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

Research output: Contribution to journalArticle

68 Citations (Scopus)

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
StatePublished - Apr 28 1998

Fingerprint

Macrophages
Peritoneal Macrophages
Plasmids
Luciferases
DNA
Poly I
Poly C
Scavenger Receptors
Dextran Sulfate
Salmon
Dextrans
Fluorescein
Reporter Genes
Gene expression
Edetic Acid
Spermatozoa
Genes
Association reactions
Gene Expression
Proteins

Keywords

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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Involvement of specific mechanism in plasmid DNA uptake by mouse peritoneal macrophages. / Takagi, Toshihide; Hashiguchi, Miwa; Mahato, Ram I; Tokuda, Hideaki; Takakura, Yoshinobu; Hashida, Mitsuru.

In: Biochemical and Biophysical Research Communications, Vol. 245, No. 3, 28.04.1998, p. 729-733.

Research output: Contribution to journalArticle

Takagi, Toshihide ; Hashiguchi, Miwa ; Mahato, Ram I ; Tokuda, Hideaki ; Takakura, Yoshinobu ; Hashida, Mitsuru. / Involvement of specific mechanism in plasmid DNA uptake by mouse peritoneal macrophages. In: Biochemical and Biophysical Research Communications. 1998 ; Vol. 245, No. 3. pp. 729-733.
@article{a7b97c772ac54f5192b8c7f520f94544,
title = "Involvement of specific mechanism in plasmid DNA uptake by mouse peritoneal macrophages",
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.",
keywords = "Mouse peritoneal macrophages, Plasmid DNA, Receptor-mediated endocytosis, Scavenger receptor",
author = "Toshihide Takagi and Miwa Hashiguchi and Mahato, {Ram I} and Hideaki Tokuda and Yoshinobu Takakura and Mitsuru Hashida",
year = "1998",
month = "4",
day = "28",
doi = "10.1006/bbrc.1998.8521",
language = "English (US)",
volume = "245",
pages = "729--733",
journal = "Biochemical and Biophysical Research Communications",
issn = "0006-291X",
publisher = "Academic Press Inc.",
number = "3",

}

TY - JOUR

T1 - Involvement of specific mechanism in plasmid DNA uptake by mouse peritoneal macrophages

AU - Takagi, Toshihide

AU - Hashiguchi, Miwa

AU - Mahato, Ram I

AU - Tokuda, Hideaki

AU - Takakura, Yoshinobu

AU - Hashida, Mitsuru

PY - 1998/4/28

Y1 - 1998/4/28

N2 - 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.

AB - 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.

KW - Mouse peritoneal macrophages

KW - Plasmid DNA

KW - Receptor-mediated endocytosis

KW - Scavenger receptor

UR - http://www.scopus.com/inward/record.url?scp=0032576968&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032576968&partnerID=8YFLogxK

U2 - 10.1006/bbrc.1998.8521

DO - 10.1006/bbrc.1998.8521

M3 - Article

VL - 245

SP - 729

EP - 733

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 3

ER -