RELATIONSHIP BETWEEN PLASMID DNA TOPOLOGICAL FORMS AND IN VITRO TRANSFECTION

Abstract

Topological isomers of plasmid DNA might be a factor affecting transfection efficiency. This study investigated the relationship between three major topoisomers of plasmid DNA, (supercoiled, open circular, and linear forms) and their biological activity. The three topoisomers of pDNA were transfected into CHO cells (Chinese Hamster Ovary) or SKnSH (human neuroblastoma) cells by (1) cationic liposomes, DOTAP/DOPE (1,2-dioleoyloxy-3-trimethylammonium propane/dioleolylphosphatidylethanolamine) or (2) electroporation. The open circular form of pCMV-luciferase demonstrated higher transfection efficiency (TE) than either supercoiled or linear while plasmid pGL3 showed no significant difference in TE for either delivery method. CHO and SKnSH cells showed similar patterns in transfection efficiency although the extent of expression was different. In order to test its stability in cell cytoplasmic lysate, pDNA was incubated at 37°C in NP40 lysate preparation. Surprisingly, supercoiled pDNA was the least stable in lysate solution (detected up to 4 min) whereas the open circular pDNA and linear form were degraded with a half-life value of 6 and 10 min respectively. To measure cellular associated pDNA, fluorescein labeled pDNA was complexed with DOTAP/DOPE liposome and transfected into CHO cells. The amount of pDNA associated with the cell was determined by flow cytometry. The flow cytometry studies demonstrated that the open circular form associated with the cell more than the linear forms when delivered with cationic liposomes. In conclusion, the three studied topoisomers of pDNA are transcriptionally active. The pattern of transgene production of the topoisoform of pDNA was independent of cell line and delivery method even though the level of expression was different.