Name: the use of mouse splenocytes to assess pathogen-associated molecular pattern influence on clock gene expression
Uploaded: 2018-07-24T15:00:00.000+00:00
Duration: 6 min 50 s
Description: Watch this Scientific Journal Video about The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression at JoVE.com

Questo lavoro è stato supportato da assegni di ricerca di facoltà dall'ufficio il College delle arti e del preside di Scienze presso l'Università di Hartford.

Durante lo studio, trattamento e cura degli animali in osservanza politica National Institutes of Health, erano in conformità con le linee guida istituzionali e sono stati approvati dal comitato di uso e cura animali istituzionale degli animali Università di Hartford.
1. trascinamento degli animali
Nota: Venti settimana-vecchio maschio B6129SF2/J topi sono usati nello studio.
<ol><li>Salire sul treno topi per una 12 ore di luce (luce standard testa bianca) / 12h scuro ciclo per 2 settimane prima dell'esperimento. Nota: Qui tempo zeitgeber (ZT) 0 corrisponde a luci su e ZT12 a luci spente, mantenendo tutti gli altri fattori ambientali (cioè, alimento, acqua e temperatura ambiente) costante 21.</li>
</ol>2. preparazione di strumenti, terreno di coltura e sfida Medium
<ol><li>Autoclave pinze e le forbici per dissezione. Avvolgere le coppie di vetrini da microscopio smerigliato in foglio di alluminio e autoclave.</li>
	<li>Preparare mezzo di coltura con l'aggiunta di siero bovino fetale (FBS) di RPMI 1640 a una concentrazione finale di 10%. Preparare 10 mL di terreno di sfida in provette da 50 mL aggiungendo il seguente PAMPs al terreno di coltura (RPMI con 10% FBS); LPS (5 µ g/mL), ODN1826 (5 µ g/mL), calore-ucciso Listeria monocytogenes (108 HKLM/mL), o un altro PAMP nella sua concentrazione suggeriti.</li>
	<li>Caldo terreno di coltura e sfida medio a 37 ° C in un bagno d'acqua e tenere circa 70 mL di soluzione salina sterile tamponata al fosfato (PBS, pH 7,2) a temperatura ambiente.</li>
	<li>Aggiungere 10 mL di terreno di coltura utilizzando una pipetta da 10 mL e pipetta aiuti per un tubo da 50 mL e posto sul ghiaccio.</li>
	<li>Aggiungere circa 30 mL di etanolo al 70% a un'estremità di Becher e posto di 100ml di forbici e forcipe di dissezione nel bicchiere graduato per evitare la contaminazione microbica.</li>
	<li>Preparare il tampone di lisi per isolamento del RNA aggiungendo 10 µ l di β-mercaptoetanolo (sotto cappa aspirante) per ogni 1 mL di Buffer RLT in una provetta da 50 mL. Fare solo l'importo che sarà necessario (600 µ l per campione, che consiste di circa 1 x 106 cellule). Nota: Il Buffer RLT è un componente del kit di estrazione di RNA che supporta l'associazione di RNA per la membrana di silice.</li>
</ol>3. Splenocyte isolamento e sfida
<ol><li>Eutanasia topi in un momento di particolare zeitgeber tramite narcosi mantenendoli nella loro gabbia originale e aggiungendo CO2 alla gabbia ad una portata di 3 L/min continua fornitura di CO2 per 1 min dopo l'arresto della respirazione.</li>
	<li>Confermare la morte tramite dislocazione cervicale posizionando il pollice e il dito indice su entrambi i lati del collo alla base del cranio. In alternativa, premere una canna alla base del cranio mentre si tira rapidamente (con l'altra mano) la base della coda o le zampe per causare la separazione delle vertebre cervicali dal cranio22.</li>
