A new reporter cell clone to determine the biological activity of type I Interferons

BÜRGI, MARÍA DE LOS MILAGROS; PRIETO, CLAUDIO; OGGERO, MARCOS; BOLLATI FOGOLÍN, MARIELA; ETCHEVERRIGARAY, MARINA; KRATJE, RICARDO

Viena

Congreso; 22nd ESACT Meeting; 2011

European Society for Animal Cell Technology

Interferons (IFNs) are potent biologically active proteins synthesized and secreted by somatic cells of all mammalian species. They play an important role in the immune response and defence against viruses because they have an antiproliferative, antiviral and immunomodulatory activity. They 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. Antiviral assays may be subject to some variability and the virus must be titrated. The elucidation of the mechanisms of cytokine-induced gene transcription led to the setting up of highly specific and sensitive bioassays, called reporter gene (RG) assays. The RG assays rely upon a cell bearing the receptor for the cytokine of interest, transfected with a plasmid carrying a reporter gene under the control of a cytokine inducible promoter. In the last few years, several specific and sensitive RG assays have been developed. In this work we generated WISH-Mx/eGFP reporter cell line to determine type I IFNs activity. WISH cells were stably transfected with the enhanced green fluorescent protein (eGFP) gene under the control of type I IFN-inducible Mx promoter. The percentage of eGFP expressing cells accurately correlates to the amount of type I IFN added to the culture and can easily be monitored. This system has several advantages when compared to antiviral activity assays and other reporter genes systems: it can determine the potency of all type I IFNs using only one cell line; it is a very fast assay, showing the highest expression of eGFP after 52 h; the expression of the eGFP reporter gene in WISH-Mx/eGFP cells is observed only in cells treated with type I IFNs, proving the specificity of the Mx promoter. It is a sensitive and safe assay, as the cell line showed a reproducible response in a dose-dependent manner between 0.32 and 750 IU/ml and 1.2 and 1,400 IU/ml for hIFN beta and alpha, respectively. The clone showed the same response along fifty generations, confirming the stability of the Mx/eGFP constructs incorporation into the cells. The inter- and intra-assay variation coefficient was lower than 20%, demonstrating the reproducibility of the assay. In conclusion, we have developed an alternative reporter system for the analysis of type I IFNs, in which the performance of the assay using WISH-Mx/eGFP line, together with its simplicity, speed, low cost, precision, sensitivity and safety make it a suitable candidate to replace conventional bioassays that are currently employed to measure IFNs potency.