Validation of an interferon stimulatory response element reporter gene assay for quantifying type I interferons.
The goal of this work was to develop a virus-free, cell-based interferon (IFN) bioassay and determine the utility of this assay on biological samples that contained IFN-τ, the trophoblast-secreted maternal recognition of pregnancy factor in ruminants. Madin-Darby bovine kidney cells were transduced with lentiviral particles that contained a firefly luciferase reporter construct driven by an IFN stimulatory response element (ISRE). Stably transduced cells were selected with the use of puromycin resistance. A linear, dose-responsive response was detected with human IFN-α and ovine IFN-τ. Interferon activity was detected in conditioned media from bovine trophoblast cells and uterine flushes collected from sheep and cattle. Activity also was detected in media collected after individual or small group culture of in vitro-produced bovine blastocysts at day 8 to 10 after fertilization. In summary, this IFN stimulatory response element-reporter assay may be used as an alternative to virus-dependent, cytopathic assays. It contains a similar sensitivity to IFNs and can be completed in a shorter time than cytopathic assays and does not require heightened biosafety conditions after cell transduction.
Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA.
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McCoski
Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA.
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Xie
Large Animal Clinical Sciences, University of Florida, Gainesville, FL, USA.
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Hall
Department of Animal Sciences, University of Florida, Gainesville, FL, USA.
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Mercadante
Center for Reproductive Biology and Department of Animal Sciences, Washington State University, Pullman, WA, USA.
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Spencer
School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland.
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Lonergan
Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA; Department of Animal Sciences, University of Florida, Gainesville, FL, USA. Electronic address: ealy@vt.edu.
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Ealy
D000818:Animals; D001681:Biological Assay; D002417:Cattle; D005260:Female; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007370:Interferon Type I / Q000235:genetics / Q000378:metabolism*; D011247:Pregnancy; D020218:Response Elements / Q000235:genetics / Q000502:physiology*; D012680:Sensitivity and Specificity
D007370:Interferon Type I
Embryo; Interferon tau; Pregnancy; Trophoblast