range of 800 V/cm to 1,200 V/cm.

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