Involvement of specific mechanism in plasmid DNA uptake by mouse peritoneal macrophages

Toshihide Takagi, Miwa Hashiguchi, Ram I. Mahato, Hideaki Tokuda, Yoshinobu Takakura, Mitsuru Hashida

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68 Scopus citations

Abstract

The binding and uptake of plasmid DNA encoding luciferase reporter gene (pCMV-Luc) were studied in vitro using cultured mouse peritoneal macrophages. A significant and time-dependent cellular association of [32P]pCMV-Luc with resident macrophages was observed at 37°C and this decreased at 4°C. The binding at 4°C was saturable and a Scatchard plot gave a maximum binding capacity of 0.81 μg/mg-protein and a dissociation constant of 0.30 μg/ml. The binding of [32P]pCMV-Luc was inhibited by polyinosinic acid, dextran sulfate and salmon sperm DNA, but not by polycytidylic acid, dextran and EDTA. A confocal microscopic study demonstrated that fluorescein-labeled pCMV-Luc was internalized at 37°C while only cell surface binding occurred at 4°C. No significant luciferase gene expression was obtained after incubation with a high concentration (100 μg/ml) of pCMV-Luc. These data suggest that plasmid DNA is taken up by macrophages via a mechanism mediated by a receptor like the macrophage scavenger receptor.

Original languageEnglish (US)
Pages (from-to)729-733
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume245
Issue number3
DOIs
Publication statusPublished - Apr 28 1998

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Keywords

  • Mouse peritoneal macrophages
  • Plasmid DNA
  • Receptor-mediated endocytosis
  • Scavenger receptor

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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