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Transgene expression of transfected supercoiled plasmid DNA concatemers in mammalian cells

✍ Scribed by Christof Maucksch; Alexander Bohla; Florian Hoffmann; Martin Schleef; Manish Kumar Aneja; Markus Elfinger; Dominik Hartl; Carsten Rudolph

Publisher
John Wiley and Sons
Year
2009
Tongue
English
Weight
271 KB
Volume
11
Category
Article
ISSN
1099-498X

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✦ Synopsis

Abstract

Background

Supercoiled topology of transfected plasmid DNA (pDNA) is critical for transgene expression in mammalian cells. In the present study, we analysed transgene expression of transfected supercoiled pDNA concatemers.

Methods

Jurkat T cells were transfected with a supercoiled 4.7‐kb monomeric and, in parallel, a 9.4‐kb dimeric pEGFP plasmid concatemer using electroporation. The absolute amounts of pDNA delivered into the cytoplasm and the nucleus were quantified by quantitative real‐time polymerase chain reaction. Further, the number and mean fluorescent intensity (MFI) of enhanced green fluorescent protein (EGFP) expressing cells and the relative amounts of TOTO‐1 fluorescently‐labeled pDNA associated with the cell, located in the cytoplasm, and in the nucleus, were analysed by flow cytometry.

Results

For both constructs, significantly higher amounts of pDNA were detected in the cytoplasm compared to the nucleus. Furthermore, from FACS analysis, we could infer the relative gene copy (E~gene~) and plasmid expression efficiency (E~plasmid~) by determining the ratio of the EGFP MFI of the transfected cells to TOTO‐1 MFI per nucleus on the single cell level. E~gene~ and E~plasmid~ were significantly 1.6‐and 3.5‐fold higher for EGFP‐dimer than for EGFP‐monomer, although the transfection rates considering the number of transfected cells were significantly lower for EGFP‐dimer than for EGFP‐monomer. Together with hydrodynamic plasmid diameter measurements, these observations suggest that concatemer arrangement increases relative gene expression efficiency, whereas plasmid size is important for cell and nucleus entry after electroporation.

Conclusions

We propose using preferably small supercoiled plasmid concatemers as the ideal plasmid vectors to maximize both transgene expression and the number of transfected target cells. Copyright © 2009 John Wiley & Sons, Ltd.

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