Time-course determination of plasmid content in eukaryotic and prokaryotic cells using real-time PCR.
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 104 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.
Carapuça
Elisabete
E
Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Lisboa, Portugal.
Azzoni
Adriano R
AR
Prazeres
Duarte M F
DM
Monteiro
Gabriel A
GA
Mergulhão
Filipe J M
FJ
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000235:genetics*; D018628:Gene Dosage / Q000235:genetics; D007700:Kinetics; D010957:Plasmids / Q000032:analysis* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors