Study of mechanisms of electric field-induced DNA transfection. V. Effects of DNA topology on surface binding cell uptake expression and integration into host chromosomes of DNA in the mammalian cell.

摘要

Neumann and coworkers (Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. EMBO J. 1:841-845) have shown that the efficiency of pulsed electric field (PEF)-induced DNA transfection of mouse L-cells by the thymidine kinase gene is several times higher for the linear DNA than for the closed circular DNA. Transfection of Escherichia coli bacteria by several plasmids indicates that the transfection efficiency was much higher for the closed circular/supercoiled (sc-) and circular/relaxed (cr-) DNA than for the linearized (In-) DNA (Xie, T. D., L. Sun, H. G. Zhao, J. A. Fuchs, and T. Y. Tsong. 1992. Biophys. J. 63:1026-1031). To resolve these conflicting observations, we have systematically examined electrotransfection of NIH3T3 mouse fibroblast by the plasmids, pRSVcat, pRSVneo, and pRSVgpt. Mg(2+)-facilitated surface binding of DNA before, and DNA uptake by 3T3 cells after treatment with PEF, were monitored by 3H-labeled plasmids. Transfection efficiency was evaluated by both the transient expression of chloramphenicol acetyltransferase (cat) activity 2-3 days after, and the permanent expression of neomycin phosphotransferase (neo) and xanthine-guanine phosphoribosyltransferase (gpt) genes in the transformants 2 weeks after the PEF treatment. Our results indicate that cell surface binding and PEF-induced cell uptake of DNA did not depend on the topology of DNA. However, both the transient and the permanent expression of the plasmids were three to five times more efficient for the cr-DNA and the sc-DNA than for the in-DNA. These results indicate that electrotransfection of cells involves several steps: the cation-dependent binding of DNA to the cell surface, the electric field-driven DNA entry into the cells, the transient expression of DNA, and the integration of DNA into the host chromosomes. For understanding mechanisms of electrotransfection, only the DNA binding to the cell surface and the electric field assisted membrane-crossing of DNA are relevant. Both the expression of the loaded DNA and the DNA integration into the host chromosomes depend more on the properties of the cell and its interactions with a foreign gene. Since these properties and interactions will be similar irrespective of the method chosen to facilitate DNA transfer, they are not relevant for the study of mechanisms of electrotransfection. Our results also support the idea that the PEF-induced cellular uptake of DNA is mainly by the electrophoresis of the surface bound DNA across the plasma membrane.