Replication efficiency of rolling - circle replicon - based 3 plasmids derived from porcine circovirus 2 in eukaryotic 4 cells

circovirus 2 in eukaryotic cells 24 25 Abstract 26 In this study, a method was developed to measure replication rates of rolling-circle replicon-27 based plasmids in eukaryotic cells. This method is based on the discriminative quantitation of 28 MboI-resistant, non-replicated input plasmids and DpnI-resistant, replicated plasmids. To do 29 so, porcine circovirus type 2 (PCV2) replicon-based plasmids were constructed. These 30 plasmids contained the PCV2 origin of replication, the PCV2 Rep promoter and the PCV2 31 Rep gene. The results show that the replication rate depends on the length of the PCV2 32 replicon-based plasmid and not on the respective position of the Rep promoter and the 33 promoter of the gene of interest that encodes the enhanced green fluorescent protein (eGFP). 34 In all cases, it was necessary to add the Rep gene encoded by a plasmid and cotransfected as a 35 replication booster. This method can evaluate the replication potential of replicon-based 36 plasmids quickly and is thereby a promising tool for the development of plasmids for vaccine 37 purposes.