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

Dette arbejde blev støttet af R01 DC013339, R03 DC011373, og Ruth L. Kirschstein National Research Service Award T32 DC000014. En del af arbejdet blev udført ved hjælp af Monell chemosensory receptorsignallering Core, som støttes delvist af finansiering fra NIH-NIDCD Core Grant P30 DC011735. Forfatterne takker C. Sezille hjælp til indsamling af data.

1.. Kultur Hana3A Cells <ol><li> Forbered M10 medier ved at supplere minimum essentielt medium (MEM) med 10% (v / v) FBS. </li><li> Kultur Vedligeholdelse <ol><li> Opretholde celler i M10 medier. BEMÆRK: ekspressionsvektorer til RTP1L, RTP2, REEP1 og G αolf give puromycinresistens til Hana3A celler, men at opretholde cellerne med dette antibiotikum påvirker ikke signifikant analyseresultater. </li><li> Subkultur i et forhold på 1:08 i 10 cm skåle hver 2-3 dage. </li><li> Inkuberes ved 37 ° C med 5% <s...

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Lugtstof identitet er kodet af olfaktoriske receptor aktivering mønstre, men receptor aktivering mønstre, herunder hvilke receptorer aktiveres og i hvilken grad, er kendt for mindre end 6% af de menneskelige olfaktoriske receptorer 1-11. Bestræbelserne på at karakterisere olfaktoriske receptorer er blevet begrænset af deres arbejdsintensive metoder eller anvendelighed til kun en delmængde af det olfaktoriske receptor familien 17,23,24,33,34. Den Hana3A heterolog ekspression system understøtt...

