Questo lavoro è stato sostenuto da una sovvenzione da parte del National Cancer Institute, CA131967.

1. Incorporazione di BrdU (Etichettatura Terminal) <ol><li> Cellule piastra per circa il 70% di confluenza di 150 mm in coltura tissutale piatto 24 ore prima dell&#39;aggiunta di BrdU. </li><li> Sostituire il supporto con nuovi mezzi completi contenenti 20 mg / ml di BrdU (Sigma) ad intervalli di tempo appropriati prima del raccolto. La lunghezza del tempo cellule vengono coltivate in mezzi con BrdU varierà con il tipo di cella e specie, tipicamente la fase G2 dura tra 2 e 5 ore (Figura 1). </li></ol> 2. Harvest Cromosoma di colture monostrato Celle <ol><li> Rimuovere terreni di coltura dalla piastra e raccogliere 10 ml in una provetta da centrifuga 15 ml. Gettare materiale rimanente. </li><li> Risciacquare le cellule una volta con 10 ml Versine [o HBSS (Sigma)]. </li><li> Aggiungere 5 ml di tripsina 0,25% in Versine (o HBSS) alla piastra. Incubare a temperatura ambiente fino a quando le cellule vengono staccate dalla piastra. </li><li> Aggiungi sospensione tripsinizzate cella a tubo contenente i 10 ml di mezzi di comunicazione a partire dal punto 2.1. &lt;/ Li&gt; <li> Centrifugare a 400 xg per 10 minuti per ottenere il pellet. Aspirare il surnatante, lasciando 0,5 ml di supporti più il pellet di cellule. </li><li> Risospendere le cellule a fondo pipettando con una pipetta Pasteur. </li><li> Aggiungere 3 gocce di soluzione ipotonica (0,075 M KCl (Sigma) riscaldato a 37 ° C); miscelare la sospensione cellulare con una pipetta Pasteur. Aggiungere 0,5 ml di soluzione ipotonica e miscelare di nuovo con una pipetta Pasteur. Portare il volume di soluzione ipotonica a 5 ml e mescolare nuovamente. Incubare a 37 ° C per 20-45 minuti, a seconda del tipo di cellula. </li><li> Centrifugare a 400 xg per 10 min. Aspirare il surnatante, lasciando 0,5 ml di supporti più il pellet di cellule. </li><li> Risospendere il pellet di cellule delicatamente muovendo il tubo. Le cellule osmoticamente gonfie sarà fragile, a questo punto, per cui occorre prestare attenzione a non disturbare le membrane citoplasmatiche. </li><li> Aggiungere 3 gocce di fissativo di Carnoy (1:3 acido acetico glaciale: Metanolo); mescolare delicatamente con una pipetta Pasteur pipette. Aggiungere 0,5 ml di fissativo e mescolare pipettando gentilmente con una pipetta Pasteur. Portare il volume di fissativo a 5 ml e mescolare ancora pipettando gentilmente con una pipetta Pasteur. Cellule fisse possono essere conservate al buio a -20 ° C per diversi mesi. </li><li> Centrifugare e aspirare, come al punto 2.8. </li><li> Risospendere il pellet cellulare in fissativo fresco; stimare il volume del pellet e aggiungere ~ 10 volte il volume del fissativo di Carnoy. </li><li> Aggiungere la sospensione cellulare e goccia a goccia sul bagnato, gelide vetrini da microscopio; tenere il vetrino a un angolo di ~ 45 ° e consentire la sospensione cellulare a scorrere sulla superficie del vetrino. Appoggiare il vetrino piatto su carta assorbente e inondare il vetrino con fissativo e lasciare asciugare all&#39;aria. Controllare vetrini per la presenza di spread mitotiche utilizzando un microscopio invertito. </li></ol> 3. RNasi trattamento e disidratazione etanolo <ol><li> Posto 200 pl di 10 pg / ml RNasi A (Sigma) in 2x SSC a ciascun vetrino; incubare a 37 &amp; deg; C per 1 ora. </li><li> Lavare i vetrini 3 volte con 2x SSC, 7,0 pH a temperatura ambiente per 3 min ciascuna. </li><li> Disidratare diapositive