	<li>Spruzzare il tronco del mouse con etanolo al 70% e asciugare con un tovagliolo di carta. Posizionare il mouse sulla sua schiena e leggermente inclinata sul fianco destro. Tagliare via la pelliccia, mediante dissezione Forbici, lato sinistro del mouse, circa a metà strada tra la parte anteriore e le zampe posteriori.</li>
	<li>Usando il forcipe, afferrare il peritoneo e incidete con attenzione per non danneggiare la milza. Rimuovere la milza con le pinzette e posto in una provetta sterile da 50 mL contenente circa 10 mL di terreno di coltura su ghiaccio. Ripetere per gli altri animali. Nota: La milza è il colore di un fagiolo, ed è più lungo e più piatta rispetto il rene.</li>
	<li>
Trasferire una milza con 2 mL di terreno di coltura per un piccolo piatto di Petri sterile.
	<ol>
<li>Omogeneizzare la milza di macinandolo fra la parte glassata di due diapositive smerigliati sterile. Se possibile, mantenere il problema e le cellule nel medium durante il processo di omogeneizzazione.</li>
		<li>Una volta accuratamente omogeneizzato, pipettare 2 mL di terreno di coltura contenente gli splenocytes attraverso un colino di cella di 40 µm in nylon in una provetta da 50 mL.</li>
		<li>Ripetere i passaggi sopra riportati per le milze rimanenti utilizzando nuove coppie di diapositive smerigliati, provette da 50 mL con terreno di coltura e filtri di cella.</li>
	</ol>
</li>
	<li>Aggiungere 8 mL di terreno di coltura fredda a ciascuna delle provette 50ml contenente gli splenocytes per un volume totale di 10 mL/tubo. Determinare il numero di cellule per millilitro utilizzando un emocitometro.</li>
	<li>Aggiungere circa 1 x 106 cellule/pozzetto in piastre di coltura 6 pozzetti. Aggiungere celle per il numero di pozzetti che corrispondono al numero di diversi PAMPs viene utilizzato per l'esperimento e includere un controllo bene. Aggiungere 3 mL di terreno di coltura o 3 mL di terreno di sfida a tutti i pozzetti.</li>
	<li>Incubare le piastre a 37 ° C in 5% CO2 per 3 h.</li>
	<li>Raschiare le cellule dal fondo del bene usando un puntale da 1.000 µ l collegato a una micropipetta P1000. Trasferire il supporto che contiene le celle utilizzando la stessa punta di dispensare 1.000 µ l in una provetta da 15 mL.</li>
	<li>Agglomerare le cellule tramite centrifugazione a 167 x g per 5 min a temperatura ambiente. Eliminare il surnatante e lavare il pellet cellulare con 5 mL di PBS.</li>
	<li>Agglomerare le cellule una seconda volta a 167 x g per 5 min, rimuovere il supernatante e aggiungere 600 µ l di tampone di lisi per il pellet cellulare per lisare le cellule. Procedere all'isolamento del RNA.</li>
</ol>4. isolamento RNA e la sintesi del cDNA
<ol><li>Isolare il RNA da splenocytes utilizzando il kit di estrazione di RNA secondo le istruzioni del produttore ed eseguire la digestione di DNA in colonna 'optional'.</li>
	<li>Prima sintesi del cDNA, calcolare la concentrazione di RNA mediante uno spettrofotometro di microvolume per verificare che la concentrazione sia all'interno della gamma ottima di RNA (fino a 2 µ g) di kit di sintesi di cDNA.</li>
	<li>
Synthesize cDNA per ciascuno dei campioni utilizzando il kit di trascrizione inversa secondo le istruzioni del produttore. Utilizzare 10 µ l di RNA per ognuno dei campioni in un volume di reazione totale di 20 µ l. Al completamento della trascrizione inversa (thermocycler esegue) aggiungere 20 μL H2O ad ogni reazione.
