title: A novel method to study insect olfactory receptor function using HEK293 cells.
abstract: The development of rapid and reliable assays to characterize insect odorant receptors (ORs) and pheromone receptors (PRs) remains a challenge for the field. Typically ORs and PRs are functionally characterized either in vivo in transgenic Drosophila or in vitro through expression in Xenopus oocytes. While these approaches have succeeded, they are not well suited for high-throughput screening campaigns, primarily due to inherent characteristics that limit their ability to screen large quantities of compounds in a short period of time. The development of a practical, robust and consistent in vitro assay for functional studies on ORs and PRs would allow for high-throughput screening for ligands, as well as for compounds that could be used as novel olfactory-based pest management tools. Here we describe a novel method of utilizing human embryonic kidney cells (HEK293) transfected with inducible receptor constructs for the functional characterization of ORs in 96-well plates using a fluorescent spectrophotometer. Using EposOrco and EposOR3 from the pest moth, Epiphyas postvittana as an example, we generated HEK293 cell lines with robust and consistent responses to ligands in functional assays. Single-cell sorting of cell lines by FACS facilitated the selection of isogenic cell lines with maximal responses, and the addition of epitope tags on the N-termini allowed the detection of recombinant proteins in homogenates by western blot and in cells by immunocytochemistry. We thoroughly describe the methods used to generate these OR-expressing cell lines, demonstrating that they have all the necessary features required for use in high-throughput screening platforms.
authors.affiliation: School 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.
authors.forename: Jacob A
authors.initials: JA
authors.lastname: Corcoran
authors.affiliation: The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland 1142, New Zealand.
authors.forename: Melissa D
authors.initials: MD
authors.lastname: Jordan
authors.affiliation: The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland 1142, New Zealand.
authors.forename: Colm
authors.initials: C
authors.lastname: Carraher
authors.affiliation: School 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. Electronic address: Richard.Newcomb@plantandfood.co.nz.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Newcomb
mesh_terms: D000818:Animals; D002352:Carrier Proteins; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008024:Ligands; D009036:Moths / Q000235:genetics / Q000378:metabolism*; D018034:Olfactory Receptor Neurons / Q000378:metabolism; D018035:Receptors, Odorant / Q000235:genetics / Q000378:metabolism*; D043563:Receptors, Pheromone / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods
chemical_list: D002352:Carrier Proteins; D008024:Ligands; D018035:Receptors, Odorant; D043563:Receptors, Pheromone
keywords: HEK293; High-throughput; Lepidoptera; Odorant receptor