Name: live-cell measurement of odorant receptor activation using a real-time camp assay
Uploaded: 2017-10-02T14:00:00
Description: Watch this Scientific Journal Video about Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay at JoVE.com

Arbeidet ble støttet av kinesiske National Science Foundation (31070972), vitenskap og teknologi kommisjonen i Shanghai kommune (16ZR1418300), programmet for nyskapende forskning Team av Shanghai kommunale utdanning provisjon, Shanghai øst Forsker Program (J50201) og nasjonale grunnforskning programmet Kina (2012CB910401).

1. Culturing og vedlikehold av Hana3A celler <ol><li>vedlikeholde celler i 10 mL av minimum viktig medium (MEM) med 10% fosterets bovin serum (FBS), 100 µg/mL penicillin-streptomycin, og 1,25 µg/mL amfotericin B i en 100 mm celle kultur parabol i en 37 ° C celle kultur inkubator med 5% CO 2. Hver annen passasje, legge til 1 µg/mL puromycin for å opprettholde stabil transfection av plasmider (se innledningen). 
	Merk: Utfører alle trinn som involverer cellekultur i en klasse II bi...

<ol>
	<li>Malakoff, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Following+the+scent+of+avian+olfaction.">Following the scent of avian olfaction.</a> Science. 286 (5440), 704-705 (1999).</li><li>Zippel, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecology+of+vertebrate+olfaction:+D.M.+Stoddart.+Chapman+and+Hall,+Andover,+Great+Britain,+1980.+£15.00,+234+pp.+ISBN+0-412-21820-8.">The ecology of vertebrate olfaction: D.M. Stoddart. Chapman and Hall, Andover, Great Britain, 1980. £15.00, 234 pp. ISBN 0-412-21820-8.</a> Behav Processes. 7 (2), 198-199 (1982).</li><li>Dryer, L., Berghard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+receptors:+a+plethora+of+G-protein-coupled+receptors.">Odorant receptors: a plethora of G-protein-coupled receptors.</a> Trends Pharmacol Sci. 20 (10), 413-417 (1999).</li><li>Zhang, X., Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+olfactory+receptor+gene+superfamily+of+the+mouse.">The olfactory receptor gene superfamily of the mouse.</a> Nat Neurosci. 5 (2), 124-133 (2002).</li><li>Glusman, G., Yanai, I., Rubin, I., Lancet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complete+human+olfactory+subgenome.">The complete human olfactory subgenome.</a> Genome Res. 11 (5), 685-702 (2001).</li><li>Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+ligands+for+odorant,+vomeronasal+and+taste+receptors.">Genes and ligands for odorant, vomeronasal and taste receptors.</a> Nat Rev Neurosci. 5 (4), 263-278 (2004).</li><li>Reed, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=After+the+holy+grail:+establishing+a+molecular+basis+for+Mammalian+olfaction.">After the holy grail: establishing a molecular basis for Mammalian olfaction.</a> Cell. 116 (2), 329-336 (2004).</li><li>Buck, L., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multigene+family+may+encode+odorant+receptors:+a+molecular+basis+for+odor+recognition.">A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.</a> Cell. 65 (1), 175-187 (1991).</li><li>McClintock, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+identification+of+eugenol-responsive+and+muscone-responsive+mouse+odorant+receptors.">In vivo identification of eugenol-responsive and muscone-responsive mouse odorant receptors.</a> J Neurosci. 34 (47), 15669-15678 (2014).</li><li>Trimmer, C., Snyder, L. L., Mainland, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+analysis+of+mammalian+olfactory+receptors:+measurement+of+receptor+activation+via+luciferase+activity.">High-throughput analysis of mammalian olfactory receptors: measurement of receptor activation via luciferase activity.</a> J. Vis. Exp. (88), e51640 (2014).</li><li>Saito, H., Chi, Q., Zhuang, H., Matsunami, H., Mainland, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+coding+by+a+Mammalian+receptor+repertoire.">Odor coding by a Mammalian receptor repertoire.</a> Sci Signal. 2 (60), ra9 (2009).</li><li>Jiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profiling+of+activated+olfactory+neurons+identifies+odorant+receptors+for+odors+in+vivo.">Molecular profiling of activated olfactory neurons identifies odorant receptors for odors in vivo.</a> Nat Neurosci. 18 (10), 1446-1454 (2015).</li><li>Malnic, B., Hirono, J., Sato, T., Buck, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+receptor+codes+for+odors.">Combinatorial receptor codes for odors.</a> Cell. 96 (5), 713-723 (1999).