Validation of a HeLa Mx2/Luc reporter cell line for the quantification of human type I interferons.
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 Mx2 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-alpha2a or IFN-alpha2b treatment with HeLa cells. We found that the Mx2 gene increased the most by treatment with both IFN-alpha2a and IFN-alpha2b. Based on this result, we designed a reporter cell line, HeLa-Mx2, suitable for determination of IFN-alpha. HeLa cells were stably transfected with the luciferase gene under the control of Mx2 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-alpha, interleukin (IL)-1beta, IL-2, IL-5, IL-6 and GM-CSF, or type II interferon (IFN-gamma) were not observed. Moreover, the robustness of our cell line is demonstrated by the lack of an effect of the HeLa-Mx2 cell culture's age on the performance of the reporter gene assay. The reporter gene assay demonstrated reproducible dose-response curves for IFN-alpha2a 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-Mx2 cell line, which has advantages with regard to simplicity, selectivity, and reliability over conventional cytopathic effect reduction assays used to quantify IFN-alpha activity.
Seo
Young-Jun
YJ
Advanced Therapy Products Research Division, National Institute of Food and Drug Safety Evaluation, Korea Food and Drug Administration, Seoul 122-704, Republic of Korea.
Kim
Gi-Hyun
GH
Kwak
Ho-Jung
HJ
Nam
Ju-Sun
JS
Lee
Hwa Jeong
HJ
Suh
Soo-Kyung
SK
Baek
Kyung-Min
KM
Sohn
Yeo-Won
YW
Hong
Seung-Hwa
SH
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D018076:DNA, Complementary; D004305:Dose-Response Relationship, Drug; D020869:Gene Expression Profiling / Q000379:methods; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000008:administration & dosage / Q000235:genetics*; D008156:Luciferases / Q000235:genetics*; D008163:Luminescent Measurements; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D014162:Transfection; D014709:Vero Cells
D018076:DNA, Complementary; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D008156:Luciferases