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Methods of identifying, isolating and using odorant and aroma receptors

An odor and receptor technology, applied in chemical instruments and methods, biochemical equipment and methods, receptors/cell surface antigens/cell surface determinants, etc., can solve problems such as incompatibility and inability to function normally

Pending Publication Date: 2016-02-17
FIRMENICH SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, a systematic approach—in which hundreds to thousands of different cell lines have unique OR proteins that can be used to assess odorant activity—is essential for comprehensively decoding the combinatorial interactions between odorants and ORs. Inadequate approach for the role of cells because many or most receptors do not function properly in such cell lines

Method used

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  • Methods of identifying, isolating and using odorant and aroma receptors
  • Methods of identifying, isolating and using odorant and aroma receptors
  • Methods of identifying, isolating and using odorant and aroma receptors

Examples

Experimental program
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Embodiment 1

[0095] Screening of mouse olfactory sensory neurons to identify receptors that bind indole and / or skatole

[0096] The olfactory epithelium containing native olfactory nerves from mice was isolated and dissociated into single cells, then transferred to dissociation buffer for gentle mechanical and enzymatic dissociation. To optimize the dissociation method, the general procedure for preparing fresh dissociation buffer described in Araneda et al. (2004), A Pharmacological Profile of the Aldehyde Receptor Repertoire in Rat Olfactory Epithelium. Journal of Physiology: 555:743-756 was followed with the following modifications: DNase II was replaced by DNase I, Ca 2+ The concentration was raised to 5 mM and the pH was adjusted to 7.35 at 32°C. Dissociated neurons were then seeded on coverslips to allow selection of >2500 cells using fluorescence microscopy. Cells were then loaded with a calcium sensitive dye (Fura-2AM) for 30 minutes at 31°C and transferred to a microscope ready ...

Embodiment 2

[0099] Isolation and amplification of mRNA from indole- and skatole-sensitive cells to generate the cDNA sequence of the "stink" gene for indole- and skatole-sensitive receptors

[0100] The activated olfactory neurons of Example 1 were isolated using glass micropipettes and then pooled into one sample for subsequent identification of odorant receptor genes expressed as mRNA in responding cells. Odor receptor gene sequences were identified from indole- and skatole-sensitive cells by placing four groups of more than five (5) olfactory neurons activated by one or more malodorous compounds ( 6, 10, 15 and 15 cells each) were isolated. Micropipettes were designed from borosilicate glass (Catalog No. B150-86-10, Sutter Instruments) and pulled using a microelectrode puller (Model P-1000, Sutter Instruments) under the following conditions: heat, ramp temperature +20°C ; Pull=0; Vel=120; Pressure=500. Pulled pipette tips were manually broken and mounted to a Newport micromanipulator...

Embodiment 3

[0104] Identification of mouse and human malodour receptors

[0105] Identification of a subset of 5 mouse indole and skatole ORs out of approximately 1200 ORs in the mouse genome by coupled calcium imaging and NGS (Examples 1-2) enabled in silico additional mouse and new Rapid identification of human indole and skatole OR. As ORs with similar overall amino acid identities may display similar odor response profiles [Adipietro et al. (2012)], mouse OR sequences obtained by NGS analysis of indole / skatole-sensitive olfactory sensory neurons isolated using calcium imaging The OR list of candidate indole and skatole was expanded by querying public mouse and human genome databases. The identity of the mouse malodorous olfactory receptor was determined by comparing the NGS results to a reference genome sequence of the same species. Putative malodor receptors would be the most abundant mRNA in olfactory neuron-derived NGS samples or present in more than one independent biological re...

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Abstract

Provided here are new methods to identify specific families of mammalian odorant receptors for odorants or aroma, particularly indole and skatole malodors and their use in assays that may be used to discover compounds that modulate (blocking, enhancing, masking or mimicking compounds) their activity. Orphan mouse odorant receptors are identified from olfactory sensory neurons that respond to target compounds. The resulting receptors as well as their human counterparts can be screened in assays against test compounds to confirm their identity as odorant or aroma receptors, particularly malodor receptors and subsequently discover for example modulators that inhibit the perception of the malodor in humans.

Description

technical field [0001] The technical field relates to odorant and aroma receptors, and analytical methods useful for identifying odorous and / or aroma compounds, and more particularly to inhibitors or counteractants of malodorous compounds such as indole or skatole. Background technique [0002] Olfaction is one of the most complex and least understood human sensory systems. There are still many steps from olfactory receptor (OR) activation to perception that require further investigation. If we can understand how the OR codes used for individual odors and mixtures translate into perception, then we can use this knowledge to bring significant benefits to several fields. These areas include: odor modifiers, such as malodorous counteractants that block the perception of unpleasant odors; new flavoring and fragrance ingredients that replace non-biodegradable or toxic compounds; Source Probe Compound Dependence. The combinatorial example of the "olfactory code" centers on the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C07K14/705G01N33/50G01N33/566
CPCC12Q1/6809G01N33/5058C12Q2600/136G01N2500/04C07K14/705G01N2570/00G01N2333/705
Inventor P·普菲斯特M·E·罗杰斯K·J·帕里斯
Owner FIRMENICH SA
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