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.
|Number of pages||8|
|Journal||Journal of Controlled Release|
|Publication status||Published - 2004|
- Animals Base Sequence COS Cells Cercopithecus aethiops *Gene Dosage Genetic Vectors Plasmids/administration & dosage/genetics/*pharmacokinetics Transfection/*methods