Abstract
The type of a nucleic acid and the type of the cell to be transfected generally affect the efficiency of electroporation, the versatile method of choice for gene regulation studies or for recombinant protein expression. We here present a combined square pulse electroporation strategy to reproducibly and efficiently transfect eukaryotic cells. Cells suspended in a universal buffer system received an initial high voltage pulse that was continuously combined with a subsequent low voltage pulse with independently defined electric parameters of the effective field and the duration of each pulse. At comparable viable cell recoveries and transfection efficiencies of up to 95% of all cells, a wide variety of cells especially profited from this combined pulse strategy by high protein expression levels of individual cells after transfection. Long-term silencing of gene expression by transfected small interfering RNA was most likely due to the uptake of large nucleic acid amounts as shown by direct detection of fluorochromated small interfering RNA. The highly efficient combined pulse electroporation strategy enables for external regulation of the number of naked nucleic acid molecules taken up and can be easily adapted for cells considered difficult to transfect.
Highlights
In the early 1980ies, upcoming recombinant techniques required efficient introduction of nucleic acids into eukaryotic cells
Transfection efficiencies improved under optimized conditions applying combined high voltage (HV)/low voltage (LV) square pulses using a standard universal phosphate buffer system as shown for preadipocytes (Fig. 2)
How might combined HV/LV square pulses become advantageous for distinct cell populations? The high field strength of short HV pulses is thought to be sufficient to create pores that overall increase cell membrane permeability for large molecules [24,25]
Summary
In the early 1980ies, upcoming recombinant techniques required efficient introduction of nucleic acids into eukaryotic cells. Physical or virus-based transfection and transduction strategies were developed [1,2]. Dependent on type and/or cell cycle state of the target cell population, these techniques might provide low transfer efficacies, be not suitable for non-adherent cells, require toxic reagents, give limited access to the nucleus, or are time-consuming [3,4,5,6]. Ever since the first description of myeloma cell transfection [8], electroporation became the method of choice for efficient transient transfection for numerous types of eukaryotic cells. The so-called nucleofection combines electric pulses and chemical transduction to direct for the nucleus [9]
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