attraverso un (Sigma) serie EtOH (70%, 90% e 100%) a temperatura ambiente per 3 min ciascuna. </li><li> Far asciugare i vetrini a temperatura ambiente. </li></ol> 4. Preparazione di cocktail di misura per BAC Inoltre cromosomica specifica Centromero Enumerazione Probe (CEP) o per le vernici intero cromosoma ibridazione in situ <ol><li> Per la BAC / CEP ibridazione simultanea, preparare due cocktail sonda separata per la pre-ibridazione. Abbiamo trovato che la pre-ibridazione le sonde BAC e CEP separatamente in diversi risultati stringencies in meno background e intensità di segnale maggiore. Abbiamo anche trovato che Fosmids individuali possono anche essere utilizzati al posto di cloni BAC. La formulazione cocktail descritto di seguito rappresenta i volumi per una singola diapositiva. Se l&#39;elaborazione più diapositive utilizzando le sonde stesse semplicemente aumentare la appropr volumi<html> iately. * BAC DNA della sonda cocktail: 11,5 microlitri tampone di ibridazione (50% formammide (Sigma) / 2x SSC / destrano solfato (Sigma)) 2 microlitri dH 2 O 2,5 microlitri BAC/Cot1 DNA in dH 2 O (vedi sotto per l&#39;etichettatura di protocollo) volume di 15 microlitri totale * CEP sonda cocktail: 7 CEP microlitri tampone di ibridazione (65% Formamide/2x SSC / destrano solfato) 2 microlitri dH 2 O 1 DNA microlitri CEP/Cot1 (acquistato prelabeled con Spectrum Orange/Cy3/Texas Rosso) 10 microlitri di volume totale * Dopo la denaturazione e pre-ibridazione (punto 5.1) mix BAC sonda con CEP sonda ad un rapporto 3:2 25 cocktail sonda pl / diapositive e mescolare. O </li><li> Aliquotare 20 pl / slide di sonda tutto vernice Cromosoma (Cy3/Texas rosso) in una provetta. Procedere alla denaturazione e pre-ibridazione passo. </li></ol>&quot;&gt; 5. Slide e sonda Denaturazione e ibridazione in situ <ol><li> Denaturare sonda cocktail (s) a 75 ° C per 10 min. Pre-ibridizzano a 37 ° C per 30 minuti per consentire la Cot1 DNA di ibridarsi a sequenze ripetitive e prevenire ibridazione successiva alla metafase cromosomi. </li><li> Diapositive denaturate in vaschette Coplin nel 70% Formamide/2x SSC, pH 7,0 a 72 ° C per 3 min. </li><li> Immediatamente disidratare diapositive attraverso una serie EtOH (70%, 90% e 100%) in vaschette Coplin a 4 ° C per 3 min ciascuna. </li><li> Lasciare i vetrini per asciugare a temperatura ambiente. </li><li> Pre-riscaldare vetrini a 45 ° C sul vetrino più caldo per gli ultimi 10 minuti della sonda pre-hybridzation (passo 5.1). Mescolare pre-ibridizzati cocktail sonda se necessario, e aggiungere 25 ul per ogni diapositiva, posizionare con cura un coprioggetto su ogni diapositiva e la guarnizione lungo tutti i bordi con mastice. Incubare per una notte a 37 ° C in una camera umidificata per evitare l&#39;evaporazione. </li></ol><ol><li> Lavare i vetrini in vaschette Coplin 3 volte nel 50% formamide/2x SSC, pH 7,0 a 38-40 ° C per 3 minuti ciascuno. La temperatura dei lavaggi può variare tra sonde. Se c&#39;è ibridazione fondo elevato è possibile aumentare la temperatura dei lavaggi. Viceversa, se il segnale è debole e non c&#39;è sfondo allora si può diminuire la temperatura di questi lavaggi. </li><li> Lavare i vetrini 1 volta in tampone PN a temperatura ambiente per 3 min, e procedere alla fase di rilevamento BrdU. </li></ol> 7. BrdU rilevamento <ol><li> Vetrini con tampone di blocco PNM per 10 min (200 pl / vetrino) a temperatura ambiente al buio. </li><li> Scaricare diapositive attivando bordo su un tovagliolo di carta. Aggiungere 100 ml