title: WISH cell line: from the antiviral system to a novel reporter gene assay to test the potency of human IFN-α and IFN-β.
abstract: Interferons (IFNs) are potent biologically active proteins that are widely used as biopharmaceuticals, so their potency must be correctly identified. Usually, the biological activity is quantified by a bioassay based on its capacity to induce an antiviral state in target cells, but this type of assays is subject to virus manipulation-related issues and they show considerable intra- and inter-assay variability. In this work, we generated a reporter gene assay (RGA) supported on the WISH-Mx/eGFP reporter cell line to determine human type I IFN activity. WISH cells were stably transfected with the enhanced green fluorescent protein (eGFP) gene under the control of type I IFN-inducible Mx2 promoter. This system implies the use of a standardized cell line for human IFN-potency analysis such as WISH cells and the simultaneous use of the sensitive reporter gene eGFP, having also several advantages when compared to antiviral activity assays and other RGAs: it can determine the potency of hIFN-α and hIFN-β using only one cell line showing the highest expression of eGFP after 28h and being only observed in cells treated with type I IFNs due to the specificity of the Mx promoter. It is a sensitive assay and it represents a safe alternative when compared with the conventional antiviral tests. The cell line showed the same sensitivity along 57 generations, allowing its use during two months of successive culture. The inter- and intra-assay coefficients of variation were lower than 20%, demonstrating its reproducibility. In addition, this reporter cell line can be used for the conventional antiviral assay, either for hIFN-α or hIFN-β. In conclusion, we have developed an alternative reporter system for the analysis of type I IFNs, in which its performance make it a suitable candidate to replace or complement conventional bioassays that are currently employed to measure IFN potency.
authors.affiliation: Cell Culture Laboratory, School of Biochemistry and Biological Sciences, Universidad Nacional del Litoral, Pcia. de Santa Fe, Argentina.
authors.forename: María de Los Milagros
authors.initials: Mde L
authors.lastname: Bürgi
authors.forename: Claudio
authors.initials: C
authors.lastname: Prieto
authors.forename: Marina
authors.initials: M
authors.lastname: Etcheverrigaray
authors.forename: Ricardo
authors.initials: R
authors.lastname: Kratje
authors.forename: Marcos
authors.initials: M
authors.lastname: Oggero
authors.forename: Mariela
authors.initials: M
authors.lastname: Bollati-Fogolín
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods; D019204:GTP-Binding Proteins / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology*; D016899:Interferon-beta / Q000494:pharmacology*; D051379:Mice; D064547:Myxovirus Resistance Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D014162:Transfection; D014721:Vesicular stomatitis Indiana virus / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D016898:Interferon-alpha; C579070:Mx1 protein, mouse; C579067:Mx2 protein, mouse; D064547:Myxovirus Resistance Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D016899:Interferon-beta; D019204:GTP-Binding Proteins