Study of mechanisms of electric field-induced DNA transfection. IV. Effects of DNA topology on cell uptake and transfection efficiency

• Published in: Biophysical journal Vol. 63; no. 4; pp. 1026 - 1031
• Main Authors: Xie, T.D., Sun, L., Zhao, H.G., Fuchs, J.A., Tsong, T.Y.
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 01.10.1992; Biophysical Society; The Biophysical Society
• Subjects: Ampicillin Resistance - genetics; Biological and medical sciences; Biological Transport; Diverse techniques; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; Electric Stimulation - methods; Escherichia coli - drug effects; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Kinetics; Magnesium Chloride - pharmacology; Molecular and cellular biology; Nucleic Acid Conformation; Plasmids; Transfection; Plasmid; Electric field; Transfection; Escherichia coli; Pulsed field; Bacteria; Electroporation; Performance analysis; Method; Enterobacteriaceae
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(92)81675-2

Electric parameters and solvent conditions are known to influence the efficiency of DNA transfection of cells by a pulsed electric field (PEF). A previous study (Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. EMBO (Eur. Mol. Biol. Organ.) J. 1:841–845) has indicated that DNA topology is also an important determinant. We report an investigation of the PEF induced uptake, stability, and expression of three different topological isomers, circular supercoiled (scDNA), circular relaxed (crDNA), and linearized (lnDNA) forms of the plasmid pBR322, by Escherichia coli strain JM105. Monomeric pBR322 prepared by the electroelution from an agarose gel was in the supercoiled form. Treatment of the scDNA with wheat germ topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form. Treatment of scDNA by a restriction endonuclease, EcoRI or Hind III, linearized the DNA. The MgCl2-dependent bindings of all three forms of DNA to the cell surface were indistinguishable. So was the PEF induced cell uptake. In contrast, the transfection efficiency (TE) for the scDNA and the crDNA were high (approximately 2 x 10(8) micrograms-1 DNA at neutral pH), whereas that for the lnDNA was approximately five orders of magnitude lower (less than 1 x 10(3) micrograms-1 DNA). Analysis by agarose gel electrophoresis indicated that the PEF loaded lnDNA was degraded by the host cell within 3 h. However, the loaded scDNA and the crDNA were stable and expressed in the cytoplasm. We conclude that first, the PEF induced DNA entry into E. coli did not depend on the topology of the DNA.