STUDY OF MECHANISMS OF ELECTRIC-FIELD-INDUCED DNA TRANSFECTION .5. EFFECTS OF DNA TOPOLOGY ON SURFACE BINDING, CELL UPTAKE, EXPRESSION, ANDINTEGRATION INTO HOST CHROMOSOMES OF DNA IN THE MAMMALIAN-CELL

Neumann and coworkers (Neumann, E., M. Schaefer-Ridder, Y. Wang, and P . H. Hofschneider. 1982. EMBO J. 1:841-845) have shown that the effici ency of pulsed electric field (PEF)-induced DNA transfection of mouse L-cells by the thymidine kinase gene is several times higher for the l inear DNA than for the closed circular DNA. Transfection of Escherichi a coli bacteria by several plasmids indicates that the transfection ef ficiency was much higher for the closed circular/supercoiled (sc-) and circular/relaxed (cr-) DNA than for the linearized (ln-) 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 s ystematically examined electrotransfection of NIH3T3 mouse fibroblast by the plasmids, pRSV(cat), pRSV(neo), and pRSV(gpt). Mg2+-facilitated surface binding of DNA before, and DNA uptake by 3T3 cells after trea tment with PEF, were monitored by H-3-labeled plasmids. Transfection e fficiency was evaluated by both the transient expression of chloramphe nicol acetyltransferase (cat) activity 2-3 days after, and the permane nt expression of neomycin phosphotransferase (neo) and xanthine-guanin e phosphoribosyltransferase (gpt) genes in the transformants 2 weeks a fter 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 p lasmids were three to five times more efficient for the cr-DNA and the sc-DNA than for the ln-DNA. These results indicate that electrotransf ection of cells involves several steps: the cation-dependent binding o f DNA to the cell surface, the electric field-driven DNA entry into th e cells, the transient expression of DNA, and the integration of DNA i nto the host chromosomes. For understanding mechanisms of electrotrans fection, only the DNA binding to the cell surface and the electric fie ld assisted membrane-crossing of DNA are relevant. Both the expression of the loaded DNA and the DNA integration into the host chromosomes d epend more on the properties of the cell and its interactions with a f oreign gene. Since these properties and interactions will be similar i rrespective of the method chosen to facilitate DNA transfer, they are not relevant for the study of mechanisms of electrotransfection. Our r esults also support the idea that the PEF-induced cellular uptake of D NA is mainly by the electrophoresis of the surface bound DNA across th e plasma membrane.