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Infection and Immunity, May 2001, p. 3110-3119, Vol. 69, No. 5
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.5.3110-3119.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Stable Transfection of the Bovine NRAMP1 Gene into Murine RAW264.7 Cells: Effect on Brucella abortus Survival

Robert Barthel,1 Jianwei Feng,1 Jorge A. Piedrahita,2 David N. McMurray,3 Joe W. Templeton,1 and L. Garry Adams1,*

Department of Veterinary Pathobiology1 and Department of Veterinary Anatomy and Public Health,2 College of Veterinary Medicine, and Department of Medical Microbiology and Immunology, College of Medicine,3 Texas A&M University, College Station, Texas 77843

Received 1 November 2000/Returned for modification 8 January 2001/Accepted 20 February 2001

Genetically based natural resistance to brucellosis in cattle provides for novel strategies to control zoonotic diseases. Bovine NRAMP1, 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 bovine NRAMP1 as stable transgenes under the regulatory control of the bovine NRAMP1 promoter in the murine RAW264.7 macrophage cell line (Bcgs) to analyze the regulation of the NRAMP1 gene and its role in macrophage function. We demonstrated that the 5'-flanking region of bovine NRAMP1, 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 bovine NRAMP1 and the control of in vitro replication of Brucella abortus but not Salmonella enterica serovar Dublin. We did not observe any differences in the production of NO by resting or gamma interferon (IFN-gamma )- and IFN-gamma -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.

* Corresponding author. Mailing address: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4467. Phone: (979) 845-5092. Fax: (979) 845-5088. E-mail: gadams{at}cvm.tamu.edu.

Infection and Immunity, May 2001, p. 3110-3119, Vol. 69, No. 5
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.5.3110-3119.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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