di anti-BrdU-FITC (50 pg / ml Roche o Millipore) in tampone PNM per 30 min a 37 ° C. </li><li> Lavare i vetrini tre volte in tampone PN a temperatura ambiente per 3 min ciascuna. </li><li> Drenareeccesso di tampone PN sulle diapositive uno alla volta ruotando sul bordo su un tovagliolo di carta. Aggiungere 20 microlitri DAPI / antifade soluzione di montaggio (Invitrogen). Coprire il vetrino con un tovagliolo di carta e premere verso il basso sul vetrino per far fuoriuscire le bolle d&#39;aria e l&#39;eccesso di soluzione di montaggio. Procedere con l&#39;analisi delle immagini. </li></ol> 8. Catturare Immagini e Quantificazione incorporazione BrdU <ol><li> Le immagini vengono acquisite con un microscopio a fluorescenza Olympus BX61 e Olympus CCD, obiettivo 100x, filtro automatico ruote e software Cytovision (Applied Imaging). BrdU viene catturato con un FITC filtro; vernici cromosomiche (Texas Red, Metasystems o Cytocell), BAC (Cy3) e CEP (Vysis Spectrum Orange) sonde sono state acquisite utilizzando un rosso Texas o Cy3 filtro; DAPI viene catturato con un filtro DAPI (vedi le figure 2 e 3). </li><li> Cromosomi individuali di interesse sono identificati con BAC / CEP o cromosoma sonde specifiche vernici (Figure 2B e 3B). Utilizzandoil software Cytovision, ciascun cromosoma di interesse &quot;cut-out&quot; dalla diffusione metafase nel suo complesso, una linea disegnata attraverso il lungo la lunghezza del cromosoma dal braccio corto a braccio lungo (Figure 2C e 3C). Il software Cytovision viene utilizzato per quantificare il profilo di intensità di pixel lungo la lunghezza di ciascun cromosoma. Il software Cytovision calcola l&#39;area occupata dai pixel e quantifica l&#39;intensità dei pixel rappresentate dai segnali BrdU o DAPI di ciascun cromosoma isolato. L&#39;intensità dei pixel media rappresentata da ciascun cromosoma viene quindi moltiplicata per l&#39;area occupata da quei pixel per ottenere il numero totale di pixel (Tabella 1 e Figura 4). Inoltre, questo protocollo permette la visualizzazione delle ultime regioni replicazione dei cromosomi. Di conseguenza, il modello a bande di incorporazione di BrdU permette l&#39;individuazione delle regioni replicazione attiva di cromosomi, e differenze di temporizzazione replica tra chromosome coppie sono visti come differenze in questo pattern di bande (figure 2 e 3). Infine, le differenze nei modelli di legame può essere paragonato a mappe replica noti temporizzazione per ciascun cromosoma 6,7, che consente una stima della differenza temporale tra replica cromosomi omologhi o bracci cromosoma 4. Ad esempio, i due cromosoma 6 è in Figura 3A presentano una differenza in bande schema di&gt; 2 ore rispetto ai tempi di replica normale del cromosoma 6 4. Inoltre, una procedura simile a quella descritta qui è stato usato con successo per lo studio della temporizzazione replica asincrona tra i cromosomi X inattiva ed attiva 8,9. I cromosomi X attivi e inattivi sono stati identificati utilizzando vernici cromosoma X. Le immagini sono state acquisite utilizzando Quips mFISH software (Vysis) e il numero di pixel occupati dai cromosomi X e il numero di pixel occupati da fluorescentemente laBeled BrdU sono stati poi calcolati utilizzando NIH Image ( <a href="http://rsb.info.nih.gov/nihimage" target="_blank">http://rsb.info.nih.gov/nih-image/index.html</a> ). </li></ol> 9. Nick Traduzione di BAC del DNA per l&#39;etichettatura fluorescente (Vysis) <ol><li> Aggiungere la seguente miscela di sonde refrigerate