Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency
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It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.Keywords:
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Gene delivery, a process of introducing foreign functional nucleic acids into target cells, has proven to be a very promising tool for inducing specific gene expression in host cells. Many different technologies have been developed for efficient gene delivery. Among them, electroporation has been adopted in gene delivery for decades, and it is currently widely used for transfection of different types of cells. Despite of the success achieved by bulk electroporation (BEP) for gene delivery in vitro and in vivo, it has significant drawbacks such as unstable transfection efficacy and low cell viability. In recent years, there is an emerging interest in understanding how individual cell accepts and responds to exogenous gene materials using single cell based micro-/nano-electroporation (MEP/NEP) technologies. In this review, the authors provide an overview of the recent development of MEP/NEP and their advantages in gene delivery. Additionally, the future perspectives of gene delivery with the application of electroporation are discussed. Keywords: Electroporation, gene delivery, nano-electroporation, nanocarriers, microfluidics.
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Objective: To compare the transfection efficiency of electroporation and chemical transfection agents in multidrug-resistant(MDR) gastric cancer cells.Methods: MDR gastric cancer SGC7901/VCR cells were transfected with plasmids bearing tumor suppressor gene WTX through electroporation method and two chemical agents(Lipofectamine2000 and Attractene), respectively. Then, the expression of enhanced green fluorescent protein(e GFP) and the WTX m RNA in the cells after transfection were examined.Results: Results of e GFP analysis showed that in SGC7901/VCR cells compared with the parental non-drug-resistant SGC7901 cells, the transection efficiencies of both chemical methods were significantly decreased(both P0.05), but the transfection efficiency of electroporation exhibited no significant decrease(P0.05), which was significantly higher than that of either chemical agent(both P0.05). Results of RT-PCR showed that the WTX m RNA expression level in SGC7901/VCR cells undergoing electroporation transfection was significantly higher than that in those transfected by the two chemical agents(both P0.05). Conclusion: In MDR gastric cancer cells, electroporation transfection will not be influenced easily by their cell membrane components, and can maintain its transfection efficiency.
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To avoid safety issues such as immune response and cytotoxicity associated with viruses and liposomes, physical methods have been widely used for either in vivo or ex vivo gene delivery. They are, however, very invasive and often provide limited efficiency. Using pEGFP and pSEAP plasmids and NIH 3T3 fibroblasts as models, we demonstrate a new electroporation-based gene delivery method, called membrane sandwich electroporation (MSE). The MSE method is able to provide better gene confinement near the cell surface to facilitate gene transport into the cells and thus shows significant improvement over transgene expression of mammalian cells compared to current electroporation techniques.
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Objective:To find the best parameters to transfect plasmid pG into 32DP210 cells.Methods:LipofectamineTM2000,transfast,liposome DMRIC-E transfection and electroporation were compared for transfecting plasmid pG,a plasmid that expressed G418 resistance protein and green fluorescent protein(GFP),into 32DP210 cells.The expression of GFP was observed and the transient transfection efficiency was measured after the transfection by fluorescence microscope.The stable transfected cells were screened by G418.Results:The optimum conditions for laboratory electroporation-mediated 32DP210 cell transfection were:power transfer voltage 270 V,capacitance of 950 μF,DNA dose of 14 μg.Transfection efficiency of electroporation after 48 h was 50.81%,while the other three methods were less than 3%.Conclusion:Electroporation is the best way to stably transfect plasmid pG into 32DP210 cells.
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