title: Sequence requirements for plasmid nuclear import.
abstract: The nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of nonviral gene delivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366-bp fragment containing the SV40 origin of replication and early promoter. In this report, we show that, although fragments throughout this region can support varying degrees of nuclear import, the 72-bp repeats of the SV40 enhancer facilitate maximal transport. The functions of the promoter and the origin of replication are not needed for nuclear localization of plasmid DNA. In contrast to the import activity of the SV40 enhancer, two other strong promoter and enhancer sequences, the human cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus LTR, were unable to direct nuclear localization of plasmids. The inability of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent protein (GFP) in microinjected cells. At times before cell division, as few as 3 to 10 copies per cell of cytoplasmically injected plasmids containing the SV40 enhancer gave significant GFP expression, while no expression was obtained with more than 1000 copies per cell of plasmids lacking the SV40 sequence. However, the levels of expression were the same for both plasmids after cell division in cytoplasmically injected cells and at all times in nuclear injected cells. Thus, the inclusion this SV40 sequence in nonviral vectors may greatly increase their ability to be transported into the nucleus, especially in nondividing cells.
authors.affiliation: College of Medicine, University of South Alabama, Mobile, Alabama 36688, USA. dean@sungcg.usouthal.edu
authors.forename: D A
authors.initials: DA
authors.lastname: Dean
authors.forename: B S
authors.initials: BS
authors.lastname: Dean
authors.forename: S
authors.initials: S
authors.lastname: Muller
authors.forename: L C
authors.initials: LC
authors.lastname: Smith
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008845:Microinjections; D009685:Nuclear Envelope / Q000378:metabolism*; D010957:Plasmids / Q000493:pharmacokinetics*; D011401:Promoter Regions, Genetic / Q000502:physiology; D018741:Replication Origin / Q000502:physiology; D013539:Simian virus 40 / Q000235:genetics
chemical_list: D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins