Volume 5 Supplement 1

The 4th Recombinant Protein Production Meeting: a comparative view on host physiology

Open Access

Determination of plasmid content in eukaryotic and prokaryotic cells using Real-Time PCR

  • Adriano Azzoni1,
  • Elisabete Carapuça1,
  • D Miguel F Prazeres1,
  • Gabriel A Monteiro1 and
  • Filipe Mergulhão1, 2
Microbial Cell Factories20065(Suppl 1):P50

https://doi.org/10.1186/1475-2859-5-S1-P50

Published: 10 October 2006

Background

Determination of the plasmid content in prokaryotic cells during plasmid DNA (pDNA) production and in eukaryotic cells after transfection is crucial for DNA vaccine development. In Escherichia coli, pDNA is usually determined after plasmid extraction, either by UV absorbance or by densitometry of ethidium bromide-stained agarose gels. Fluorescence microscopy techniques are mainly used in eukaryotic cells. These techniques are time-consuming and labour-intensive and can not be used for process control. Thus, a Real-Time PCR method was developed to monitor the plasmid content of E. coli and Chinese Hamster Ovary (CHO) cells.

Results

Real-Time PCR with a 108 bp amplicon enabled the detection of quantities as low as 4 copies of pDNA per cell in E. coli and 100 copies/cell in CHO cells transfected with high copy-number plasmids (see figure 1). The procedure can be performed in less than 30 minutes and requires no sample pre-treatment. Analysis of pDNA number in E. coli harbouring a ColE1 plasmid revealed that copy number reached a maximum during exponential phase of growth and that this number decreased up to 80% upon entry into stationary phase. Additionally, the half-life of pDNA in transfected CHO cells was 20 hours and around 100 copies of plasmid were still detected 6 days after transfection.
Figure 1

Variation of threshold cycle numbers using plasmid-free E. coli (left) and CHO cells (right) spiked with pDNA. In E. coli, 5 × 104 (□), 2.5 × 105 () and 3.5 × 106 () cells were spiked with 8.5 × 10-4 to 8.5 ng of pDNA. With CHO, 1.2 × 104 cells were spiked with pDNA masses ranging from 5 pg to 100 ng. A linear working range was obtained from 5 pg to 2.5 ng and was subsequently used for quantitation.

Conclusion

Monitoring the pDNA content on producing and recipient cells is crucial for DNA vaccine development. The Real-Time PCR method developed on this work provides quasi-online results and is suitable for process control and optimisation. The procedure was first developed for E. coli and was quickly adapted to CHO cells. It is therefore likely that it can be modified for application with other prokaryotic and eukaryotic systems.

Authors’ Affiliations

(1)
Centro de Engenharia Biológica e Química, Instituto Superior Técnico
(2)
Chemical Engineering Department, LEPAE, Faculty of Engineering of the University of Porto

Copyright

© Azzoni et al; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd.

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