The effects of plasmid copy number and sequence context upon transfection efficiency.
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.
Walker
Wendy E
WE
Molecular Medicine Centre, Department of Medicine, University of Edinburgh, Western General Hospital, Crewe Road, EH4 2XU, UK.
Porteous
David J
DJ
Boyd
A Christopher
AC
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*