</li>
	<li>Costruire la curva standard utilizzando una micropipetta P10 o P20 per piscina mRNA da alcuni campioni di controllo (ad esempio, 5 µ l da 2 campioni per un totale di 10 µ l) in una provetta 0,5 mL per preparare il cDNA.<ol>
<li>Aggiungere 10 µ l 2 x mix master della trascrittasi inversa.</li>
		<li>Al completamento della trascrizione inversa, aggiungere 10 µ l di H2O al tubo di reazione, che servirà come la concentrazione iniziale ("1") della serie di diluizione per la curva standard.</li>
		<li>Eseguire una serie di diluizioni 10 volte (da 1 a 10-4) con l'aggiunta di 45 µ l di acqua utilizzando una micropipetta P100 o P200 nelle quattro provette 0,5 mL indicate 10-1, 10-2, 10-3e 10-4.<ol>
<li>Aggiungere 5 µ l di concentrazione iniziale "1" nel primo tubo (10-1) usando una micropipetta P20, mix pipettando su e giù parecchie volte, quindi trasferire 5 µ l dalla provetta 10-1 nel tubo indicato 10-2 e mix.</li>
			<li>Trasferire 5 µ l utilizzando una micropipetta P20 dal 10-2 tubo nel tubo indicato 10-3 e mescolare. Trasferire 5 µ l usando una micropipetta P20 dal 10-3 tubo nel tubo indicato 10-4 e mescolare.</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. quantitativo reazione a catena della polimerasi (qPCR)
<ol><li>Determinare la quantificazione relativa dei livelli di mRNA di qPCR. All'interno l'allestimento sperimentale, selezionare "Quantificazione – relativa curva Standard" e "TaqMan" chimica. Cambiare il volume di reazione a 10 µ l. Immettere le informazioni rilevanti nel layout del piatto (ad es., gene bersaglio, curva standard, giornalista, ecc.).</li>
	<li>Preparare la reazione, in modo che esso contiene 0,5 µ l del dosaggio primer/sonda, 5 µ l di mix master di espressione genica, 2 µ l di H2O e 2,5 µ l di cDNA (10 – 100 ng).<ol>
<li>Preparare un mix master moltiplicando ciascuno dei reagenti precedenti (tranne il cDNA) per il numero di reazioni, assicurandosi di includere quelli dalla curva standard, controlli negativi, esibendosi ogni reazione in duplicato e 2 reazioni supplementare che rappresenteranno per variazione di pipettaggio.</li>
		<li>Dispensare 7,5 µ l del mix master preparati in pre-determinati pozzetti in una piastra di reazione a 96 pozzetti con una micropipetta o una pipetta multicanale. Dispensare 2,5 µ l di cDNA nel pozzo appropriato per le incognite e gli standard e 2,5 µ l di H2O nei pozzetti di controllo negativo.</li>
		<li>Sono saggi di primer/sonda per vari geni orologio molecolare e includono un'analisi per un controllo endogeno (ad es., β-actina).</li>
	</ol>
</li>
	<li>Al fine di determinare i valori relativi di espressione, calcolare la quantità relativa media per ogni replica, quindi, per ogni campione, dividere la quantità relativa media per il gene dell'obiettivo di quantità relativa media per β-actina.</li>
</ol>6. elaborazione statistica
<ol><li>Utilizzando software di analisi statistica, inserire i dati nel programma sotto l'opzione di statistiche di colonna. Selezionare un one-way ANOVA con il test post-hoc di Dunnett per valutare le differenze tra i valori medi di espressione del gene dell'orologio dopo la sfida PAMP contro il controllo.</li>
</ol>

