NTU Undergraduates' research April 2014 - Biosciences | Page 83

Optimisation of HEK293 transfection using PEI
Jack Firkin

Abstract
Transfection describes the process of introducing foreign genetic material into mammalian cells. In
the study of mammalian genes and proteins, transfection is advantageous over transformation of
bacterial cells as cells maintain biological activity and protein folding specific to mammalian cells.
Furthermore, transfection can be used to produce large quantities of recombinant proteins.
Recombinant proteins are widely used in therapeutics such as large-scale production of human
insulin. There is therefore a need for low-cost, high transfection efficiency protocols.
Polyethylenimine (PEI) is a cationic polymer capable of delivering genetic material into cells via
formation of polyplexes and endocytosis. The use of PEI has been adapted for the use in industrial
scale transfection processes due to its low cost and relatively high transfection efficiency. To achieve
high transfection efficiency with any transfection agent, the conditions of the process have to be
optimised. In this study the ratio of plasmid DNA to PEI and number of cells were varied to optimise
the transfection efficiency. Transfections were performed using DNA:PEI (w/v) ratios of 1:6, 1:1 and
6:1 to transfect samples containing 1x105, 3x105 and 6x105 HEK293 cells. The plasmid DNA used for
transfection was pCDH-NRF1-GFP. This plasmid contains the green-fluorescence gene as a
selectable marker to monitor transfection efficiency via flow cytometry. The highest transfection
efficiency was recorded in transformation of 3x105 cells with PEI using a 1:6 (w/v) ratio. This
illustrates the requirement of an excess of PEI to transfect cells and the importance of cell confluence
at the time of transfection.
Keywords: Polyethylenimine (PEI), optimisation, HEK293, transfection efficiency, N/P

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Jack Firkin