Exhaustive in vivo labelling of plasmid DNA with BrdU for intracellular detection in non-viral transfection of mammalian cells.
The study of the non-viral gene delivery process at the molecular level, e.g. during the transfection of mammalian cells, is currently limited by the difficulties of specifically detecting the transfected plasmid DNA within the cells. Here we describe the in vivo production of 5-bromodeoxyuridine (BrdU)-labelled plasmid DNA by a thymine-requiring Escherichia coli strain leading to 92 +/- 15% BrdU incorporation while minimizing plasmid structure alteration. The labelled plasmid is produced on the milligram scale in a two-stage cultivation process. The relevance of this approach for plasmid DNA visualisation in the field of gene delivery is demonstrated by localising the BrdU-labelled plasmid DNA via immunodetection/fluorescence microscopy in CHO-K1 cells after electroporation with naked, BrdU-labelled plasmid DNA and after polyfection with polyethylenimine/BrdU-labelled plasmid complexes.
Jérôme
Valérie
V
Chair for Process Biotechnology, University of Bayreuth, 95440 Bayreuth, Germany.
Heider
Andreas
A
Schallon
Anja
A
Freitag
Ruth
R
D000818:Animals; D001973:Bromodeoxyuridine / Q000737:chemistry*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D004247:DNA; D001973:Bromodeoxyuridine