Electroporation efficiency

Resealing characteristics determine the viability as well as nature and size of exogenous molecules for incorporation into electroporated cells [21-24]. The composition of the membrane differs from one cell type to another, and consequently, the obtained electroporation efficiency of cells. In addition, it is evident from Equation 26.3 that the magnitude of induced membrane potential depends on the shape and dimension of the cell. 

Electroporation efficiency depends on the parameters of electric pulses that are delivered to the treated cells using specially designed electrodes and electronic devices. In vitro experiments usually employ parallel plate types of electrodes made of inert metals like stainless steel or platinum but needle types of electrodes are also used for tissue electroporation [24,25,27,28] as well as for tumor treatment apphcations [29-32]. There are two types of electroporator devices available devices with voltage output and those with current output. However, a voltage output device seems to be preferable, which is widely used for diverse applications. 

For the identical experimental conditions, electroporation efficiency depends on the type of cells the composition of the membrane, shape, and size of cells strongly influences the electroporation efficiency [40-42]. In electroporation of bacteria, the growth phase of cell has significant influence on transformation efficiency, which is higher for cells harvested and electroporated from mid-log phase. However, cells from stationary phase can also be transected with reasonably good efficiency. Mammalian cell can be electroporated at relatively lower fields but pulse length controls the entry of external molecules into cells. 

Most adherent cell types can be efficiently transfected with lipid reagents. Even notoriously hard to transfect cells such as neurons, certain primary cells, and embryonic stem cells can be successfully transfected under optimized conditions with the appropriate reagents, presumably because siRNAs only need to be delivered to the cytoplasm (unlike DNA plasmids that must enter the nucleus for expression). For reasons that are not yet clear, suspension cells tend to be refractory to lipid-based transfection and typically require electroporation or viral-based methods for delivery of siRNA. While electroporation efficiently delivers naked siRNA directly to the cytosol for access to RISC, thereby bypassing the endosomal compartment, the extensive cell death that results and large amounts of siRNA required are prohibitive for broader-range applications. 

Lawrenz, M. B., Kawabata, H., Purser, J. E., and Norris, S. J. (2002) Decreased electroporation efficiency in Borrelia burgdorferi containing linear plasmids lp25 and lp56 impact on transformation of infectious Borrelia. Infect. Immun. 70, 4851 858. 

The efficiency of antibody incorporation into the cells electroporated using this method can be determined several ways. The concentration of antibody can be detected by Western blotting of the cell extracts, using an anti-mouse antibody for monoclonal and anti-rabbit antibody for polyclonal antibody (Fig. 1), and quantitated by densitometry. Electroporation efficiency can also be determined by flow cytometry. Fluorescein isothiocyanate (FITC)-labeled antibody can be used for electroporation, and the percentage of fluorescent cells can be determined with a fluorescence-activated cell sorter. 

The electroporation conditions and parameters described here were optimized for a mouse lymphoma cell line (L5178YD10/R) that grows in suspension and two adherent cell lines, HeLa and a human fibroblast (HT-5). Approximately 80-90% cell viability and 85-95% electroporation efficiency were achieved using the above... 

The number of resultant colonies is dependent on the number of cells electroporated, the amount of DNA used and the type of targeting strategy employed. Most of the selection vectors we use are devoid of polyadenylation signals and yield 100-300 colonies per experiment Identical constructs bearing polyadenylation signals typically yield 5-10 times more colonies. The promoter used to drive positive selectable markers can also affect the electroporation efficiency we use the PGK promoter, which routinely yields 2-5 times more colonies than comparable vectors driven by SV40 enhancer sequences... 

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. 

As an alternative to the chemical transfection systems, the primary cells can be effectively transfected by electroporation (Appendix 5). Amaxa has developed a nucleofection (electroporation) system that is very effective for delivering expression vectors into cells at high efficiency and low cellular mortality. The step-by-step conditions for the transfection of numerous primary cell types, including human airway epithelial cells, can be found at http //www.amaxa.com/primary-cells.html. 

Therefore alternative techniques for gene transfer are necessary. While progress has been made in electroporation of Desulfovibrio (Rousset et al. 1991, 1998), the development of reliable and efficient procedures deserves further attention. Evidence has been obtained for the presence of a restriction barrier that limits transformation in D. vulgaris (Fu and Voordouw 1997). The efficiency of electroporation of plasmids into D. vulgaris was increased from an undetectable level to lO transformants... 

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. 

The floxed stop cassette of the integrated construct can be deleted in ES cells by transient transfection with a Cre-expressing plasmid (available, e.g., at http //www.addgene. org/) and subsequent low-density plating as selection for recombined clones is impossible. After electroporation, we plate 500-2,000 cells on feeders in a 10 cm dish and analyze 24 clones for recombination by PCR or Southern blot. At least two recombined clones should be analyzed for knockdown efficiency. 

Because JC8679 competent cells for electroporation are not commercially available, we had to produce our own. We routinely prepare competent cells following the protocol reported previously (14). Although JC8679 competent cells for electroporation of 10 -10 cfii/pg pUC19 are routinely prepared, the apparent transformation efficiency for the ORF trap is much lower (typically 50-100 colonies per 30 pL of transformation culture). 

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). 

Pretreatment of the muscle with enzymes such as hyaluronidase enhances the electroporation effect thus allowing a reduction in the field strength and consequently reducing the myofiber damage associated with electroporation (251). This raises the efficiency to the equivalent of the best that can be achieved with local delivery using the best viral vector. 

Van Tendeloo, V.F., Ponsaerts, P, Lardon, F., et al. (2001). Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood, 98, 49-56. 

Available methods for carrying DNA into an animal cell vary in efficiency and convenience. Some success has been achieved with spontaneous uptake of DNA or electroporation, techniques roughly comparable to the common methods used to transform bacteria. They are inefficient in animal cells, however, transforming only 1 in 100 to 10,000 cells. Microiqjection—the injection of DNA directly into a nucleus, using a very fine needle—has a high success rate for skilled practitioners, but the total number of cells that can be treated is small, because each must be injected individually. 

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