<ol>
	<li>Bell-Pedersen, D., 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=Circadian+rhythms+from+multiple+oscillators:+lessons+from+diverse+organisms.">Circadian rhythms from multiple oscillators: lessons from diverse organisms.</a> Nat. Rev. Genet. 6, 544-556 (2005).</li><li>Mohawk, J. A., Green, C. B., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+and+Peripheral+Circadian+Clocks+in+Mammals.">Central and Peripheral Circadian Clocks in Mammals.</a> Annu. Rev. Neurosci. 35, 445-462 (2012).</li><li>Balsalobre, A., Damiola, F., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+serum+shock+induces+circadian+gene+expression+in+mammalian+tissue+culture+cells.">A serum shock induces circadian gene expression in mammalian tissue culture cells.</a> Cell. 93, 929-937 (1998).</li><li>Yoo, S. -. H., 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=PERIOD2::LUCIFERASE+real-time+reporting+of+circadian+dynamics+reveals+persistent+circadian+oscillations+in+mouse+peripheral+tissues.">PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.</a> Proc. Natl. Acad. Sci. U. S. A. 101, 5339-5346 (2004).</li><li>Yamazaki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+Central+and+Peripheral+Circadian+Oscillators+in+Transgenic+Rats.">Resetting Central and Peripheral Circadian Oscillators in Transgenic Rats.</a> Science. 288, 682-685 (2000).</li><li>Lowrey, P. L., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+circadian+rhythms+in+mammalian+model+organisms.">Genetics of circadian rhythms in mammalian model organisms.</a> Adv. Genet. 74, 175-230 (2011).</li><li>Curtis, A. M., Bellet, M. M., Sassone-Corsi, P., O'Neill, L. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+Clock+Proteins+and+Immunity.">Circadian Clock Proteins and Immunity.</a> Immunity. 40, 178-186 (2014).</li><li>Keller, 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=A+circadian+clock+in+macrophages+controls+inflammatory+immune+responses.">A circadian clock in macrophages controls inflammatory immune responses.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 21407-21412 (2009).</li><li>Silver, A. C., Arjona, A., Hughes, M. E., Nitabach, M. N., Fikrig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+expression+of+clock+genes+in+mouse+macrophages,+dendritic+cells,+and+B+cells.">Circadian expression of clock genes in mouse macrophages, dendritic cells, and B cells.</a> Brain. Behav. Immun. 26, 407-413 (2012).</li><li>Kawai, T., Akira, 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+role+of+pattern-recognition+receptors+in+innate+immunity+update+on+Toll-like+receptors.">The role of pattern-recognition receptors in innate immunity update on Toll-like receptors.</a> Nat. Publ. Gr. 11, 373-384 (2010).</li><li>Marpeg&#225;n, L., Bekinschtein, T. A., Costas, M. A., Golombek, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+responses+to+endotoxin+treatment+in+mice.">Circadian responses to endotoxin treatment in mice.</a> J. Neuroimmunol. 160, 102-109 (2005).</li><li>Okada, 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=Injection+of+LPS+Causes+Transient+Suppression+of+Biological+Clock+Genes+in+Rats.">Injection of LPS Causes Transient Suppression of Biological Clock Genes in Rats.</a> J. Surg. Res. 145, 5-12 (2008).</li><li>Haimovich, 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=In+vivo+endotoxin+synchronizes+and+suppresses+clock+gene+expression+in+human+peripheral+blood+leukocytes.">In vivo endotoxin synchronizes and suppresses clock gene expression in human peripheral blood leukocytes.</a> Crit. Care Med. 38, 751-758 (2010).</li><li>Curtis, A. 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=Circadian+control+of+innate+immunity+in+macrophages+by+miR-155+targeting+Bmal1.">Circadian control of innate immunity in macrophages by miR-155 targeting Bmal1.</a> Proc. Natl. Acad. Sci. U. S. A. 112, 7231-7236 (2015).</li><li>Silver, A. C., Arjona, A., Walker, W. E., Fikrig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Circadian+Clock+Controls+Toll-like+Receptor+9-Mediated+Innate+and+Adaptive+Immunity.">The Circadian Clock Controls Toll-like Receptor 9-Mediated Innate and Adaptive Immunity.</a> Immunity. , (2012).</li><li>Gibbs, J. 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=The+nuclear+receptor+REV-ERB+&#945;+mediates+circadian+regulation+of+innate+immunity+through+selective+regulation+of+inflammatory+cytokines.">The nuclear receptor REV-ERB &#945; mediates circadian regulation of innate immunity through selective regulation of inflammatory cytokines.</a> PNAS. 109, 582-587 (2012).</li><li>Zee, P. C., Attarian, H., Videnovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+abnormalities.">Circadian rhythm abnormalities.</a> Contin. Lifelong Learn. Neurol. 19, 132-147 (2013).</li><li>Bovbjerg, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+disruption+and+cancer:+sleep+and+immune+regulation.">Circadian disruption and cancer: sleep and immune regulation.</a> Brain. Behav. Immun. 17, 48-50 (2003).</li><li>Fu, L., Lee, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+clock:+pacemaker+and+tumour+suppressor.">The circadian clock: pacemaker and tumour suppressor.</a> Nat. Rev. Cancer. 3, 350-361 (2003).</li><li>Germain, A., Kupfer, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CIRCADIAN+RHYTHM+DISTURBANCES+IN+DEPRESSION.">CIRCADIAN RHYTHM DISTURBANCES IN DEPRESSION.</a> Hum. Psychopharmacol. 23, 571-585 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Mouse+Care+and+Maintenance.">Basic Mouse Care and Maintenance.</a> JoVE. , (2018).</li><li>Leary, 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=AVMA+Guidelines+for+the+Euthanasia+of+Animals+:+2013+Edition.">AVMA Guidelines for the Euthanasia of Animals : 2013 Edition.</a> AVMA. , (2013).</li><li>Silver, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen-associated+molecular+patterns+alter+molecular+clock+gene+expression+in+mouse+splenocytes.">Pathogen-associated molecular patterns alter molecular clock gene expression in mouse splenocytes.</a> PLoS One. , 12-15 (2017).</li><li>Silver, A. 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=Daily+oscillations+in+expression+and+responsiveness+of+Toll-like+receptors+in+splenic+immune+cells.">Daily oscillations in expression and responsiveness of Toll-like receptors in splenic immune cells.</a> Heliyon. , 00579 (2018).</li></ol>

