title: Characterisation of gamma-interferon responsive promoters in fish.
abstract: Reporter constructs of three interferon (IFN)-gamma-induced rainbow trout genes were generated to examine specificity to type I or type II IFN. Constructs included gammaIP-10, LMP2 and TAP2 and were used to transfect trout fibroblast cells (RTG-2) which were then exposed to rainbow trout rIFNs. The gammaIP-10 construct showed high reporter activity even in the absence of rIFNs. The LMP2 promoter contained one GAS element and two double ISRE elements, of four constructs made, only those with ISRE elements showed significant reporter activity following rIFN-gamma stimulation. The TAP2 regulatory region contained two GAS, two ISRE and one C/EBP element from which four constructs were made. Reporter expression for the construct containing all five elements showed an 11- and 2-fold increase in response to rIFN-gamma and type I rIFN, respectively. Constructs containing only the GAS elements did not respond to rIFNs. The TAP2 construct with two ISRE and the C/EBP gave the greatest dose-dependent reporter response to rIFN-gamma, with no significant response to type I rIFN. These data suggest that the ISRE elements, or elements nearby, are essential for the induction of type II IFN responsive genes in trout. The TAP2 construct is a candidate to develop a IFN-gamma reporter stable cell line.
authors.affiliation: Scottish Fish Immunology Research Centre, University of Aberdeen, Aberdeen, Scotland, UK.
authors.forename: Rosario
authors.initials: R
authors.lastname: Castro
authors.forename: Samuel A M
authors.initials: SA
authors.lastname: Martin
authors.forename: Steve
authors.initials: S
authors.lastname: Bird
authors.forename: Jesús
authors.initials: J
authors.lastname: Lamas
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Secombes
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D000818:Animals; D002460:Cell Line; D054357:Chemokine CXCL10 / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000235:genetics; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D007371:Interferon-gamma / Q000494:pharmacology*; D008156:Luciferases / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D020218:Response Elements / Q000235:genetics; D017384:Sequence Deletion; D013997:Time Factors
chemical_list: D018528:ATP-Binding Cassette Transporters; D054357:Chemokine CXCL10; D029941:Fish Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C071313:LMP-2 protein; D007371:Interferon-gamma; D008156:Luciferases; D003546:Cysteine Endopeptidases