</li><li>Touhara, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+identification+and+reconstitution+of+an+odorant+receptor+in+single+olfactory+neurons.">Functional identification and reconstitution of an odorant receptor in single olfactory neurons.</a> Proc Natl Acad Sci U S A. 96 (7), 4040-4045 (1999).</li><li>Kajiya, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+bases+of+odor+discrimination:+Reconstitution+of+olfactory+receptors+that+recognize+overlapping+sets+of+odorants.">Molecular bases of odor discrimination: Reconstitution of olfactory receptors that recognize overlapping sets of odorants.</a> J Neurosci. 21 (16), 6018-6025 (2001).</li><li>von der Weid, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+transcriptional+profiling+of+chemosensory+neurons+identifies+receptor-ligand+pairs+in+vivo.">Large-scale transcriptional profiling of chemosensory neurons identifies receptor-ligand pairs in vivo.</a> Nat Neurosci. 18 (10), 1455-1463 (2015).</li><li>D'Hulst, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MouSensor:+A+Versatile+Genetic+Platform+to+Create+Super+Sniffer+Mice+for+Studying+Human+Odor+Coding.">MouSensor: A Versatile Genetic Platform to Create Super Sniffer Mice for Studying Human Odor Coding.</a> Cell Rep. 16 (4), 1115-1125 (2016).</li><li>Lu, M., Echeverri, F., Moyer, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum+retention,+degradation,+and+aggregation+of+olfactory+G-protein+coupled+receptors.">Endoplasmic reticulum retention, degradation, and aggregation of olfactory G-protein coupled receptors.</a> Traffic. 4 (6), 416-433 (2003).</li><li>McClintock, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+of+olfactory-adrenergic+receptor+chimeras+and+intracellular+retention+of+heterologously+expressed+olfactory+receptors.">Functional expression of olfactory-adrenergic receptor chimeras and intracellular retention of heterologously expressed olfactory receptors.</a> Brain Res Mol Brain Res. 48 (2), 270-278 (1997).</li><li>Krautwurst, D., Yau, K. W., Reed, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+ligands+for+olfactory+receptors+by+functional+expression+of+a+receptor+library.">Identification of ligands for olfactory receptors by functional expression of a receptor library.</a> Cell. 95 (7), 917-926 (1998).</li><li>Saito, H., Kubota, M., Roberts, R. W., Chi, Q., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RTP+family+members+induce+functional+expression+of+mammalian+odorant+receptors.">RTP family members induce functional expression of mammalian odorant receptors.</a> Cell. 119 (5), 679-691 (2004).</li><li>Zhuang, H., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergism+of+accessory+factors+in+functional+expression+of+mammalian+odorant+receptors.">Synergism of accessory factors in functional expression of mammalian odorant receptors.</a> J Biol Chem. 282 (20), 15284-15293 (2007).</li><li>Li, Y. R., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+state+of+the+M3+muscarinic+acetylcholine+receptor+modulates+mammalian+odorant+receptor+signaling.">Activation state of the M3 muscarinic acetylcholine receptor modulates mammalian odorant receptor signaling.</a> Sci Signal. 4 (155), ra1 (2011).</li><li>Durocher, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reporter+gene+assay+for+high-throughput+screening+of+G-protein-coupled+receptors+stably+or+transiently+expressed+in+HEK293+EBNA+cells+grown+in+suspension+culture.">A reporter gene assay for high-throughput screening of G-protein-coupled receptors stably or transiently expressed in HEK293 EBNA cells grown in suspension culture.</a> Anal Biochem. 284 (2), 316-326 (2000).</li><li>Liberles, S. D., Buck, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+second+class+of+chemosensory+receptors+in+the+olfactory+epithelium.">A second class of chemosensory receptors in the olfactory epithelium.</a> Nature. 442 (7103), 645-650 (2006).</li><li>Saraiva, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+effects+of+odorants+on+mouse+behavior.">Combinatorial effects of odorants on mouse behavior.</a> Proc Natl Acad Sci U S A. 113 (23), E3300-E3306 (2016).</li><li>Nara, K., Saraiva, L. R., Ye, X., Buck, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale+analysis+of+odor+coding+in+the+olfactory+epithelium.">A large-scale analysis of odor coding in the olfactory epithelium.</a> J Neurosci. 31 (25), 9179-9191 (2011).