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All'interno di questo protocollo, uno spettrofotometro di microvolume può essere utilizzato per quantificare e valutare la purezza del RNA utilizzato nel determinare l'espressione genica. Acidi nucleici assorbono UV luce a 260 nm, proteine in genere assorbono la luce a 280 nm, mentre altri potenziali contaminanti utilizzati durante una procedura di estrazione di RNA (ad es., fenolo) sono rilevabili a 230 nm. Pertanto, valutando l'assorbanza (A) rapporto a 260/280 nm (RNA alla proteina) e 260/230 nm (RNA a prote...

Il master clock nei mammiferi, che orchestra oscillazioni 24h per quasi tutti gli aspetti della fisiologia e del comportamento, si trova all'interno del nucleo soprachiasmatico (SCN) dell'ipotalamo1,2. Oltre alla regolazione di processi biologici a livello organico, il master clock sincronizza anche periferici Orologi Cellulari in tutto il corpo3,4,5. Mentre il macchinario di orologio molecolare è costituito da almeno tre cicli di feedback trascrizionale traslazionale ad incastro, il nucleo è composto il periodo (Per1-3), crittocromo (Cry1-2), Bmal1, e orologio geni6,7. Oltre a mantenere la tempistica accurata dell'orologio molecolare core, anche alcuni prodotti del gene orologio accessorie (ad es., Rev-erbα e Dbp) regolano l'espressione dei geni dell'orologio non, cioè, orologio geni controllati (GCC)6 , 7.
Orologi molecolari funzionali sono state descritte in vari tessuti immuni (ad es., milza e linfonodi)8 e cellule (ad es., B cellule, cellule dendritiche, macrofagi)8,9. Queste cellule rilevare e rispondere ai pattern molecolari associati (PAMPs), conservati componenti microbici, attraverso recettori di riconoscimento immune innata come recettori Toll-like (TLR)10. Ad oggi, sono stati descritti 13 funzionale TLR, che riconoscono costituenti microbici quali componenti della parete cellulare batterica, proteina fustigatrice e acidi nucleici microbici10. La PAMP, lipopolisaccaride (LPS), un componente della parete cellulare dei batteri gram-negativi, riconosciuto da TLR4, è stato indicato per alterare i ritmi circadiani a livello sia organismica e molecolare. Ad esempio, in vivo sfida di LPS indotto da ritardi di fase fotica-come misurata dall'attività in topi11 e portato a orologio ridotta espressione genica nel SCN e nel fegato come determinato mediante ibridazione in situ e PCR quantitativa, rispettivamente, in ratti12. Dopo una sfida in vivo con LPS, analisi del sangue periferico umano leucociti13 e tessuto adiposo sottocutaneo14 ha rivelato alterata espressione di diversi geni dell'orologio come misurato tramite qPCR. Infine, ex vivo sfide LPS di macrofagi umani e macrofagi peritoneali del topo, inoltre ha condotto ad alterata espressione dell'orologio come misurato da qPCR14.
Qui, descriviamo un protocollo per valutare l'influenza dei LPS PAMPs, ODN1826 (oligonucleotidi sintetici contenenti unmethylated CpG motivi) e calore-ucciso Listeria monocytogenes (HKLM), riconosciuto da TLR4, TLR9 e TLR2, rispettivamente, il espressione genica di orologio molecolare negli splenocytes del topo. Il protocollo comprende lo splenectomy mouse, isolamento splenocyte e sfida, RNA estrazione, sintesi del cDNA e qPCR per valutare l'espressione di diversi geni dell'orologio. Questo protocollo permette l'acquisizione tempestiva di un gran numero di cellule del sistema immunitario con molto piccolo animale o manipolazione cellulare, che può quindi essere contestato ex vivo con vari PAMPs. L'orologio molecolare è stato indicato per modulare i vari aspetti della risposta immunitaria8,15,16, pertanto, rottura dell'orologio molecolare molto probabilmente potrebbe compromettere la corretta variazione dipendente dal tempo della risposta immunitaria. Inoltre, poiché le interruzioni dei ritmi circadiani possono portare a gravi patologie17,18,19,20, può essere di interesse per i ricercatori di sfidare gli splenocytes con una vasta gamma di molecole e valutare la loro influenza sull'orologio.

