Generation and characterization of inducible nitric oxide synthase deficient macrophage cell lines.
Stable inducible nitric oxide synthase deficient mouse macrophage cell lines were generated by the antisense technology. A 666 bp fragment of a mouse inducible nitric oxide synthase cDNA was cloned in antisense orientation into a mammalian expression vector behind the CMV promoter. This construct was transfected into J774.1A cells, a mouse macrophage cell line. The inducible nitric oxide synthase antisense lines showed up to 84% reduction of nitric oxide production in response to lipopolysaccharide stimulation and 66% reduction of nitric oxide production in response to interferon-gamma and a combination of interferon-gamma and lipopolysaccharide stimulation. The deficiency in inducible nitric oxide synthase expression had no impact on lipopolysaccharide induced tumor necrosis factor alpha and interleukin-1 secretion. The stable and specific inhibition of inducible nitric oxide synthase expression by antisense DNA vectors allows a direct analysis of contribution of inducible nitric oxide synthase activity to macrophage regulatory and immune defence functions.
Rothe
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H
Diabetes Research Institute, Heinrich-Heine-University of Düsseldorf, Germany.
Bosse
G
G
Fischer
H G
HG
Kolb
H
H
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D016373:DNA, Antisense; D018076:DNA, Complementary / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000172:deficiency* / Q000235:genetics; D009573:Nitrites / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
D016373:DNA, Antisense; D018076:DNA, Complementary; D007375:Interleukin-1; D008070:Lipopolysaccharides; D009573:Nitrites; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase