Effects of the dilution rate on cell cycle distribution and PEI-mediated transient gene expression by CHO cells in continuous culture

Li, Weiwei; Liu, Shao-Jiao; Chen, Yantian; Zheng, Chen; Qi, Nianmin

doi:10.1016/j.procbio.2013.07.002

Cited by 3 publications

(1 citation statement)

References 43 publications

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“…In order to analyze transfection efficiency, certain reporter genes have been co-expressed with target proteins, and many studies have used green fluorescent protein (GFP) to mark positive cells after transfection. Transfection efficiency has been enhanced by various strategies based on reporter genes, or by optimization or addition of conditions or factors such as transfection reagents, 5 DNA topology of plasmid, 11 cell cycle distribution of host cells, 12 and non-encoding carrier DNA. 13 The Epstein-Barr virus nuclear antigen-1 (EBNA-1) gene was shown to accelerate gene delivery and promote transcription.…”

Section: Discussionmentioning

confidence: 99%

A simple, rapid method for evaluation of transfection efficiency based on fluorescent dye

et al. 2016

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Enhanced transfection efficiency of transient gene expression (TGE) and electroporation is a useful approach for improvement of recombinant therapeutic proteins in mammalian cells. A novel method is described here in which CHO cells expressing recombinant FVII (rFVII) were labeled with fluorescent dye and analyzed by confocal microscopy. Cells with or without rFVII encoding gene were detectable by flow cytometry. Thus, we were able to distinguish positive cells (with rFVII encoding gene) and quantify their percentages. We evaluated the effects of varying electroporation conditions (voltage, number of repetitions, plasmid amount, carrier DNA) in order to optimize transfection efficiency. The highest transfection efficiency achieved was ∼86%. The method described here allows rapid evaluation of transfection efficiency without co-expression of reporter genes. In combination with appropriate antibodies, the method can be extended to evaluation of transfection efficiency in cells expressing other recombinant proteins.

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