Stable transfection of the bovine NRAMP1 gene into murine RAW264.7 cells: Effect on Brucella abortus survival

Citation
R. Barthel et al., Stable transfection of the bovine NRAMP1 gene into murine RAW264.7 cells: Effect on Brucella abortus survival, INFEC IMMUN, 69(5), 2001, pp. 3110-3119
Citations number
61
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Immunology
Journal title
INFECTION AND IMMUNITY
ISSN journal
0019-9567 → ACNP
Volume
69
Issue
5
Year of publication
2001
Pages
3110 - 3119
Database
ISI
SICI code
0019-9567(200105)69:5<3110:STOTBN>2.0.ZU;2-S
Abstract
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 contr olling the in vivo resistant phenotype. We developed an in vitro model for expression of resistance- and susceptibility-associated alleles of bovine N RAMP1 as stable transgenes under the regulatory control of the bovine NRAMP 1 promoter in the murine RAW264.7 macrophage cell line (Bcg;) to analyze th e regulation of the NRAMP1 gene and its role in macrophage function. We dem onstrated that the 5'-flanking region of bovine NRAMP1, despite the lack of TATA and CAAT boxes, has a functional promoter capable of driving the expr ession of a transgene in murine macrophages. A polymorphism within a micros atellite 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 differen ces in the production of NO by resting or gamma interferon (IFN-gamma)- and IFN-gamma -lipopolysaecharide (LPS)-treated transfected cell lines, yet th e resistant transfected cell lines produced significantly less NO than othe r cell lines, following stimulation with LPS at 24 and 48 h.