Cell plasma membrane electroporation

Physical methods of gene transfer. Genes can often be transferred without the use of a cloning vehicle. This is especially important for certain plant cells, such as those of cereal grains, for which transfer of genes via the Ti plasmid has been difficult.167 If DNA, which may be in a plasmid, is coprecipitated with calcium phosphate, it can often be taken up directly either by animal cells or by plant protoplasts.168 169 Polycations also facilitate DNA uptake cationic liposomes seem to be especially effective.170 In the widely used electroporation technique a short electrical pulse of a few hundred volts / cm is applied to create transient pores in the plasma membrane through which the DNA can enter a cell.111 8,171 175 Chromosomes can be transferred by cell fusion and either... 

Figure 6.35. Electroporation. Foreign DNA can be introduced into plant cells by electroporation, the application of intense electric fields to make their plasma membranes transiently permeable.

When cells are placed in external apphed electric fields, they experience an electric force. Electroporation involves the use of short, high voltage pulses to overcome barrier of the cell membrane. When a cell is submitted to an external electric field of high intensity and short duration (kV/cm, p,s), transient and dramatic increase in the permeability of the plasma membrane occurs beyond a point. This phenomenon is popularly called electroporation or electropermeabilization, which allows entry of otherwise impermeable exogenous molecules into the cell interior. This phenomenon has been an active area of research in biology and bioelectrochemistry for more than three decades [3,4] and has found many apphcations in cell biology. 

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. 

Interpretation of the data is complicated by the presence of nonapoptotic cells with damaged membranes. Such cells may have phosphatidylserine exposed on plasma membrane and, therefore, similar to apoptotic cells, bind annexin V. Mechanical disaggregation of tissues to isolate individual cells extensive use of proteolytic enzymes to disrupt cell aggregates, remove adherent cells from cultures, or to isolate cells from tissue mechanical removal of the cells from tissue culture flasks (e.g., by a rubber policeman) and cell electroporation, may affect the binding of annexin V. Such treatments, therefore, may introduce experimental bias in subsequent analysis of apoptosis by this method. 

In both methods, cultured animal cells must be treated to facilitate their initial uptake of a recombinant plasmid vector. This can be done by exposing cells to a preparation of lipids that penetrate the plasma membrane, Increasing Its permeability to DNA. Alternatively, subjecting cells to a brief electric shock of several thousand volts, a technique known as electroporation, makes them transiently perme able to DNA. Usually the plasmid DNA Is added In sufficient concentration to ensure that a large proportion of the cultured cells will receive at least one copy of the plasmid... 

Electroporation has been studied in different systems as artificial lipid bilayers, lipid vesicles, and animal and plant cells, and the effect has been considered at the level of plasma membranes. During the last two decades nanoelectroporation has been developed. The extremely short pulses applied in this approach allow the poration to occur at the level of subcellular structures. 

Since 1982, electroporation has been a promising approach for both in vitro and in vivo gene deUvery [61]. Electroporation causes disruption of the plasma membrane and formation of membrane-associated DNA aggregates, which enter the cytoplasm through transient pores [62], These complexes have been shown to enter the cytoplasm 30 min after administration of current, and proceed to perinuclear locations by 24 h postadministration [62]. Electroporation has shown promising results in the administration of complexes to skeletal muscles, indicating a possible role for the technique in future in vivo muscular applications [ 63 ]. Electroporation can be combined with chemical transfection methods, including dendrimers, to increase the overall efficiency of transfection [64], A similar approach, termed nucleofection, utilizes an electrical current in conjunction with specific solutions to deliver DNA not only into the cell but also into the cell nucleus [65]. 

DNA can transfect a cell without a delivery vector when simply injected intramuscularly or intravenously. However, this technique has a much lower efficiency than other methods. To improve transfection efficiency, physical methods can be used to improve gene delivery. Electroporation using an electric current or calcium ions temporarily increases the permeability of the plasma membrane and allows DNA to pass into the cell (Neumann et al., 1982). Sonoporation uses ultrasonic sound to... 

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