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Updated: May 3, 2026
High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes
Published on: March 24, 2015
Validation of an interferon stimulatory response element reporter gene assay for quantifying type I interferons
S R McCoski1, M Xie1, E B Hall2
1Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA.
Insights
A new interferon (IFN) bioassay using reporter cells offers a faster, safer alternative to traditional methods. This virus-free assay effectively detects ovine interferon-tau (IFN-τ), crucial for pregnancy recognition in ruminants.
Area of Science:
- Biotechnology
- Animal Reproduction
- Molecular Biology
Background:
- Traditional interferon (IFN) bioassays often rely on virus-dependent methods, posing biosafety concerns and requiring longer assay times.
- Interferon-tau (IFN-τ) is a critical hormone for maternal recognition of pregnancy in ruminants, but its detection can be challenging.
- Developing a robust, virus-free bioassay for IFN-τ is essential for reproductive research and diagnostics in livestock.
Purpose of the Study:
- To develop a novel, virus-free, cell-based interferon bioassay.
- To validate the assay's utility for detecting ovine interferon-tau (IFN-τ) in various biological samples.
- To establish a sensitive and rapid alternative to conventional cytopathic IFN assays.
Main Methods:
- Madin-Darby bovine kidney cells were genetically modified using lentiviral vectors to express a firefly luciferase reporter gene under the control of an IFN stimulatory response element (ISRE).
- Stable cell lines were selected using puromycin.
- The assay's performance was evaluated using known concentrations of human IFN-α and ovine IFN-τ, and its activity was measured in conditioned media from bovine trophoblast cells and uterine flushes from sheep and cattle.
Main Results:
- The developed ISRE-luciferase reporter assay demonstrated a linear, dose-dependent response to both human IFN-α and ovine IFN-τ.
- Significant interferon activity was detected in conditioned media from bovine trophoblast cells and uterine flushes from ruminants.
- The assay successfully detected IFN activity in media from in vitro-cultured bovine blastocysts.
Conclusions:
- The ISRE-reporter assay provides a sensitive, virus-free, and rapid alternative to traditional virus-dependent interferon bioassays.
- This assay is suitable for detecting ovine IFN-τ and can be applied to various biological samples relevant to ruminant reproduction.
- The assay simplifies biosafety requirements post-transduction, making it a more practical tool for research and diagnostics.
Abstract:
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.