</li><li>Zhuang, H., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+cell-surface+expression+and+measuring+activation+of+mammalian+odorant+receptors+in+heterologous+cells.">Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells.</a> Nat Protoc. 3 (9), 1402-1413 (2008).</li><li>Fan, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+genetically+encoded+biosensors+using+firefly+luciferase.">Novel genetically encoded biosensors using firefly luciferase.</a> ACS Chem Biol. 3 (6), 346-351 (2008).</li><li>Fan, B. F., Wood, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-Cell+Luminescent+Assays+for+GPCR+Studies:+Combination+of+Sensitive+Detection+and+Real-Time+Analysis+Expands+Applications.">Live-Cell Luminescent Assays for GPCR Studies: Combination of Sensitive Detection and Real-Time Analysis Expands Applications.</a> Genetic Engineering &amp; Biotechnology News. 29, 30-31 (2009).</li><li>Geithe, C., Andersen, G., Malki, A., Krautwurst, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Butter+Aroma+Recombinate+Activates+Human+Class-I+Odorant+Receptors.">A Butter Aroma Recombinate Activates Human Class-I Odorant Receptors.</a> J Agric Food Chem. 63 (43), 9410-9420 (2015).</li><li>Noe, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OR2M3:+A+Highly+Specific+and+Narrowly+Tuned+Human+Odorant+Receptor+for+the+Sensitive+Detection+of+Onion+Key+Food+Odorant+3-Mercapto-2-methylpentan-1-ol.">OR2M3: A Highly Specific and Narrowly Tuned Human Odorant Receptor for the Sensitive Detection of Onion Key Food Odorant 3-Mercapto-2-methylpentan-1-ol.</a> Chem Senses. 42 (3), 195-210 (2016).</li><li>Geithe, C., Noe, F., Kreissl, J., Krautwurst, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+broadly+tuned+odorant+receptor+OR1A1+is+highly+selective+for+3-methyl-2,4-nonanedione,+a+key+food+odorant+in+aged+wines,+tea,+and+other+foods.">The broadly tuned odorant receptor OR1A1 is highly selective for 3-methyl-2,4-nonanedione, a key food odorant in aged wines, tea, and other foods.</a> Chem Senses. 42 (3), 181-193 (2016).</li><li>Duan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crucial+role+of+copper+in+detection+of+metal-coordinating+odorants.">Crucial role of copper in detection of metal-coordinating odorants.</a> Proc Natl Acad Sci U S A. 109 (9), 3492-3497 (2012).</li><li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smelling+Sulfur:+Copper+and+Silver+Regulate+the+Response+of+Human+Odorant+Receptor+OR2T11+to+Low-Molecular-Weight+Thiols.">Smelling Sulfur: Copper and Silver Regulate the Response of Human Odorant Receptor OR2T11 to Low-Molecular-Weight Thiols.</a> J Am Chem Soc. , (2016).</li><li>Wu, L., Pan, Y., Chen, G. Q., Matsunami, H., Zhuang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-transporting+protein+1+short+(RTP1S)+mediates+translocation+and+activation+of+odorant+receptors+by+acting+through+multiple+steps.">Receptor-transporting protein 1 short (RTP1S) mediates translocation and activation of odorant receptors by acting through multiple steps.</a> J Biol Chem. 287 (26), 22287-22294 (2012).</li><li>Shirasu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+receptor+and+neural+pathway+responsible+for+highly+selective+sensing+of+musk+odors.">Olfactory receptor and neural pathway responsible for highly selective sensing of musk odors.</a> Neuron. 81 (1), 165-178 (2014).</li><li>Block, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implausibility+of+the+vibrational+theory+of+olfaction.">Implausibility of the vibrational theory of olfaction.</a> Proc Natl Acad Sci U S A. 112 (21), E2766-E2774 (2015).</li><li>Sato-Akuhara, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+Specificity+and+Evolution+of+Mammalian+Musk+Odor+Receptors:+Effect+of+Single+Receptor+Deletion+on+Odor+Detection.">Ligand Specificity and Evolution of Mammalian Musk Odor Receptors: Effect of Single Receptor Deletion on Odor Detection.</a> J Neurosci. 36 (16), 4482-4491 (2016).</li><li>Gane, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+vibration-sensing+component+in+human+olfaction.">Molecular vibration-sensing component in human olfaction.</a> PLoS One. 8 (1), e55780 (2013).</li><li>Young, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+evolutionary+processes+shaped+the+mouse+and+human+olfactory+receptor+gene+families.">Different evolutionary processes shaped the mouse and human olfactory receptor gene families.</a> Hum Mol Genet. 11 (5), 535-546 (2002).</li></ol>

Nøyaktig måle en OR aktivisering ved eksponering til en viss odorant er det første trinnet i å tyde koding av olfactory informasjon. Eksperimentene vist i denne studien viser et eksempel på hvordan man kan identifisere, bruke en i vitro OR uttrykk system, forståelsesfull ORs blant menneskelige OR repertoaret for illeluktende kjemiske av interesse og senere karakteriserer reseptoren farmakologi ved hjelp av ulike konsentrasjoner av kjemiske. Resultatene bekrefter OR5AN1 som en bona fide reseptor fo...

Luktesans spiller en viktig rolle i dyrenes overlevelse som de er avhengige av sine olfactory evner til å skaffe mat, unngå rovdyr og fare, skille arter og velg kompis1,2. Realisering av disse funksjonene avhenger odorant receptors (ORs), som er individuelt på ciliary overflaten av olfactory sensoriske neurons (OSNs) i olfactory epitel (OE). ORs utgjør den største familien av G-protein kombinert reseptor (GPCR) gruppe med ca 400 og 1200 forskjellige OR gener i menneskelig og mus, henholdsvis3,4,5. ORs aktivert ved odorants føre til økt intracellulær leiren nivå via sekvensiell aktivering av olfactory G-protein (Golf) og type III adenylyl cyclase (ACIII). Den resulterende intensivering av intracellulær leiren kan fungere som en andre budbringer, som åpner nukleotid-gated kanal på celleoverflaten, utløser tilstrømningen av kasjoner inkludert Ca2 + og handling potensialer, og til slutt starte Nevro-potensielle overføring og olfactory oppfatning. Oppdage og diskriminerende mange odorants av ORs regnes som det første trinnet olfactory oppfatning6,7.
Siden Buck og Axel8 først ble klonet odorant reseptorer og belyst mekanismen av olfactory oppfatning initiert av ORs, ble deorphanization av OR familien en av hotspots i dette feltet. Ulike i vivo, ex vivo og i vitro metoder for å måle OR aktivisering har vært rapportert9,10,11,12. En tradisjonell metode som brukes Ca2 + imaging etterfulgt av encellede RT PCR på OSNs aktivert identifikasjon av ulike ORs til alifatisk odorants13,14,15. Flere nylig, bruk av store transcriptome analyser promoterte utviklingen av flere høy gjennomstrømming i vivo metoder. Kentucky analysen identifiserte eugenol - og muscone-responsive musen ORs med bruk av S100a5-tauGFP reporter musen belastning og microarray analyse9. DREAM teknologien basert på nedgangen i OR mRNA nivåer etter odorant eksponering, og ansatt en transcriptomic tilnærming til å bestemme OR aktivisering profiler i begge vertebrate og ikke-virveldyr16. Tilsvarende gitt fosforylering av S6 i neuronal aktiveringer, Matsunami gruppe sekvensert mRNAs fra fosforylert ribosom immunoprecipitations å identifisere forståelsesfull ORs12. Til slutt, gruppen Feinstein rapportert super snuse mus som kan tjene som en plattform for å studere lukt koding i vivo, kjent som MouSensor teknologi17.
I området i vitro gjør utfordringen med dyrking OSNs et heterologous uttrykk system som etterligner OR funksjonelle uttrykk i vivo en ideell løsning å gjennomføre omfattende screening av luktende kjemikalier for ORs. Likevel, siden kulturperler linjer av ikke-olfactory opprinnelse avvike fra opprinnelige OSNs eller proteiner er beholdt i det endoplasmatiske retikulum og ikke trafikk til plasma membranen, som resulterer i OR fornedrelse og tap av reseptor funksjonen18 , 19. for å løse dette problemet, omfattende verk er gjort å gjenskape OR funksjonelle uttrykk i cellemembranen i heterologous linjer. Krautwurst et al. først knyttet de første 20 aminosyrene av rhodopsin (Rho-tag) til N-terminalen OR protein og dette forfremmet celle-overflate uttrykk for noen ORs i menneskelige embryonale nyre (HEK) celler20. Ved å gjennomføre en seriell analyse av gene expression (SAGE) bibliotek analyse fra enkelt OSNs, Saito et al. først klonet reseptor-transport familiemedlemmer for protein (RTP), RTP1 og RTP2 og reseptor uttrykk enhancer protein 1 (REEP1) som muliggjorde OR smuglingen til cellemembranen og forbedret odorant-mediert svar av ORs i HEK293T celler 21. basert på disse resultatene, gruppen Matsunami opprettet Hana3A celle linjen stabilt transfekterte med RTP1, RTP2, REEP1 og Gαolf i HEK293T og transiently transfekterte med Rho-merket ORs, for effektiv eller funksjonell uttrykk. Senere studier avdekket 1) kortformen av RTP1, RTP1S, som mer robust fremme eller funksjon enn det opprinnelige RTP1 proteinet og 2) den type 3 muscarinic acetylcholin reseptoren (M3R) som kan forbedre OR aktivitet via hemming av β-arrestin-2 Rekruttering, begge ble introdusert til heterologous uttrykk systemet å optimalisere eksperimentelle utgang22,23.
Flere gjenkjenningsmetoder har blitt brukt å kvantifisere reseptor aktivisering i heterologous systemer. Utskilles placental alkalisk fosfatase (SEAP) analysen fungerer med en reporter enzymet transcriptionally regulert av leiren respons elementer (CREs), noe som gjør det til et attraktivt alternativ for å vurdere OR aktivisering. Fluorescensen oppdages lett i et utvalg av kultur medium etter inkubasjon med SEAP gjenkjenning reagens24. Bruker denne metoden, preget funksjonene ORs samt en sekundær klasse av chemosensory reseptorer uttrykt i OE-sporet Amin-assosiert reseptorer (Milton) har vært25,26,27. En annen vanlig metode, luciferase analysen, bruker et firefly luciferase reporter gen under kontroll av leiren svar elementet (Grobunn). Måle luminescence generert av luciferase produksjon gir en effektiv og robust måte å kvantifisere OR aktivisering10,11,28.
Sanntid leiren analyser har også vært mye brukt i dynamisk overvåking funksjon heterologous eller endogene GPCRs. Et eksempel på slike avanserte analyser utnytter en genetisk kodet biosensor varianten, som besitter en cAMP-bindende domene smeltet sammen til en mutant form av luciferase. Når leiren binder fører conformational endringen til aktivering av luciferase, luminescence som kan deretter måles med chemiluminescence leseren29,30. Sanntid leiren teknologien har blitt rapportert egnet for de orphaning av menneskelig odorant reseptorer i HEK293 og NxG 108CC15 cellerass = "xref" > 31,32,33, så vel som Hana3A HEK293T-avledet celler34,35. Gruppen Krautwurst også beskrevet i detalj i sanntid leiren teknologi for å være egnet for toveis store eller sortering tilnærminger32,33.
Her beskriver vi en protokoll for å måle OR aktivisering benytter en sanntid leiren analysen i Hana3A celler. I denne protokollen målt luminescence pre equilibrated levende celler kinetically i 30 min etter behandling med bestemt flyktige forbindelser, som representerer en mer effektiv og nøyaktig analyse av OR aktivisering som er mindre utsatt for gjenstander som oppstår i mobilnettet miljø med lengre tid og lukt-indusert celle toksisitet. Denne sanntids måling gir en storstilt screening ORs og ligander, samt karakteristikk av OR-ligand datamaskinpar rundt. Bruker denne metoden, identifisert vi har OR5AN1 som reseptoren for moskus sammensatte muscone ved å utføre en screening mot 379 menneskelige ORs og deretter bekrefter positive screening resultatet.