Pattedyrs olfaktoriske system har evnen til at reagere på et utal af lugtende stimuli, der giver mulighed for påvisning og diskrimination af tusindvis af lugtstoffer. Olfaktoriske receptorer (periferi) er de molekylære sensorer, som fremsættes af de olfaktoriske sensoriske neuroner i lugtepithelet 12. Mammalian lugt anerkendelse sker gennem differentieret aktivering af regioners af lugtstoffer, og OR gen familien er omfattende, med omtrent 1.000 murine og 400 humane receptorer 12-16. Tidligere funktionelle analyser af yderste periferi i olfaktoriske neuroner og i heterologe celler har vist, at forskellige lugtstoffer er anerkendt af unikke, men overlappende ensembler af regioners 10,17-20. Matchende ligander til yderste periferi er afgørende for forståelsen af ​​olfaktoriske kode og afgørende for at opbygge levedygtige modeller af lugtesansen. På grund af vanskeligheder, der udtrykker periferi i heterologe systemer samt det store antal af både lugtstoffer og periferi, har disse data været stort set fraværende fra field; ja, mindre end 6% af de menneskelige yderste periferi har en offentliggjort ligand 1-11. Denne protokol beskriver anvendelsen af ​​et luciferase assay for at karakterisere lugtstof / ELLER interaktioner. Denne analyse gør det muligt for high-throughput karakterisering af yderste periferi, en opgave, der er nødvendigt for at forstå lugtstof / eller interaktioner samt udvikle en model af lugt kodning. High-throughput studier af den yderste periferi står over for tre store udfordringer. Først blev periferi udtrykt i heterologe celler bevares i Skadestuen og efterfølgende nedbrydes i proteasomet 21,22, hvilket forhindrer den yderste periferi i at interagere med lugtstoffer i assaysystemet 23-25. Dette problem blev løst ved opdagelsen af accessoriske proteiner, der fremmer en stabil celleoverfladeekspression af en bred vifte af yderste periferi 19,26,27. Receptor-transporter-proteiner 1 og 2 (RTP1 og 2) fremme eller celleoverfladeekspression og aktivering som reaktion på lugtstof stimulation 19. Baseret på dette arbejde, var HEK293T cellermodificeres til stabilt at udtrykke RTP1 lange (RTP1L) og RTP2 receptor ekspression-forøgende protein 1, og G αolf, hvilket resulterer i Hana3A cellelinie 19,27. Desuden type 3 muskarine acetylcholin-receptor (M3-R) samvirker med den yderste periferi på celleoverfladen og forbedrer aktivering som reaktion på lugtstoffer 26. Co-transfektion af en OR med RTP1S og M3-R i Hana3A celler resulterer i robust, konsekvent og funktionelle udtryk for en bred vifte af yderste periferi på celleoverfladen 27. Andet pattedyr eller repertoirer er temmelig store. Hos mennesker, for eksempel, OR repertoire er en størrelsesorden mere talrige end smagssansen receptor repertoire, og 2 størrelsesordener mere talrige end den visuelle receptor repertoire. Selv klone et enkelt eller er en forholdsvis enkel protokol, er betydelig up-front indsats, der kræves for at generere et omfattende bibliotek. For det tredje, selv om vi ved, at i et syn, bølgelængde udmønter sig i farve ogi audition frekvens udmønter sig i tonehøjde, er tilrettelæggelsen af ​​lugte dårligt forstået, hvilket gør det svært for forskerne at interpolere fra en repræsentativ stikprøve af lugtstoffer. Selvom der er gjort på denne front 10,28 visse fremskridt, kortet over den olfaktoriske landskab er stadig ufuldstændig. Screening titusinder af molekyler mod hundredvis af yderste periferi er en skræmmende opgave; high-throughput skærme i dette domæne kræver nøje definerede kampagner. De største resterende udfordringer er dem af logistik og omkostninger snarere end problemer forbundet med teknikken. Selvom heterolog screening ikke har været meget anvendt til at identificere ligander med akademiske grupper har et privat selskab brugt den samme teknik til at identificere ligander for 100 menneskelige yderste periferi 29. Desværre er der stadig disse data proprietære. High-throughput luciferaseassayet beskrevet her, har flere fordele i forhold til alternative metoder anvendes til evaluering eller aktivering. Selvom ansvarses af indfødte olfaktoriske sensoriske neuroner er blevet målt ved hjælp af elektrofysiologi og calcium imaging, disse teknikker har svært ved at drille hinanden, hvilket ELLER fører til en neuron reaktion på grund af overlapningen i respons egenskaber for olfaktoriske neuroner. Selv banke-på et GFP-mærket receptortype 30,31, der leverer specifikke receptorer via adenovirus for murine olfaktoriske neuroner 32,33, eller udfører RT-PCR efter optagelser 17,24,33 kan linke optagelser til en enkelt receptor typer, disse metoder er lav-throughput og ikke egnet til store skærme. Heteroloqe screening systemer er mere skalerbar og to store former er fundet i litteraturen: cAMP pathway reportere og inositoltriphosphat (IP3) pathway journalister. Ved lugt stimulering, aktivere periferi en G αolf transduktion signaleringskaskade, som resulterer i produktionen af cyklisk AMP (cAMP) 12.. Ved co-transfektion af en ildflue-luciferase-reportergen under kontrol af acAMP-responselement (CRE), kan måles luciferase produktionen som en funktion af lugt respons, giver mulighed for kvantificering af OR-aktivering. Eller aktivering kan også være knyttet til IP3 vej ved co-udtrykker G-proteiner, såsom G α15/16 eller en G α15-olf kimære 24,25,34. Vi har valgt analysen præsenteres her baseret på tre faktorer: (1) co-ekspression af RTP1 med Rho-mærkede olfaktoriske receptorer forbedrer udtryk for olfaktoriske receptorer på celleoverfladen 19,27; (2) brug af en cAMP-responsiv reporter gen giver mulighed for måling af OR aktivering via den kanoniske anden messenger vej; og (3) assayet er velegnet til high-throughput skærme. Denne high-throughput luciferaseassayet er anvendelig til en række undersøgelser værdifulde til området for lugtesansen. For det første kan et stort antal periferi screenes mod en enkelt lugtstof for at bestemme receptor aktivering mønster for en specific duftstof. Denne type undersøgelse identificerede OR7D4 som eller er ansvarlige for at reagere på den steroid lugtstof androstenon 8. Omvendt kan en eller screenes mod et panel af lugtstoffer for at bestemme receptor reaktionsprofil 10. Når kandidat olfaktoriske lugtstof / ELLER par er identificeret via disse skærme, kan interaktion bekræftes ved at gennemføre en dosis-respons eksperiment undersøger svaret fra OR til stigende koncentrationer af lugtstof. Dosisreaktionskurver kan også vurdere, hvordan den genetiske variation i en OR påvirker in vitro lugtstof respons 8,9,11,35, og disse undersøgelser kan udvides til interspecifikke ELLER variation, der giver mulighed for undersøgelse af receptor-evolution på tværs af arter og kausale mutationer i evolution 36,37, Endelig, dette assay kan anvendes til screening for lugt-antagonister der er i stand til at antagonisere eller reaktion på en bestemt lugtstof til en kendt lugtstof / receptor par 38,39. Sammenfattende denne høje-Throughput luciferaseassayet er anvendelig til en række undersøgelser, der vil hjælpe karakterisere eller aktivering mønstre og give en bedre forståelse af lugt kodning i det olfaktoriske system.

