In:
Pharmacology, S. Karger AG, Vol. 84, No. 3 ( 2009), p. 135-144
Kurzfassung:
Although antiviral assays have been the most widely available biological assays for interferons (IFNs), they are less sensitive and provide considerable interassay variation. In this study, we demonstrate a new reporter cell line, which is based on HeLa cells transfected with a plasmid containing a human 〈 i 〉 Mx2 〈 /i 〉 promoter driving a luciferase (Luc) cDNA. To characterize the specific gene expression profiles induced by interferon alpha, we analyzed the microarray results of interferon response gene expression induced by IFN-α2a or IFN-α2b treatment with HeLa cells. We found that the 〈 i 〉 Mx2 〈 /i 〉 gene increased the most by treatment with both IFN-α2a and IFN-α2b. Based on this result, we designed a reporter cell line, HeLa- 〈 i 〉 Mx2 〈 /i 〉 , suitable for determination of IFN-α. HeLa cells were stably transfected with the luciferase gene under the control of 〈 i 〉 Mx2 〈 /i 〉 promoter. The expression of luciferase can be easily measured for luminescence using a 96-well luminometer and has been correlated with the concentration of added IFN and cell density. In the validation results, our reporter cell line had specificity for type I IFN, but the significant effects of a number of other cytokines such as tumor necrosis factor-α, interleukin (IL)-1β, IL-2, IL-5, IL-6 and GM-CSF, or type II interferon (IFN-γ) were not observed. Moreover, the robustness of our cell line is demonstrated by the lack of an effect of the HeLa- 〈 i 〉 Mx2 〈 /i 〉 cell culture’s age on the performance of the reporter gene assay. The reporter gene assay demonstrated reproducible dose-response curves for IFN-α2a in the range of 1–10,000 IU/ml. The 95% confidential limit and total coefficient of variation estimates ranged between 96 and 116 and 10.51% in the reducible range mentioned above, respectively. In conclusion, we established a stable IFN-responsible HeLa- 〈 i 〉 Mx2 〈 /i 〉 cell line, which has advantages with regard to simplicity, selectivity, and reliability over conventional cytopathic effect reduction assays used to quantify IFN-α activity.
Materialart:
Online-Ressource
ISSN:
0031-7012
,
1423-0313
Sprache:
Englisch
Verlag:
S. Karger AG
Publikationsdatum:
2009
ZDB Id:
1483550-2
SSG:
15,3