<table><tbody><tr><td>Frosted slides</td><td>Fisher</td><td>12-550-343</td><td></td></tr><tr><td>Cell strainers</td><td>Fisher</td><td>22363547</td><td></td></tr><tr><td>Lipopolysaccharide&amp;nbsp;</td><td>InvivoGen</td><td>ltrl-eklps</td><td></td></tr><tr><td>ODN1826</td><td>InvivoGen</td><td>Tlrl-1826-1</td><td></td></tr><tr><td>HKLM</td><td>InvivoGen</td><td>Tlrl-hklm</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>20012-043</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104 or 74106</td><td></td></tr><tr><td>RNase-Free DNase Set</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>6-well cell culture plate</td><td>Denville</td><td>T1006</td><td></td></tr><tr><td>50 mL tubes</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>15 mL tubes</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit</td><td>ThermoFisher</td><td>4368814</td><td></td></tr><tr><td>TaqMan Gene Expression Assays b-actin</td><td>ThermoFisher</td><td>Mm00607939_s1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Per2</td><td>ThermoFisher</td><td>Mm00478113_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Rev-erba</td><td>ThermoFisher</td><td>Mm00520708_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Bmal1</td><td>ThermoFisher</td><td>Mm00500226_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Dbp</td><td>ThermoFisher</td><td>Mm00497539_m1</td><td></td></tr><tr><td>qPCR machine StepOnePlus</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>ThermoFisher</td><td>4369016</td><td></td></tr><tr><td>MicroAmp Fast 96-well reaction plate (0.1 mL)</td><td>ThermoFisher</td><td>4346907</td><td></td></tr><tr><td>Statistical Analysis Software</td><td>Prism 7.0a</td><td></td><td></td></tr></tbody></table>

the use of mouse splenocytes to assess pathogen-associated molecular pattern influence on clock gene expression

Dal comportamento di espressione genica, i ritmi circadiani regolano quasi tutti gli aspetti della fisiologia. Qui, presentiamo una metodologia per sfidare splenocytes del topo con il pattern molecolari associati (PAMPs) lipopolysaccharide (LPS), il ODN1826 e il calore-ucciso Listeria monocytogenes ed esaminare il loro effetto su molecolare circadiano orologio. In precedenza, studi si sono concentrati sull'esame l'influenza di LPS sull'orologio molecolare usando una varietà di approcci in vivo ed ex vivo da un assortimento di modelli (ad es., topo, ratto e umani). Questo protocollo descrive l'isolamento e la sfida di splenocytes, nonché la metodologia per valutare la post-sfida espressione di gene di orologio tramite PCR quantitativa. Questo approccio può essere utilizzato per valutare non solo l'influenza di componenti microbiche sull'orologio molecolare ma anche altre molecole che può alterare l'espressione dell'orologio. Questo approccio potrebbe essere utilizzato per prendere in giro a pezzi il meccanismo molecolare di come l'interazione di PAMP-Toll-like receptor influenza espressione dell'orologio.