una provetta per microcentrifuga: 70 microlitri (4 mg DNA) in dH 2 O 10 microlitri 0,2 mM dUTP-arancione o verde 20 pl 0,1 mM dTTP 20 pl 10x Nick Traduzione Buffer 40 microlitri NT dNTP (meno dTTP) 40 ml nick traduzione enzima 200 microlitri di volume totale Incubare @ 16 ° CO / N. Fermare la reazione mediante riscaldamento a 70 ° C per 10 min. Raffreddare in ghiaccio per 5 min. </li><li> Precipitare il DNA con etanolo aggiungendo la seguente miscela di sonde: 200 microlitri soluzione della sonda 48 microlitri 3 M NaOAc 160 microlitri Cot1DNA (0,25 mg / ul) 1200 microlitri EtOH 100% </li><li> Conservare a -80 ° C per 10 min a O / N. </li><li> Spin in fredda a 12.000 xg per 20 min. Decantare EtOH. Lavare 1 volta con il 70% EtOH. Spin come prima. Decantare EtOH. Asciugare solo fino evapora EtOH. Risospendere in 40 microlitri dH 2 O per la concentrazione finale di 100 ng / ml di BAC DNA. </li></ol>

<ol>
	<li>Payer, B., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X+chromosome+dosage+compensation:+how+mammals+keep+the+balance.">X chromosome dosage compensation: how mammals keep the balance.</a> Annu. Rev. Genet. 42, 733-772 (2008).</li><li>Breger, K. S., Smith, L., Turker, M. S., Thayer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionizing+radiation+induces+frequent+translocations+with+delayed+replication+and+condensation.">Ionizing radiation induces frequent translocations with delayed replication and condensation.</a> Cancer Research. 64, 8231-8238 (2004).</li><li>Smith, L., Plug, A., Thayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+Replication+Timing+Leads+to+Delayed+Mitotic+Chromosome+Condensation+and+Chromosomal+Instability+of+Chromosome+Translocations.">Delayed Replication Timing Leads to Delayed Mitotic Chromosome Condensation and Chromosomal Instability of Chromosome Translocations.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 13300-13305 (2001).</li><li>Stoffregen, E. P., Donley, N., Stauffer, D., Smith, L., Thayer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+autosomal+locus+that+controls+chromosome-wide+replication+timing+and+mono-allelicexpression.">An autosomal locus that controls chromosome-wide replication timing and mono-allelicexpression.</a> Hum. Mol. Genet. 20, 2366-2378 (2011).</li><li>Breger, K. S., Smith, L., Thayer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+translocations+with+delayed+replication:+evidence+for+cis+control+of+chromosome+replication+timing.">Engineering translocations with delayed replication: evidence for cis control of chromosome replication timing.</a> Hum. Mol. Genet. 14, 2813-2827 (2005).</li><li>Camargo, M., Cervenka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+DNA+replication+of+human+chromosomes.+II.+Replication+map+and+replication+model.">Patterns of DNA replication of human chromosomes. II. Replication map and replication model.</a> Am. J. Hum. Genet. 34, 757-780 (1982).</li><li>Cohen, S. M., Cobb, E. R., Cordeiro-Stone, M., Kaufman, D. G. <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+chromosomal+bands+replicating+early+in+the+S+phase+of+normal+human+fibroblasts.">Identification of chromosomal bands replicating early in the S phase of normal human fibroblasts.</a> Exp. Cell Res. 245 (98), 321-329 (1998).</li><li>Diaz-Perez, 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=The+element(s)+at+the+nontranscribed+Xist+locus+of+the+active+X+chromosome+controls+chromosomal+replication+timing+in+the+mouse.">The element(s) at the nontranscribed Xist locus of the active X chromosome controls chromosomal replication timing in the mouse.