27 December 2007 Enhancement of reverse transfection efficiency by combining stimulated DNA surface desorption and electroporation
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Abstract
Transfection cell microarrays (TCMs) are a high-throughput, miniaturised cell-culture system utilising reverse transfection, in which cells are seeded onto a DNA array resulting in localised regions of transfected cells. TCMs are useful for the analysis of gene expression, and can be used to identify genes involved in many cellular processes. This is of significant interest in fields such as tissue engineering, diagnostic screening, and drug testing[1, 2]. Low transfection efficiency has so far limited the application and utility of this technique. Recently, the transfection efficiency of TCMs was improved by an application of a high voltage for a short period of time to the DNA array resulting in the electroporation of cells attached to the surface[3, 4]. Furthermore, application of a low voltage for a longer period of time to the DNA array was shown to improve the transfection efficiency by stimulating the desorption of attached DNA, increasing the concentration of DNA available for cellular uptake[5]. In the present study, the optimisation of the uptake of adsorbed DNA vectors by adherent cells, utilising a voltage bias without compromising cell viability was investigated. This was achieved by depositing negatively charged DNA plasmids onto a positively charged allylamine plasma polymer (ALAPP) layer deposited on highly doped p-type silicon wafers either using a pipettor or a microarray contact printer. Surface-dependant human embryonic kidney (HEK 293 line) cells were cultured onto the DNA vector loaded ALAPP spots and the plasmid transfection events were detected by fluorescence microscopy. Cell viability assays, including fluorescein diacetate (FDA) / Hoechst DNA labelling, were carried out to determine the number of live adherent cells before and after application of a voltage. A protocol was developed to screen for voltage biases and exposure times in order to optimise transfection efficiency and cell viability. Cross-contamination between the microarray spots carrying different DNA vectors was also investigated. By application of a voltage of 286 V/cm for 10 ms, transfection efficiency was doubled compared to using only transfection reagent, whilst maintaining a cell viability of 60-70% of the positive control.
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Rhiannon Creasey, Rhiannon Creasey, Andrew Hook, Andrew Hook, Helmut Thissen, Helmut Thissen, Nicolas H. Voelcker, Nicolas H. Voelcker, } "Enhancement of reverse transfection efficiency by combining stimulated DNA surface desorption and electroporation", Proc. SPIE 6799, BioMEMS and Nanotechnology III, 67991D (27 December 2007); doi: 10.1117/12.759382; https://doi.org/10.1117/12.759382
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