Stable Transfection of the BovineNRAMP1Gene into Murine RAW264.7 Cells: Effect onBrucella abortusSurvival

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Abstract
Genetically based natural resistance to brucellosis in cattle provides for novel strategies to control zoonotic diseases. BovineNRAMP1, the homologue of a murine gene (Bcg), has been identified as a major candidate for controlling the in vivo resistant phenotype. We developed an in vitro model for expression of resistance- and susceptibility-associated alleles of bovineNRAMP1as stable transgenes under the regulatory control of the bovineNRAMP1promoter in the murine RAW264.7 macrophage cell line (Bcgs) to analyze the regulation of theNRAMP1gene and its role in macrophage function. We demonstrated that the 5′-flanking region of bovineNRAMP1, despite the lack of TATA and CAAT boxes, has a functional promoter capable of driving the expression of a transgene in murine macrophages. A polymorphism within a microsatellite in the 3′ untranslated region critically affects the expression of bovineNRAMP1and the control of in vitro replication ofBrucella abortusbut notSalmonella entericaserovar Dublin. We did not observe any differences in the production of NO by resting or gamma interferon (IFN-γ)- and IFN-γ–lipopolysaccharide (LPS)-treated transfected cell lines, yet the resistant transfected cell lines produced significantly less NO than other cell lines, following stimulation with LPS at 24 and 48 h.

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