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A novel method to study insect olfactory receptor function using HEK293 cells.

Jacob A Corcoran1, Melissa D Jordan2, Colm Carraher2

  • 1School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand; The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland 1142, New Zealand.

Insect Biochemistry and Molecular Biology
|September 2, 2014
PubMed
Summary
This summary is machine-generated.

Developing a new high-throughput screening method for insect odorant receptors (ORs) using human embryonic kidney cells (HEK293). This robust assay enables rapid identification of ligands and potential pest management tools.

Keywords:
HEK293High-throughputLepidopteraOdorant receptor

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Area of Science:

  • Molecular biology
  • Insect olfactory research
  • Assay development

Background:

  • Characterizing insect odorant receptors (ORs) and pheromone receptors (PRs) is crucial for understanding olfaction and developing pest control strategies.
  • Current methods for OR/PR functional characterization, such as in vivo assays in Drosophila or in vitro expression in Xenopus oocytes, are not suitable for high-throughput screening.
  • A need exists for a practical, robust, and consistent in vitro assay for high-throughput screening of OR/PR ligands and potential pest management compounds.

Purpose of the Study:

  • To develop a novel, high-throughput in vitro assay for functional characterization of insect odorant receptors (ORs).
  • To establish a method utilizing human embryonic kidney (HEK293) cells for rapid screening of OR ligands.
  • To demonstrate the utility of this assay for identifying compounds for olfactory-based pest management.

Main Methods:

  • Utilized human embryonic kidney (HEK293) cells transfected with inducible receptor constructs for OR functional characterization.
  • Employed a 96-well plate format with a fluorescent spectrophotometer for assay readout.
  • Generated stable HEK293 cell lines expressing specific ORs (EposOrco and EposOR3) from Epiphyas postvittana, incorporating epitope tags for detection.
  • Used Fluorescence-Activated Cell Sorting (FACS) for single-cell sorting to select isogenic cell lines with maximal responses.

Main Results:

  • Successfully generated HEK293 cell lines with robust and consistent responses to OR ligands.
  • Demonstrated the ability to detect recombinant OR proteins using western blot and immunocytochemistry via N-terminal epitope tags.
  • Confirmed that the developed cell lines possess the necessary characteristics for high-throughput screening platforms.
  • Validated the assay using ORs from the pest moth Epiphyas postvittana.

Conclusions:

  • A novel, high-throughput assay for insect OR functional characterization has been developed using engineered HEK293 cells.
  • This method provides a robust and consistent platform for screening OR ligands and potential pest management agents.
  • The developed assay overcomes limitations of traditional methods, enabling efficient screening of large compound libraries.