Electroporation cells

Remove the electroporated cells as quickly as possible and incubate them in a microcentrifuge tube with 1 mL of SOC medium at 37°C for 1 h. Plate different volumes of cells (90-300 pL) on agar medium supplemented with ampicillin and the relevant peptide in order to select PRP-resistant mutant clones (see Note 4). 

ES cell culture Resuspend the electroporated cells with ES media to 2.5 x 105 cells/4 mL, and add 4 mL of the ES cells to each 6-cm EF plate. Two plates are labeled with single selection (G418), and all the others with double selection (G418 + ganciclovir). Also, set up one plate as a control in which ES cells have not been electroporated. 

Electroporation. Cells are subjected to a high-voltage pulse, which temporarily disrupts the membrane and allows the vector to enter the cell. This is the most efficient method of delivering genes to bacterial cells. 

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. 

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. 

Fill four sterile tubes with 1 mL LB medium and prepare four successive 1/10 dilutions starting from the electroporated cells. Spread 50-pL aliquots of each dilution onto a plate, using a triangular-shaped, sterile glass rod. Incubate the plates at 37°C overnight. 

Hibino, M., Shigemori, M., Itoh, H., Nagayama, K., and Kinosita, K. (1991) Membrane conductance of an electroporated cell analyzed by submicrosecond imaging of transmembrane potential. Biophysical Journal, 59 (1), 209-220. 

Tekle, E., Asmmian, R.D., and Chock, P.B. (1994) Selective and asymmetric molecular-transport across electroporated cell membranes. Proceedings of the National Academy of Sciences of the USA, 91 (24),... 

During electroporation, cells or tissues are exposed to an electric field with high voltage (up to 1 kV). Short, rapid pulses cause transient membrane instability and the formation of pores with a mean lifetime of minutes [30]. Soluble DNA constructs that have been added to the culture medium or injected into the tissue may thereby enter the cell and ultimately reach the nucleus. The basic principle is also known to pharmacists as iontophoresis and is used for transdermal drug apphcation [31]. This gene transfection method has so far been well accepted by patients and is safe, especially due to the low risk of infection. To date, mainly liver, muscle, and skin cells have been transformed this way, mostly via the transdermal route [32]. 

Fig. 3. Cytosolic Ca + increase induced by cAMP in electroporated cells loaded with fura-2-dextran at 4 h development. Mean R340/380 SEM of nine cells tested at 6.5 h is plotted versus time R340/380 was taken as a measure for [Ca +J.. The time of addition of cAMP (f uM) was at 45 sec. The experiment was done in FI5-buffer containing 1 mM Ca + (Redrawn from ref. 4).

T. (1995) Introduction of macromolecules into living Dictyostelium cells by electroporation. Cell Struct. Funct. 20, 185-190. 

Following electroporation, incubate the cell suspension at 21°C for 15 min with 4 pL of healing buffer and then add the mixture of 10 mL of buffer with 10 mL of low-osmotic-strength buffer to the electroporated cell suspension (rrrNote 7). 

A series of innovative strategies have been developed to electroporate cells on a microfluidic platform. In these methods, high-density microelectrodes or structures with subcellular dimensions are required. Lu et al. presented... 

After 3h, the embryos are photographed as in section 3.5.3 to visualize the location of the electroporated cells if fluorescent constructs are onployed for labehng (Fig. 8B Note 24)... 

Cells are routinely passaged two days prior to electroporating. Cells are ready for electroporating when their density is optimal. Usually one 10-cm plate at approx 80% confluency will provide enough cells for 1-2 electroporations. Our standard electroporation protocol is given below. 

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. 

Count cells positive for FITC fluorescence in the cytoplasm under a fluorescence microscope in a defined area on the coverslip in relation to all cells in this area. Correct this rate for the number of cells that survived the electroporation using the control experiment. Optimal voltage is determined from a diagram of number of successfully electroporated cells versus voltage. This procedure can also be used to optimize other variables influencing the efficiency of electroporation experiments mentioned above. 

Two days after electroporation cells are screened for T-antigen expression by indirect immunofluorescence. Efficiency is expressed as the percentage of T-antigen-positive cells. 

Electroporate cells at a brief pulse of 750 V/cm and duration of 0.7-0.9 msec at room temperature. Cell porator settings should be an initial voltage of 300, the resistance switch at low position, and capacitances of 50-/xF for suspension cells and 60 //F for adherent cells. 

The choice of electroporation medium and temperature is also important for this technique. A reduced serum-containing medium, Opti-MEM/5%FBS, was found to be most suitable for the cell types chosen for electroporation. Although prechilled cells were used to counteract the local heating by electric pulse, room temperature electroporation was more favorable than 4°C for the rapid recovery of the electroporated cells (Chakrabarti et al., 1989 Chu et al., 1987). At lower temperatures, membrane pores tend to stay open longer (Stopper et al., 1987) which may have impacts on cell survival. 

Electroporated cells can be used only for short-term metabolic studies, as the intracellular antibody concentration remained intact only for 72 hr. 

Aliquot the electroporated cells to 100-mm plates with feeder layers (note that the feeder cell medium must first be changed to complete ES medium) The number of plates required depends on the number of resultant colonies, crowding will... 

Electrocompetent Escherichia coli XL-1 Blue and BL-21 (DE3) were used for plasmid synthesis and protein synthesis respectively. Aliquots (100 p,L) of electrocompetent cells were thawed on ice, and 20-100 ng of plasmid was added and mixed. Cells were kept on ice for 5 min before transferring to an electroporation cuvette, where an electrical pulse is applied to the cells. Immediately following electroporation, cells were transferred to 9(X) p L of LB medium and incubated at 37 °C with agitation for 1 h. Typically, 1(X) pL of cells were then plated onto LA plates enriched with the appropriate antibiotic (30 pg/mL Kanamycin or Chloramphenicol). Plates were incubated overnight at 37 °C, with transformation of the cells confirmed by presence of colonies, which were picked and grown for plasmid synthesis (XL-1 Blue) or protein synthesis (BL-21 (DE3)). 

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