Transfection efficiency depending on distance between a double-strand break site and EGFP-gene in plasmid DNA

We have established an experimental procedure of transfection and expression of irradiated plasmid DNA encoding fluorescent protein, EGFP, in eukaryotic cells. In this assay, the number of cells expressing the fluorescent protein increases with the incubation of cells as DNA damage is repaired. When a DSB-induced linear plasmid DNA arises after irradiation, it has been thought that the plasmid could be enzymatically digested immediately after transfection or delivery into nucleus. Previously we found that the linear plasmid DNA obtained by restriction enzyme treatments shows slightly expresses EGFP [1]. However, it has not yet been clarified how the linear DNA expresses after transfection. In this study, we aim to elucidate the efficiency of the linear plasmid DNA expression of EGFP in human cells. The linear plasmids were prepared by treatments with various restriction enzymes with different recognition sequences. We cleaved the plasmid around the EGFP gene with three blunt end restriction enzymes at the upstream side, the downstream side, and a 1.2 kbp distance away from the downstream sides of the EGFP gene. Preliminary results indicate that EGFP-expressing was observed for the linear plasmid transfections. The farther the cleavage site from the EGFP gene induced the higher transfection efficiency, suggesting that the plasmids might progressively be digested from the unstable terminal ends when they are transfected into cells. [1] Obata et al. Rad. Res. (2021) In press.日本放射線影響学会第64回大