Cloning of fox (Vulpes vulpes) Il2, Il6, Il10 and IFNgamma and analysis of their expression by quantitative RT-PCR in fox PBMC after in vitro stimulation by Concanavalin A.
The immune response in the fox (Vulpes vulpes), despite the success of the oral rabies vaccine is not well characterised, and specific immunological tools are needed. A quantitative RT-PCR using SyBR Green to investigate fox cytokine expression after antigen PBMC in vitro re-stimulation is presented here. First, we cloned by homology with dog cytokine sequences the fox IL2, IL6, IL10, IFNgamma and a partial 18S sequence. Fox specific primers were then defined and used to set up a species-specific quantitative RT-PCR assay using SyBR Green and 18S housekeeping gene as internal standard. The technique was validated using total RNA from fox PBMC stimulated with a polyclonal activator, Concanavaline A.
Rolland-Turner
Magali
M
AFSSA Nancy, Laboratoire d'Etudes et de Recherches sur la Rage et la Pathologie des Animaux Sauvages, B.P. 9, 54220 Malzéville, France. m.rolland@afssa.fr
Farré
Guillaume
G
Boué
Franck
F
D000595:Amino Acid Sequence; D000818:Animals; D002415:Cats; D003001:Cloning, Molecular; D003208:Concanavalin A / Q000494:pharmacology*; D004285:Dogs; D005589:Foxes / Q000235:genetics*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D007371:Interferon-gamma / Q000737:chemistry / Q000235:genetics*; D016753:Interleukin-10 / Q000737:chemistry / Q000235:genetics*; D007376:Interleukin-2 / Q000737:chemistry / Q000235:genetics*; D015850:Interleukin-6 / Q000737:chemistry / Q000235:genetics*; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D007376:Interleukin-2; D015850:Interleukin-6; D003208:Concanavalin A; D016753:Interleukin-10; D007371:Interferon-gamma