<table border="0" cellpadding="0" cellspacing="0" style="width:685px;" width="685">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Name of Material/ Equipment</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:265px;">Hana3A cells</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:265px;">RTP1S-pCI</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</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

Lugtstoffer skabe unikke og overlappende mønstre af olfaktoriske receptor-aktivering, så en familie på cirka 1.000 murine og 400 menneskelige receptorer til at genkende tusindvis af lugtstoffer. Lugtstof ligander er blevet offentliggjort for mindre end 6% af de menneskelige receptorer 1-11. Denne mangel på data er dels på grund af vanskeligheder funktionelt udtrykker disse receptorer i heterologe systemer. Her beskriver vi en fremgangsmåde til at udtrykke et flertal af det olfaktoriske receptor familien i Hana3A celler, efterfulgt af en høj-throughput vurdering af olfaktoriske receptor-aktivering ved hjælp af en luciferase reporter assay. Dette assay kan anvendes til (1) screen paneler af lugtstoffer mod paneler af olfaktoriske receptorer; (2) bekræfte lugtstof / receptor interaktion via dosisreaktionskurver; og (3) sammenligne receptor aktivering niveauer blandt receptor varianter. I vores eksempeldata blev 328 olfaktoriske receptorer afskærmet mod 26 duftstoffer. Lugtstof / receptor par med varierende respons scoringer var selektivted og testet i dosisrespons. Disse data indikerer, at en skærm er en effektiv metode til at berige for odorant / receptor par, der vil passere en dosis-respons eksperiment, dvs receptorer, der har en bona fide reaktion på et lugtstof. Derfor er denne high-throughput luciferaseassayet er en effektiv metode til at karakterisere olfaktoriske receptorer-et vigtigt skridt i retning af en model af lugt kodning i pattedyrs olfaktoriske system.

En primær skærm testet 328 periferi mod 26 lugte i en koncentration på 100 uM. Denne lugt koncentration er blevet demonstreret til effektivt at aktivere en stor del af den yderste periferi med kendte ligander 10. Først blev normaliseret luciferaseaktivitet beregnes ved at dividere ildflueluciferase læsning af Renilla luciferase læsning. Derefter blev baselined værdier beregnes ved at trække de normaliserede luciferase aflæsninger til ingen lugt kontrol fra de normaliserede luciferase aflæsn...

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

Olfaktoriske receptor aktivering mønstre indkode lugt identitet, men manglen på offentliggjorte data, der identificerer lugtstof ligander for pattedyr olfaktoriske receptorer hindrer udviklingen af ​​en omfattende model af lugt kodning. Denne protokol beskriver en fremgangsmåde til at lette højkapacitetsidentifikation af olfaktoriske receptorligander og kvantificering af receptor-aktivering.

High-throughput analyse af Mammale Olfaktoriske Receptors: Måling af receptor-aktivering via luciferaseaktivitet

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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A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

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In vivo Electroporation of Developing Mouse Retina

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

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

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Funktionel Interrogation Adult Hypothalamus Neurogenese med Focal Radiological Hæmning

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Hele Mount immunolabeling af olfaktoriske receptor neuroner i<em&gt; Drosophila</em&gt; Antenne

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

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

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