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.