Mammalian cells 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. 

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...

Transfection, DNA uptake in eukaryotic systems, often is more problematic then bacterial transformation the mode of DNA uptake is poorly understood and efficiency is much lower. In yeast, cell walls can be digested with degradative enzymes to yield fragile protoplasts, which are then able to take up DNA. Cell walls are resynthesized after removal of the degrading enzymes. Mammalian cells take up DNA after precipitation onto their surface with calcium phosphate [Fugene 6 (Roche) Lipofectin (Life Technologies) Effectene (Qiagen)]. Electroporation is often more efficient for transfection in eukaryotic cell systems, especially in yeasts. 

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... 

Bertling W, Hunger-Bertling K, Cline MJ (1987), Intranuclear uptake and persistence of biologically active DNA after electroporation of mammalian cells, J. Biochem. Biophys. Methods 14 223-232. 

Chu, G., Hayakawa, H., and Berg, P. (1987). Electroporation for the Efficient Transfection of Mammalian Cells with DNA Nucleic Adds Res 15 1311. 

The continuous interest and growth of the various new industrial processes related to the life sciences will also require significant contributions from membrane engineering. It is hoped that future research would provide deeper insight on the precise mechanisms involved, which would show new direction for research in membrane science and applications. It is, however, important to recognize that applicability of electroporation has been demonstrated in a variety of bacteria, yeast, and mammalian cells and some applications are ready for exploitation while many new technologies seem potentially possible. 

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. 

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. 

To produce stable and long-lasting knockdown, viral vectors have been developed to efficiently express siRNAs or short hairpin RNAs (shRNAs) in a wide variety of mammalian cells (Fig. 8). Expression vectors include standard plasmids as well as those made from adenoviruses, adeno-associated viruses (AAVs), onco-retroviruses, and lentiviruses. The viral expression systems have the added benefit of being able to deliver siRNA, shRNA, or miRNA expression cassettes efficiently into cell types that are otherwise difficult to transfect via common transfection or electroporation protocols.f ... 

In the previous two sections we discussed the electrodeformation and electroporation of vesicles made of single-component membranes in water. In this section, we consider the effect of salt present in the solutions. The membrane response discussed above was based on data accumulated for vesicles made of phosphatidylcholines (PCs), the most abundant fraction of lipids in mammahan cells. PC membranes are neutral and predominantly located in the outer leaflet of the plasma membrane. The inner leaflet, as well as the bilayer of bacterial membranes, is rich in charged lipids. This raises the question as to whether the presence of such charged lipids would influence the vesicle behavior in electric fields. Cholesterol is also present at a large fraction in mammalian cell membranes. It is extensively involved in the dynamics and stability of raft-hke domains in membranes [120]. In this section, apart from considering the response of vesicles in salt solutions, we describe aspects of the vesicle behavior of fluid-phase vesicles when two types of membrane inclusions are introduced, namely cholesterol and charged lipids. 

As is known to molecular biologists, DNA electrotransfer to bacteria is a standard method, and a small pulse generator is everyday equipment in most laboratories. However, whereas the molecular biologist would not hesitate to let billions of bacteria succumb to side effects of high-field electroporation, this view is of course not shared by those working with mammalian cells-and doctors working in the clinic would have safety as the major priority [2]. 

In order to work with mammalian cells, pulse parameters need to be much better controlled, and this is efficiently done with a square wave electroporator [3]. Here, the pulse amplitude and duration may be individually controlled, allowing a much better adaptability to the target cells or tissues as well as to the molecule that is to be transferred. Another important stage of the development has been the availabihty of square wave pulse generators designed and approved for chnical use. 

In addition to calcium phosphate transfection, methods for DNA transfer into mammalian cells by electroporation [31, 32] and by transfection mediated through cationic lipids, liposome [33-35], biohstics [36] and polymers [37, 38] have been developed. Most of these techniques have been reported to mediate higher transfection efficiencies as compared to calcium phosphate-mediated DNA transfer. Such claims must be regarded with some caution, as all DNA transfer techniques estabhshed so far suffer from high variabihty due to technical difficulties. Other factors to cause major variations in transfection efficiency are the type of cells used and the condition of the cells prior to transfection. 

Delivery of DNA vaccines (or any DNA plasmids) to mammalian cells can be achieved by physical methods such as electroporation [18], in which electric pulses cause a transient increase of cell membrane permeability to DNA, and the gene-gun technology [19], where DNA is coated onto gold particles and forced into cells... 

Chu G, Hayakawa H, Berg P. Electroporation for the efficient transfection of mammalian cells w/ith DNA. Nucleic Acids Res 1987 15 1311-1326. 

Wang H-Y, Lu C (2006) Electroporation of mammalian cells in a microfluidic channel with geometric variation. Anal Chem 78 5158-5164... 

Knutson, J. C., and Yee, D. (1987) Electroporation Parameters affecting transfer of DNA into mammalian cells. Anal. Biochem. 164, 44-52. 

Duration, form, and number of pulses applied The duration of a single electrical pulse is typically varied between a few microseconds and several milliseconds. When low voltages are used usually long pulses are applied, and vice versa, to obtain good results. In most applications the pulse form is exponential (Fig. 2) but square-shaped pulses also have been used successfully (Presse et al., 1988). The number of pulses applied to the cell chamber is usually limited technically to one. More than one pulse (achieved with the EMBL device) may improve transfection efficiency of DNA in mammalian cells. Reports show increased efficiency of electroporation and cell fusion with high-frequency (20-100 kHz) ac pulse trains (Chang, 1989). 

Concentration of molecules and cells Cells are prepared at concentrations of 10 to 10 per milliliter for mammalian cells and up to 10 ° to 10 per milliliter for bacterial cells. In our laboratory, typically 40-50 fjt[ of cell solution is used per transfection experiment. DNA concentrations of 5 /tg/ml already give good transformation efficiencies. Higher DNA concentrations improve the results and should be used when sufficient amounts of DNA are available. For electroporation of proteins, antibodies and dyes are used at concentrations ranging from 10 to 10 M. 

Culture conditions and electroporation buffer Cells are cultured under normal culture conditions. A medium of low ionic strength should be used as buffer solution for electroporation. PBS or HBS is preferred for mammalian cells and pure distilled water or 10% glycerol for bacteria. Successful use of chemical stimulators added to the electroporation buffer has been reported (Satyabhama and Estonia, 1988). Additives increasing cell surface binding of the molecules to be electroporated may be taken into consideration. For the electroporation, cells are kept either on ice or at room temperature. To avoid contamination, sterile working conditions are important for mammalian cells. 

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