Topi sono stati sacrificati a ZT13, splenocytes sono stati isolati e contestato ex vivo con il LPS PAMPs, ODN1826 o HKLM. Dopo 3 h, RNA è stato isolato e qPCR è stato utilizzato per valutare i livelli di espressione relativa dei geni orologio molecolare orologio, Per2, Dbp e Rev-erbα rispetto alle cellule di controllo incontrastato. Dopo la sfida PAMP, i livelli di espressione di Clock non erano significativamente differenti che l'e...

Watch this Scientific Journal Video about The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression at JoVE.com

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Questo protocollo descrive una tecnica usando splenocytes del topo per scoprire pattern molecolari associati che alterano l'espressione genica di orologio molecolare.

L'uso del Mouse Splenocytes per valutare Pathogen-associated Molecular Pattern influenza sull'espressione del Gene dell'orologio

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse ...

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Universidad San Sebastian

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JoVE Journal

Genetics

7.4K Views.  University of Hartford. Questo protocollo descrive una tecnica usando splenocytes del topo per scoprire pattern molecolari associati che alterano l'espressione genica di orologio molecolare.

Video: The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Misura parallela di circadiano Clock espressione genica e la secrezione dell&#39;ormone in colture cellulari primarie umane

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

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Genetica

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

In Vitro Dosaggio di bioluminescenza per caratterizzare il ritmo circadiano in cellule epiteliali mammarie

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

Temporale ordinazione di espressione Dynamic Data di espressione spaziale dettagliata Mappe

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Biologia dello sviluppo

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

A seguito in tempo reale l&#39;impatto dei fattori di virulenza pneumococco in un acuto mouse Polmonite modello tramite batteri bioluminescenti

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

Immunologia e infezioni

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Un modello di topo per patogeni indotta infiammazione cronica a siti locali e sistemici

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal allophycocyanin (APC) fluorescence intensity, are conjugated to antibodies specific to a particular analyte. Next, a selected panel of defined capture bead sets is incubated with a biological sample containing target analytes specific to the capture antibodies. A biotinylated detection antibody cocktail is added, which leads to the formation of capture bead-analyte-detection antibody sandwiches.
Finally, streptavidin-phycoerythrin (SA-PE) is added, which binds to biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE fluorescent signal of analyte-specific beads regions is quantified using flow cytometry, and the concentrations of particular analytes are determined using data analysis software and the standard curve generated in the assay.
In this experiment, we use a mouse T helper cytokine panel to simultaneously quantify the concentration of 13 separate cytokine targets in tissue culture supernatants collected from mouse splenocytes cultured under various stimulatory conditions.

This protocol describes the quantification of multiple cytokine targets simultaneously in tissue culture supernatants collected from stimulated mouse splenocytes using multiplex bead based immunoassay platform and a flow cytometer.

Multiplex Cytokine Profilatura degli Splenocytes stimolati Mouse utilizzando una piattaforma basata su Cytometric Bead Immunoassay

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Utilizzando un agente patogeno batterico alla sonda per le risposte cellulari e organismica-livello Host

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

In Vivo Monitoraggio dell'espressione genica orologio circadiano nel nucleo soprachiasmatico Mouse mediante fluorescenza reporter

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

Neuroscienze

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

Imaging di In Situ Interferon Gamma Production nella Spleen del topo dopo l'infezione da Listeria monocytogenes

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

L'uso del Mouse Splenocytes per valutare Pathogen-associated Molecular Pattern influenza sull'espressione del Gene dell'orologio

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Misura parallela di circadiano Clock espressione genica e la secrezione dell'ormone in colture cellulari primarie umane