Name: high-throughput analysis of mammalian olfactory receptors: measurement of receptor activation via luciferase activity
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Description: Watch this Scientific Journal Video about High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity at JoVE.com

This work was supported by R01 DC013339, R03 DC011373, and Ruth L. Kirschstein National Research Service Award T32 DC000014. A portion of the work was performed using the Monell&#160;Chemosensory Receptor Signaling Core, which is supported, in part, by funding from the NIH-NIDCD Core Grant P30 DC011735. The authors thank C. Sezille for help with data collection.

1. Culture of Hana3A Cells
<ol>
	<li>Prepare M10 media by supplementing minimum essential medium (MEM) with 10% (v/v) FBS.</li>
	<li>Culture Maintenance
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		<li>Maintain cells in M10 media. NOTE: The expression vectors for RTP1L, RTP2, REEP1, and G&#945;olf confer puromycin resistance to Hana3A cells, but maintaining the cells with this antibiotic does not significantly affect assay results.</li>
		<li>Subculture at a ratio of 1:8 in 10 cm dishes every 2-3 days.</li>
		<li>Incubate at 37 &#176;C with 5% ...

<ol>
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Odorant identity is encoded by olfactory receptor activation patterns, but receptor activation patterns, including which receptors are activated and to what degree, are known for fewer than 6% of human olfactory receptors1-11. Efforts to characterize olfactory receptors have been limited by their labor-intensive methods or applicability to only a subset of the olfactory receptor family17,23,24,33,34. The Hana3A heterologous expression system supports the robust expression of the majority of tested o...

