Establishment of an IFN-gamma specific reporter cell line in fish.
An interferon (IFN)-gamma responsive stable cell line RTG-3F7 has been developed for rainbow trout by modifying the RTG-2 cell line through transfection with a plasmid construct (pGL4.14[luc2/hygro]-PrTAP2) containing a promoter element from the IFN-gamma responsive gene TAP2 linked to a luciferase reporter gene and a hygromycin resistance gene. Following transfection single clones were selected in 96 well plates using hygromycin B, and those showing specific activation after rIFN-gamma stimulation were maintained. Five clones that showed the highest reporter activity to rIFN-gamma were incubated with different stimuli to examine specificity. No significant induction of luciferase was observed following exposure to recombinant type I IFN, LPS, PHA or poly I:C. The cell line was responsive to rIFN-gamma at concentrations between 150 pg and 20 ng ml(-1). Supernatants of primary cultures of head kidney leucocytes stimulated with PHA, known to induce IFN-gamma gene expression, were also used to assess the reporter activity of the stable cell line. A dose-dependent induction of the promoter activity was observed with these supernatants indicating the presence of IFN-gamma. These results indicate that the stable cell line RTG-3F7 is an excellent tool for monitoring the presence of trout IFN-gamma in biological samples, and in addition, enables the study of intracellular signalling pathways of IFNs, their receptor interactions, and other closely related signalling networks.
Castro
Rosario
R
Scottish Fish Immunology Research Centre, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, AB24 2TZ Scotland, UK. mrcastrosequ@jouy.inra.fr
Martin
Samuel A M
SA
Zou
Jun
J
Secombes
Christopher J
CJ
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D002452:Cell Count; D002460:Cell Line; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics*; D007371:Interferon-gamma / Q000235:genetics* / Q000276:immunology*; D008221:Lymphoid Tissue / Q000166:cytology; D017686:Oncorhynchus mykiss / Q000276:immunology; D010835:Phytohemagglutinins / Q000494:pharmacology; D011070:Poly I-C / Q000494:pharmacology; D013997:Time Factors
D000276:Adjuvants, Immunologic; D010835:Phytohemagglutinins; D007371:Interferon-gamma; D011070:Poly I-C