<table width="1240">
	<tbody>
		<tr height="25">
			<td height="25" style="width:405px;height:25px;">Amphotericin B</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;">A2942</td>
			<td style="width:623px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">FBS</td>
			<td>Gibco</td>
			<td>10099-141</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">GloSensor cAMP reagent</td>
			<td>Promega</td>
			<td>E1290</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">pGloSensor-20F cAMP plasmid</td>
			<td>Promega</td>
			<td>E1171</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Hana3A cells</td>
			<td></td>
			<td></td>
			<td>available from authors upon request</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">HBSS, w/o calcium chloride and magnesium chloride</td>
			<td>GIBCO</td>
			<td>14175095</td>
			<td style="width:623px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">HEPES</td>
			<td>Hyclone</td>
			<td>SH30237</td>
			<td style="width:623px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Lipofectamine2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">M3R plasmid</td>
			<td></td>
			<td></td>
			<td>cloned into a mammalian expression vector such as pCI</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MEM, w/EBSS and w/L-glutamine</td>
			<td>Hyclone</td>
			<td>SH30024</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Muscone</td>
			<td>Santa Cruz</td>
			<td>sc-200528</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Musk tibetene</td>
			<td>Sigma-Aldrich</td>
			<td>S359165</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">OR plasmids</td>
			<td></td>
			<td></td>
			<td>cloned with a Rho-tag into a mammalian expression vector such as pCI</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">PBS, w/o calcium or magnesium</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Penicillin-streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Plasmid miniprep kit</td>
			<td>Tiangen</td>
			<td>DP103-03</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Puromycin</td>
			<td>Sigma</td>
			<td>P8833</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">RTP1S plasmid</td>
			<td></td>
			<td></td>
			<td>cloned into a mammalian expression vector such as pCI</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Trypsin-EDTA</td>
			<td>Hyclone</td>
			<td>SH30236</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">0.2-mL PCR tube</td>
			<td>Axygen</td>
			<td>PCR-02-C</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">1.5-mL Eppendorf tube</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">15-mL 17 mm x 120 mm conical tube</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">8-well and/or 12-well multichannel pipetman</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">96-well flat-bottomed white cell culture plate</td>
			<td>Greiner</td>
			<td>655098</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">100 mm x 20 mm cell culture dish</td>
			<td>BD Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Class II biological safety cabinet with laminar flow</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell culture incubator, w/ 5% CO2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Centrifuge, with swinging bucket rotor for 15-ml conical tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Infinite F200 plate reader</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Phase-contrast microscope with x10 and x20 objectives</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Spectrophotometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Sterile reagent reservoirs for multichannel distribution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Sterile paper towel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

