Quantification of Atlantic salmon type-I interferon using an Mx1 promoter reporter gene assay.
We here describe an assay for the detection of interferon-like activity in Atlantic salmon based on the transient transfection of chinook salmon embryo cells (CHSE-214 cells) with a rainbow trout Mx1 promoter linked to a luciferase reporter. A beta-galactosidase gene under the control of a constitutively expressed beta-actin promoter was used as a transfection standard, and luciferase and beta gal expression were measured by a commercially available kit. Interferon containing supernatants from poly I:C- or CpG-stimulated leucocytes added to transfected CHSE-cells induced high luciferase expression (>60-fold induction compared to supernatants from non-stimulated cells). There was no response to supernatants from LPS- and ConA/PMA-stimulated leucocytes, demonstrating the specificity for type I interferon-like activity. Duplicate samples analysed using a cell protection assay for detection of antiviral activity correlated well with levels obtained by the Mx1 promoter reporter gene assay (R2=0.97), confirming the reporter assay as a reliable substitute for the standard antiviral assay. The Mx reporter gene assay also has advantages in terms of sensitivity, high dynamic range and reliability over the conventional cell protection assay.
Johansen
Audny
A
Fiskeriforskning, Norwegian Institute of Fisheries and Aquaculture, N-9291 Tromsø, Norway.
Collet
Bertrand
B
Sandaker
Elin
E
Secombes
Christopher J
CJ
Jørgensen
Jorunn B
JB
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002478:Cells, Cultured; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics; D007370:Interferon Type I / Q000276:immunology / Q000378:metabolism*; D007962:Leukocytes / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D016215:Macrophage-Activating Factors / Q000378:metabolism; D064547:Myxovirus Resistance Proteins; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D020317:Salmo salar / Q000276:immunology / Q000378:metabolism*; D012474:Salmon; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D017931:DNA Primers; D007370:Interferon Type I; D016215:Macrophage-Activating Factors; D064547:Myxovirus Resistance Proteins; D008156:Luciferases; D001616:beta-Galactosidase; D019204:GTP-Binding Proteins