The mammalian olfactory system has the ability to respond to a vast number of odorous stimuli, allowing for the detection and discrimination of thousands of odorants. Olfactory receptors (ORs) are the molecular sensors expressed by the olfactory sensory neurons in the olfactory epithelium12. Mammalian odor recognition occurs through differential activation of ORs by odorants, and the OR gene family is extensive, with roughly 1,000 murine and 400 human receptors12-16. Previous functional analyses of ORs in olfactory neurons and in heterologous cells have shown that different odorants are recognized by unique, but overlapping ensembles of ORs10,17-20. Matching ligands to ORs is critical for understanding the olfactory code and essential for building viable models of olfaction. Due to difficulties expressing ORs in heterologous systems as well as the large number of both odorants and ORs, this data has been largely absent from the field; indeed, fewer than 6% of human ORs have a published ligand1-11. This protocol describes the use of a luciferase assay to characterize odorant/OR interactions. This assay enables the high-throughput characterization of ORs, a task that is essential to understanding odorant/OR interactions as well as developing a model of odor coding.
High-throughput studies of ORs face three major challenges. First, ORs expressed in heterologous cells were retained in the ER and subsequently degraded in the proteasome21,22, preventing the ORs from interacting with odorants in the assay system23-25. This problem was addressed by the discovery of accessory proteins that facilitate stable cell-surface expression of a broad range of ORs19,26,27. Receptor-transporter-proteins 1 and 2 (RTP1 and 2) promote OR cell-surface expression and activation in response to odorant stimulation19. Based on this work, HEK293T cells were modified to stably express RTP1 long (RTP1L) and RTP2, receptor expression-enhancing protein 1, and G&#945;olf, resulting in the Hana3A cell line19,27. In addition, the type 3 muscarinic acetylcholine receptor (M3-R) interacts with ORs at the cell surface and enhances activation in response to odorants26. Co-transfection of an OR with RTP1S and M3-R into Hana3A cells results in the robust, consistent, and functional expression of a broad range of ORs at the cell surface27. Second, mammalian OR repertoires are quite large. In humans, for example, the OR repertoire is an order of magnitude more numerous than the gustatory receptor repertoire, and 2&#160;orders of magnitude more numerous than the visual receptor repertoire. Although cloning a single OR is a relatively straightforward protocol, significant up-front effort is required to generate a comprehensive library. Third, although we know that in vision, wavelength translates into color and in audition frequency translates into pitch, the organization of odors is poorly understood, making it difficult for researchers to interpolate from a representative sample of odorants. Although some progress has been made on this front10,28, the map of the olfactory landscape remains incomplete. Screening tens of thousands of molecules against hundreds of ORs is a daunting task; high-throughput screens in this domain require carefully defined campaigns. The major remaining challenges are those of logistics and cost rather than problems inherent to the technique. Although heterologous screening has not been widely used to identify ligands by academic groups, a private company has used the same technique to identify ligands for 100 human ORs 29. Unfortunately, these data remain proprietary.
The high-throughput luciferase assay outlined here has several advantages over alternative methods used to assess OR activation. Although the responses of native olfactory sensory neurons have been measured using electrophysiology and calcium imaging, these techniques have difficulty teasing apart which OR leads to a neuron&#39;s response due to the overlap in response properties for olfactory neurons. Although knocking-in a GFP-labeled receptor type30,31, delivering specific receptors via adenovirus to murine olfactory neurons32,33, or performing RT-PCR after recordings17,24,33 can link recordings to single receptor types, these methods are low-throughput and not suitable for large-scale screens. Heterologous screening systems are more scalable, and two major forms are found in the literature: cAMP pathway reporters and inositol triphosphate (IP3) pathway reporters. Upon odor stimulation, ORs activate a G&#945;olf transduction signaling cascade that results in the production of cyclic AMP (cAMP)12. By co-transfecting a firefly luciferase reporter gene under the control of a cAMP response element (CRE), luciferase production can be measured as a function of odor response, allowing for the quantification of OR activation. OR activation can also be linked to the IP3 pathway by co-expressing G-proteins such as G&#945;15/16 or a G&#945;15-olf chimera24,25,34. We have chosen the assay presented here based on three factors: (1) the co-expression of RTP1 with Rho-tagged olfactory receptors improves the expression of olfactory receptors at the cell surface19,27; (2) use of a cAMP-responsive reporter gene allows for the measurement of OR activation through the canonical second messenger pathway; and (3) the assay is well-suited to high-throughput screens.
This high-throughput luciferase assay is applicable to a variety of studies valuable to the field of olfaction. First, a large number of ORs can be screened against a single odorant in order to determine the receptor activation pattern for a specific odorant. This type of study identified OR7D4 as the OR responsible for responding to the steroid odorant androstenone8. Conversely, one OR can be screened against a panel of odorants in order to determine the receptor response profile10. When candidate olfactory odorant/OR pairs are identified via these screens, interaction can be confirmed by conducting a dose response experiment examining the response of the OR to increasing concentrations of odorant. Dose response curves can also assess how genetic variation in an OR affects in vitro odorant response8,9,11,35, and these studies can be extended to interspecific OR variation, allowing for the examination of receptor evolution across species and causal mutations in evolution36,37, Finally, this assay can be used to screen for odor antagonists that are able to antagonize OR response to a particular odorant for a known odorant/receptor pair38,39. In summary, this high-throughput luciferase assay is applicable to a range of studies that will help characterize OR activation patterns and provide a better understanding of odor coding in the olfactory system.