live-cell measurement of odorant receptor activation using a real-time camp assay

Enorme størrelsene av pattedyr odorant reseptor (OR) presentere vanskeligheter å finne deres beslektet ligander blant mange flyktige kjemikalier. For å effektivt og nøyaktig deorphanize ORs, kombinere vi bruk av en heterologous celle linje å uttrykke pattedyr ORs og en genetisk modifisert biosensor plasmider for å måle leiren produksjon nedstrøms av OR aktivisering i sanntid. Denne analysen kan brukes til skjermen odorants mot ORs og vice versa. Positiv odorant-reseptor interaksjoner fra skjermene kan bekreftes senere ved å teste mot ulike lukt konsentrasjoner, genererer konsentrasjon svar kurver. Her brukte vi denne metoden til å utføre en høy gjennomstrømming screening av en illeluktende sammensatte mot et menneskelig OR bibliotek uttrykt i Hana3A celler og bekreftet at positivt-svare reseptoren beslektet reseptoren for sammensatte interesse. Vi fant denne høy gjennomstrømming oppdagelsen metoden å være effektive og pålitelige vurdere OR aktivisering og våre data gir et eksempel på bruken potensielle OR funksjonelle studier.

Muscone er den viktigste aromatiske komponenten fra naturlige moskus. Nyere studier identifisert OR5AN1 som en menneskelig reseptor for muscone og andre macrocyclic musk forbindelser basert på homologi til musen eller, MOR215-1, Klon fra muscone svarer glomeruli i oppfører seg mus37,38. Screening menneskelige OR repertoaret, vår gruppe og Touhara gruppen også identifisert OR5AN1 som en stor reseptor for to macrocyclic musk for...

Watch this Scientific Journal Video about Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay at JoVE.com

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

Karakterisere funksjon odorant reseptorer serverer en uunnværlig del i deorphanization prosessen. Vi beskriver en metode for å måle aktivering av odorant reseptorer i sanntid ved hjelp av en leir analysen.

Live-celle måling av Odorant reseptor aktivisering benytter en sanntid leiren analysen

