Establishment of an IFN-γ 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 (pCL4.14[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 RTC-3F7 is an excellent tool for monitoring the presence of trout IFNI-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. (C) 2009 Elsevier Ltd. All rights reserved.