Postmitotic nuclear retention of episomal plasmids is altered by DNA labeling and detection methods.
One often overlooked aspect of nonviral gene therapy is the maintenance and localization of plasmids within a transfected cell. In this study we have quantified the nuclear retention of plasmids within microinjected cells after a single round of cell division. We employed several commercially available reagents to label plasmids with fluorophores for our microinjection tracking experiments. Interestingly, plasmids labeled with different techniques produced drastically different results. Naked plasmids microinjected directly into nuclei and later detected by in situ hybridization were found almost exclusively within the nuclei of the daughter cells after mitosis and were partitioned between the daughter nuclei with a normal, Gaussian distribution. Identical results were obtained with plasmids labeled with a fluorescent peptide nucleic acid. However, when plasmids were labeled with several commercially available fluorescent DNA labeling kits that randomly attach fluorophores to the entire plasmid and injected into HeLa cell nuclei, the modified plasmids were excluded from daughter nuclei after cell division. Taken together, these results suggest that naked, unmodified plasmids are retained in the nucleus following cell division and likely continue to express in the daughter cells. Our results demonstrate the significant alterations in episome localization that the labeling technique itself can have on plasmid trafficking.
Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, 240 E. Huron Avenue, McGaw 2336, Chicago, IL 60611, USA.
Joshua Z
JZ
Gasiorowski
David A
DA
Dean
D001692:Biological Transport; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000032:analysis / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000494:pharmacology; D018014:Gene Transfer Techniques; D005821:Genetic Techniques; D015316:Genetic Therapy / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017403:In Situ Hybridization; D008938:Mitosis; D008954:Models, Biological; D016011:Normal Distribution; D010957:Plasmids / Q000378:metabolism*; D014162:Transfection
D005456:Fluorescent Dyes; D004247:DNA