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

The overall goal of this real-time high-throughput cyclic AMP assay is to screen odorants against odorant receptors and vice versa. Since mice are useful for investigating odorant receptor functioning in that it allows for larger scale screenings of both odorant receptors and the ligands as well as pharmacological cartelizations of a receptor-ligand pairs of interest. The main advantage of this technique is that we can assess odorant-to-receptor activation efficiently and reliably.Heterologous odorant-to-receptor expression system using live cells. Begin by observing Hana3A cells under a phase-contrast microscope to ensure cell viability and to estimate confluence. Then aspirate all medium from the cell culture dish.And wash the cells by adding 10 milliliters of phosphate buffered saline onto the plate, swirling the dish, and aspirating the PBS. Next, add the three milliliters of 0.5%trypsin-EDTA onto the plate. Leave the trypsin on the cells for one minute or until all cells are afloat.Inactivate the trypsin by adding 5 milliliters of MEM with 10%FBS and pipetting up and down to break up chunks of cell mass. After performing a cell count, transfer 2x10 to the 6th cells per 96-well plate to be transfected to a 15 milliliter tube. It is important that you calculate the correct number of cells to be prepared onto 96-well plate to avoid over or undergrowth of cells prior to stimulation.Centrifuge the tubes at 200 times G for five minutes. And then aspirate the supernatant without disturbing the cell pallet. Add six milliliters of medium without antibiotics for each 96-well plate and pipette up and down without creating air bubbles to break up chunks of the cell mass.Transfer the suspended cells into a reservoir. Using a multi-channel pipette, pipette 50 microliters of cells into each well of the 96-well plates. Incubate overnight at 37 degrees Celsius with 5%carbon dioxide.Prior to transfection observe the plated cells under a phase-contrast microscope to ensure a proper confluence of approximately 30 to 50%per well and then return to the incubator. Follow the row tagged odorant-to-receptor construct, the accessory factor constructs, and the cyclic AMP biosensor variant construct. Then prepare a plasma transfection mixture in 500 microliters of MEM for each 96-well plate.Next, prepare a transfection mixture containing 18 microliters of lipid-mediated transfection reagent in 500 microliters of MEM. Mix the transfection mixture with the plasma mixture by pipetting up and down. Then incubate at room temperature for 15 minutes.After the incubation, stop the reaction by adding 5 milliliters of MEM with 10%FBS to the transfection mix. Next, spread out a thick layer of sterile paper towels in the cell culture hood. Take a 96-well plate with cells from the incubator.Gently and repeatedly, tap the plate upside down on the pile of paper towels so that the medium is completely absorbed by the paper towel. Transfer 50 microliters of the combined transfection mixture to each well of the 96-well plate and incubate overnight for 18 to 24 hours at 37 degrees Celsius with 5%carbon dioxide. You can manage the transfection and the stimulation schedule should persist on instrument for more than one 96-well plate is tested in one experiment so that all plates are mirrored after same transfection and distillers exposure interval.Before stimulation, observe the transfected cells under a phase-contrast microscope to ensure a proper confluence of 50 to 80%per well before returning to the incubator. Then prepare stimulation medium by adding 10 millimolar heaps and five millimolar glucose to Hanks'balanced salt solution. Thaw 55 microliters alloquats of real-time cyclic AMP assay substrate reagent alloquats on ice.Prepare 2%equal abration solution by mixing 55 microliters of the substrate reagent and 2, 750 microliters of stimulation medium. After spreading out a thick layer of paper towels on the bench, gently and repeatedly tap the plate upside down on the paper towels so that the transfection medium is absorbed by the paper towels. Wash the cells by pipetting 50 microliters of stimulation medium to each well.Gently and repeatedly tap out the stimulation medium from the 96-well plate. Pipette 25 microliters of 2%equillibration solution into each well and incubate at room temperature in the dark for two hours. Prior to the end of the incubation time, dilute thawed odorant stock solutions to working concentration and stimulation medium.Before odorant addition, use a chemiluminescence plate reader to measure the basal luminescence level of the plate. Quickly remove the plate from the plate reader and add 25 microliters of the odorant dilutions to each well. Then immediately start continuous luminescence measurement of all wells for 20 cycles within 30 minutes.379 unique human odorant receptors were screened against 30 micromolar muscone using the real-time cyclic AMP assay. Colored blocks along the X axis indicate different human odorant-receptor families. The Y axis represents normalized luminescence 30 minutes after odorant addition when the response reached the maximum for the positive control.The response of OR5AN1 is indicated. This figure shows real-time measurements of OR5AN1 activation by different concentrations of muscone and the result from a no-odor negative control. The arrow indicates the time point of oderant addition.Here, a concentration response curve of OR5AN1 against muscone 30 minutes after oderant addition is shown. The technique described here takes a total of three days starting with a stock plate of culture tiers on the first day. Once mastered, the process on the last day can be completed in three hours for each 96-well plate if it is performed properly.After watching this video, you should have a good understanding of how to to manage the activation of oderant receptors using a cyclic AMP assay to harness the results.

Research

JoVE Journal

Neuroscience

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 ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Samtidig Electroencephalography, Real-time Måling av laktat Konsentrasjon og Optogenetic Manipulering av neuronal aktivitet i Rodent Cerebral Cortex

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

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

Høy gjennomstrømming Analyse av Pattedyr Olfactory reseptorer: Måling av Receptor Aktivering via Luciferase aktivitet

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

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Sanntidselektro: Bruk lukket sløyfe Protokoller å probe Nevronale Dynamics og Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

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

Perforert Patch-clamp Opptak av Mouse olfactory sensoriske nevroner i Intakt neuroepithelium: Funksjonell analyse av nerveceller uttrykker en Identifiserte odoranten Receptor

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

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

En protokoll for administrasjon av Real-Time fMRI Neurofeedback opplæring

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Analyse for å måle nukleocytoplasmisk transport i sanntid innen Motor Neuron-lignende NSC-34-celler

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Lokalisering av luktningsmiddelreceptorgener i Locust Antenna av RNA<em&gt; I situ</em&gt; Hybridisering

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

SwarmSight: Real-time Tracking of Insect Antenna Movements and Proboscis Extension Reflex Using a Common Preparation and Conventional Hardware

SwarmSight: Sanntidssporing av insekt antenne bevegelser og Snabel forlengelsen Reflex bruker vanlige forberedelse og tradisjonell isenkram

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

En høyt innhold analysen for overvåking AMPA reseptor menneskehandel

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...