title: The effects of plasmid copy number and sequence context upon transfection efficiency.
abstract: It is known that large P1 artificial chromosome (PAC) vectors exhibit reduced transfection efficiency in comparison to small plasmid vectors. We investigated the dynamics of this effect, by comparing expression from a small plasmid (4.7 kb) and a PAC vector (111 kb) containing the Enhanced Green Fluorescent Protein (EGFP) reporter gene under the control of a P(CMV) promoter. EGFP expression was detected by fluorescence activated cell sorting (FACS). We found that the lower transfection efficiency of PAC vectors represents both a smaller percentage of cells expressing the transgene, and a lower level of expression per cell. We have shown that the lower number of plasmid molecules administered per cell in a PAC transfection does not explain this effect, and that this effect does not act in trans. Surprisingly, dilution of a reporter construct with an irrelevant plasmid did not appear to compromise transfection efficiency; in fact, a dilution of 1/10 slightly enhanced transfection. Therefore, it seems that the plasmid content of a liposome-DNA complex need not be 100% reporter construct for optimum transfection efficiency. This discovery has potential practical utility in a number of applications.
authors.affiliation: Molecular Medicine Centre, Department of Medicine, University of Edinburgh, Western General Hospital, Crewe Road, EH4 2XU, UK.
authors.forename: Wendy E
authors.initials: WE
authors.lastname: Walker
authors.forename: David J
authors.initials: DJ
authors.lastname: Porteous
authors.forename: A Christopher
authors.initials: AC
authors.lastname: Boyd
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D018628:Gene Dosage; D005822:Genetic Vectors; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics / Q000493:pharmacokinetics*; D014162:Transfection / Q000379:methods*