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

摘要

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 pl5A origin of replication and CHO cells were transfected with a ColEl 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.