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  • br Discussion Electroporation is a convenient and efficient


    Discussion Electroporation is a convenient and efficient way to introduce foreign material into dna synthesis inhibitors [10], [16], [17], [18]. There are a lot of different protocols for transfection with electroporation. It was reported that there are several parameters that are relevant to successful electroporation. Of these, the maximum voltage of shock, the duration of the current pulse, and the electroporation buffer are most important [1], [6], [10], [12]. In the present study, we systematically compared different electroporation parameters for CHO DHFR− cells. We evaluated the transfection efficiency of introducing plasmid DNA pSV-β-Gal into CHO DHFR− cells under the influence of: (1) voltage, capacitance and pulse duration; (2) number of pulse; and (3) electroporation buffer. Theoretically, when a strong electric voltage (>4.5KV/cm) is applied, the pores continue to exist long after the pulse, leading to irreversible damage of the cell membrane and induce apoptosis [17]. Conversely, if the electric field is relatively weak, the pores shrink and disappear once the pulse is removed, and the electrical membrane breakdown can thus be reversed, leading lower transfection efficiency [17]. In order to get higher transfection efficiency, an optimal voltage and time constant was tried by different researchers. It was reported that when the cells were exposed to high electric field strength the cell membrane permeabilization was maximal [19]. But Cegovnik and Novaković reported that a significant increase in transfection yield was achieved when the cells were treated by low-voltage pulse after the first short electroporative pulse [10]. Moreover, van den Hoff and co-workers reported that electroporation of cells in intracellular cytomix buffer combines a high efficiency of transfection with a good rate of cell survival [16]. Based on these results, we firstly measured the transfection efficiency of CHO DHFR− cells by setting different voltage and capacitance using cytomix electroporation buffer. The voltage settings were varied from 200 to 500V and the capacitance ranged from 25 to 950μF. In parallel, duration of applied electrical pulse was increased from 0.6 to 15.5ms. For a given electroporation buffer, we observed that longer pulse duration can increase the transfection efficiency. These results are consistent with previous reports that the longer the pulse duration, the higher the electroporative efficiency can be achieved [1], [20]. On the other hand, Ferreira and co-workers reported that increasing the number of pulse reduces the need for increasing the pulse amplitude for maximal MSC electroporation [19]. Our study showed that when two pulses were applied at 400V, 375μF, the transfection efficiency was increased from 0.006 to 0.289%. As indicated by several groups, the conductivity, osmolarity and components of the suspending medium during electroporation can affect the uptake of DNA, the electropermeability of the cell membrane and cellular survival [18], [21]. Moreover, it also suggested that cells electroporated in an intracellular buffer increases cell survival [16]. Our results showed that the transfection efficiency in Berg\'s and Hank\'s buffers was higher than that of in cytomix buffer under the same electroporation condition (400V, 375μF), but there is no obvious difference between Berg\'s and Hank\'s buffers. This result suggests that higher transfection efficiency was achieved in Berg\'s and Hank\'s buffers. In general, the transfection frequency, which is how many cells are transfected out of a total cell number, is between 1 in 104 to 105 for most cell types [23]. Our results indicated that the frequency of electroporation was 1 in 102 to 103 (the efficiency is 0.058 to 0.289%) for CHO DHFR− cells, which is better than that described preciously [23]. Taken together, we have determined the basic parameters for electrotransfection of CHO DHFR− cells and optimized the procedure in the present study. These results offer a guideline for effectively introducing DNA into CHO DHFR− cells by electroporation.