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			<td height="21" style="height:21px;width:265px;">Name of Material/ Equipment</td>
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			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="84" style="height:84px;width:265px;">Hana3A cells</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
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			<td height="84" style="height:84px;width:265px;">RTP1S-pCI</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
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		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:265px;">M3-R-pCI</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">pCRE-luc</td>
			<td style="width:135px;">Agilent</td>
			<td style="width:119px;">219076</td>
			<td><a href="http://www.chem.agilent.com/store/ProductDetail.aspx?productID=219076%28Agilent%29">LUC</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">pSV40-RL</td>
			<td style="width:135px;">Promega</td>
			<td style="width:119px;">E2231</td>
			<td><a href="http://www.promega.com/products/reporter-assays-and-transfection/reporter-vectors-and-cell-lines/prl-renilla-luciferase-control-reporter-vectors/">RL</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimum Essential Media, Eagle</td>
			<td>Sigma Aldrich</td>
			<td>M4655</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/m4655?lang=en&amp;region=US">MEM</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">FBS</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
			<td><a href="https://products.invitrogen.com/ivgn/product/16000044?ICID=search-16000044">FBS</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS (without Ca2+ and Mg2+)</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td><a href="http://cellgro.com/products/balanced-salt-solutions/phosphate-buffered-saline/phosphate-buffered-saline-1x-2.html">PBS</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin (0.05% Trypsin EDTA)</td>
			<td>Life Technologies</td>
			<td>25300</td>
			<td><a href="https://products.invitrogen.com/ivgn/product/25300054?ICID=search-product">Trypsin</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">CD293</td>
			<td>Life Technologies</td>
			<td>11913-019</td>
			<td><a href="http://products.invitrogen.com/ivgn/product/11913019?ICID=search-11913019">CD293</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">96 well PDL white/clear plate</td>
			<td style="width:135px;">BD BioCoat</td>
			<td style="width:119px;">356693</td>
			<td><a href="http://www.thomassci.com/Supplies/Tissue-Culture-Cellware/_/BD-Biocoat-Poly-D-Lysine-Culture-Dishes?q=356693">plates</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:265px;">Lipid transfection reagent: Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668-019</td>
			<td><a href="https://products.invitrogen.com/ivgn/product/11668019?ICID=search-11668019">Lipofectamine</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:265px;">Firefly luciferase substrate, firefly luciferase quencher/Renilla luciferase substrate: Dual-Glo Assay</td>
			<td style="width:135px;">Promega</td>
			<td style="width:119px;">E2980</td>
			<td><a href="http://www.promega.com/products/reporter-assays-and-transfection/reporter-assays/dual_glo-luciferase-assay-system/?__utma=1.1932750276.1376486173.1376486173.1376486173.1&amp;__utmb=1.1.10.1376486173&amp;__utmc=1&amp;__utmx=-&amp;__utmz=1.1376486173.1.1.utmcsr=google|utmc">dual glo</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synergy S2&#160;</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">SLAD</td>
			<td><a href="http://www.biotek.com/products/microplate_detection/synergy2_multimode_microplate_reader.html">BioTek S2</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:265px;">Microplate reader software: Gen5 Data Analysis Software</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">Gen5</td>
			<td><a href="http://www.biotek.com/products/microplate_software/gen5_data_analysis_software.html">Gen5</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">BIOSTACK</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">BIOSTACK2WR</td>
			<td><a href="http://www.biotek.com/products/microplate_software/biostack_microplate_stacker.html">BioStack</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Multiflo</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">MFP</td>
			<td><a href="http://www.biotek.com/products/liquid_handling/multiflo_microplate_dispenser.html">MultiFlo</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">300ul GripTips</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">4433</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_4_e.html">GripTips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">12.5ul GripTips</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">4414</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_4_e.html">GripTips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">300ul GripTips ViaFlo96</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">6433</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_XYZ_4_e.html">XYZ tips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">12.5ul GripTips 384 XYZ</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">6403</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_XYZ_4_e.html">XYZ tips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">384ViaFlo</td>
			<td style="width:135px;">Integra</td>
			<td>6030</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/96_384_channel_pipette.html">384ViaFlo</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TE buffer</td>
			<td>Macherey Nagel</td>
			<td>740797.1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2650-100ML</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/d2650?lang=en&amp;region=US">DMSO</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">forskolin</td>
			<td>Enzo Life Sciences</td>
			<td>BML-CN100-0010</td>
			<td><a href="http://www.thomassci.com/007635F0-AA41-4B24-B46A-05723D685997/_/FORSKOLIN-10MG?q=c002e24">FOR</a></td>
		</tr>
	</tbody>
</table>

high-throughput analysis of mammalian olfactory receptors: measurement of receptor activation via luciferase activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

A primary screen tested 328 ORs against 26 odors at a concentration of 100 &#181;M. This odor concentration has been demonstrated to effectively activate a large proportion of ORs with known ligands10. First, normalized luciferase activity was calculated by dividing the firefly luciferase reading by the Renilla luciferase reading. Next, baselined values were calculated by subtracting the normalized luciferase readings for the no odor control from the normalized luciferase readings for each well (<stro...

Watch this Scientific Journal Video about High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity at JoVE.com

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Research

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Neuroscience

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In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

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Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

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Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

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Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...