title: Replication efficiency of rolling-circle replicon-based plasmids derived from porcine circovirus 2 in eukaryotic cells.
abstract: In this study, a method was developed to measure replication rates of rolling-circle replicon-based plasmids in eukaryotic cells. This method is based on the discriminative quantitation of MboI-resistant, non-replicated input plasmids and DpnI-resistant, replicated plasmids. To do so, porcine circovirus type 2 (PCV2) replicon-based plasmids were constructed. These plasmids contained the PCV2 origin of replication, the PCV2 Rep promoter and the PCV2 Rep gene. The results show that the replication rate depends on the length of the PCV2 replicon-based plasmid and not on the respective position of the Rep promoter and the promoter of the gene of interest that encodes the enhanced green fluorescent protein (eGFP). In all cases, it was necessary to add the Rep gene encoded by a plasmid and cotransfected as a replication booster. This method can evaluate the replication potential of replicon-based plasmids quickly and is thereby a promising tool for the development of plasmids for vaccine purposes.
authors.affiliation: French Food Safety Agency (AFSSA), Viral Genetics and Biosafety Unit, BP-53, F-22440 Ploufragan, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Faurez
authors.forename: Daniel
authors.initials: D
authors.lastname: Dory
authors.forename: Aurélie
authors.initials: A
authors.lastname: Henry
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Bougeard
authors.forename: André
authors.initials: A
authors.lastname: Jestin
mesh_terms: D017925:Circovirus / Q000235:genetics*; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005057:Eukaryotic Cells; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins; D010957:Plasmids; D011401:Promoter Regions, Genetic; D018741:Replication Origin; D012093:Replicon; D014764:Viral Proteins / Q000235:genetics
chemical_list: D004279:DNA, Viral; D014764:Viral Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases