The effects of plasmid copy number and sequence context upon transfection efficiency.

Walker WE, Porteous DJ, Boyd AC

Journal Of Controlled Release : Official Journal Of The Controlled Release Society

J Control Release. 2004 Jan 8;94(1):245-52.

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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.

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