A Genetic Platform for Functionally Profiling Odorant Receptors Ex Vivo Using Olfactory Cilia

Abstract

The molecular basis for odor perception in humans remains a black box as odorant receptors (ORs) are notoriously difficult to study outside of their native environment. Efforts toward OR expression and functional profiling in heterologous systems have been met with limited success due to poor efficiency of cell surface expression and consequently reduced G-protein signal amplification. We previously reported a genetic strategy in mice to increase the number of sensory neurons expressing specific ORs, which transforms the 10 million neurons of the mouse nose into a bioreactor producing large quantities of fully functional OR protein. We now describe the isolation of cilia from these bioreactors for two human ORs. Cilia are known to contain all components of the olfactory signal transduction machinery and can be placed into an ex vivo well-plate assay to rapidly measure robust, reproducible odor-specific responses. Our OR1A1 and OR5AN1 isolated cilia reveal 10-100fold more sensitivity than existing assays. Tissue from a single animal produces up to 4,000 384-well assay wells, and isolated olfactory cilia can be stored frozen and thus preserved for long term usage. This pipeline offers a sensitive, highly scalable ex vivo odor screening platform that opens the door for decoding human olfaction.

Commonality of Odorant Receptor Choice Mechanism Revealed by Analysis of a Highly Represented Odorant Receptor Transgene

“…We confirmed the DESeq findings by RT-qPCR analysis of RNA levels for the Class I OR, Olfr690 and the Class II OR, Olfr358, both of which showed a ~4-fold decrease consistent with the ~6fold DESeq data (Figure S1). Interestingly, A second OR1A1 line (Omura, 2021) with significant dorsal expression produced with IRES-Gcamp6f also revealed the similar ~4-fold decrease in expression for Olfr358. On the other hand, 325/348 DEGs with diminished representation are OR genes, with a subset representing all the known dorsal classes (6 TAARs, 90 Class I ORs, 179 Class II ORs|DVIs 1.05-1.90).…”