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

• Type: Article in International Scientific Journal
• Date: 2007

Authors

Elisabete Carapuca (Author) Other; Adriano R Azzoni (Author) Other; Duarte M F Prazeres (Author) Other; Gabriel A Monteiro (Author) Other; Filipe J M Mergulhao (Author) FEUP

Journal

Title: Molecular Biotechnology

Vol. 37

Pages: 120-126

ISSN: 1073-6085

Publisher: Springer Nature

Indexing

ISI Web of Knowledge - 36 Citations

ISI Web of Science

Scopus - 39 Citations

Scientific classification

FOS: Natural sciences > Biological sciences

CORDIS: Technological sciences > Technology > Biotecnology

Other information

Authenticus ID: P-004-79G

DOI: 10.1007/s12033-007-0007-3

Abstract (EN): A Real-Time PCR method was developed to monitor the plasmid copy number (PCN) in Escherichia coli and Chinese hamster ovary (CHO) cells. E. coli was transformed with plasmids containing a ColE1 or p15A origin of replication and CHO cells were transfected with a ColE1 derived plasmid used in DNA vaccination and carrying the green fluorescent protein (GFP) reporter gene. The procedure requires neither specific cell lysis nor DNA purification and can be performed in <30 min with dynamic ranges covering 0.9 pg-55 ng, and 5.0 pg-2.5 ng of plasmid DNA (pDNA) for E. coli and CHO cells, respectively. Analysis of PCN in E. coli batch cultures revealed that the maximum copy number per cell is attained in mid-exponential phase and that this number decreases on average 80% towards the end of cultivation for both types of plasmids. The plasmid content of CHO cells determined 24 h post-transfection was around 3 x 10(4) copies per cell although only 37% of the cells expressed GFP one day after transfection. The half-life of pDNA was 20 h and around 100 copies/cell were still detected 6 days after transfection.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 7