</a> Genetics. 171, 663-672 (2005).</li><li>Diaz-Perez, S. V., 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+deletion+at+the+mouse+Xist+gene+exposes+trans-effects+that+alter+the+heterochromatin+of+the+inactive+X+chromosome+and+the+replication+time+and+DNA+stability+of+both+X+chromosomes.">A deletion at the mouse Xist gene exposes trans-effects that alter the heterochromatin of the inactive X chromosome and the replication time and DNA stability of both X chromosomes.</a> Genetics. 174, 1115-1133 (2006).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2415-2420 (2008).</li><li>Schlesinger, S., Selig, S., Bergman, Y., Cedar, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+inactivation+of+rDNA+loci.">Allelic inactivation of rDNA loci.</a> Genes Dev. 23, 2437-2447 (2009).</li><li>Branzei, D., Foiani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+checkpoint+response+to+replication+stress.">The checkpoint response to replication stress.</a> DNA Repair (Amst). 8, 1038-1046 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

La preparazione degli spread cromosoma è un passo fondamentale per il successo di replica-timing saggio descritto qui. L&#39;inclusione di una fase di pretrattamento Colcemid prima del trattamento ipotonico può facilitare la frequenza e la diffusione delle cellule mitotiche. Abbiamo tipicamente esporre le cellule ad colcemdi per 1-3 ore prima del raccolto, e utilizzare Colcemid ad una concentrazione finale di 10 ug / ml. Tuttavia, l&#39;inclusione di una fase di pretrattamento Colcemid può modificare la lunghezza di ...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Anti-BrdU-FITC </td><td> Roche Millipore </td><td> 11202593001 MAB326F </td><td> 50 pg / pl </td></tr><tr><td> Nick Traduzione Kit </td><td> Abbott Molecular (Vysis) </td><td> 07J00-0001 </td><td></td></tr><tr><td> Spectrum Arancione dUTP </td><td> Abbott Molecular (Vysis) </td><td> 02N33-050 </td><td></td></tr><tr><td> CEP </td><td> Abbott Molecular (Vysis) </td><td> Varia </td><td></td></tr><tr><td> LSI / WCP ibridazione tampone </td><td> Abbott Molecular (Vysis) </td><td> 06J67-011 </td><td></td></tr><tr><td> CEP ibridazione tampone </td><td> Abbott Molecular (Vysis) </td><td> 07J36-001 </td><td></td></tr><tr><td> Cromosoma painti </td><td> MetaSystems Gruppo </td><td> D-14NN-050-TR </td><td></td></tr><tr><td> Olympus BX61 microscopio a fluorescenza </td><td> Olimpo </td><td> BX61TRF-1-5 </td><td></td></tr><tr><td> Microscopio di imaging del software di sistema </td><td> Applied Imaging </td><td> Cytovision 3.93.1 </td><td></td></tr><tr><td> Macchina fotografica digitale </td><td> Olimpo </td><td> UCMAD3 </td><td></td></tr><tr><td></td><td></td><td></td><td> RICETTE IN ibridazione in situ Soluzioni Formammide 70% Formamide/2x SSC 35 ml di formammide * (Sigma) 10 ml 10x SSC 5 ml d 2 H 2 0 pH a 7,0 con HCl (Sigma) * E &#39;importante utilizzare formammide che è stato conservato a -20 ° C. Prolungata conservazione a temperatura ambiente genera acido formico e il w pHill essere troppo bassa. 50% Formamide/2x SSC 25 ml formamide (Sigma) 10 ml 10x SSC 15 ml dH 2 O pH a 7,0 con HCl (Sigma) SSC 20x, 4 L 702 g NaCl (Sigma) 358 g Na citrato (Sigma) dH 2 O a volume PN Buffer [0,1 M NaP0 4 0,1% NP_40 (Sigma)] Effettuare una soluzione 0,1 M di fosfato di sodio ogni (Filtro sterilizzare e conservare in 500 ml aliquote). 0,1 M NaH 2 P0 4, 1 L 13,8 g di NaH 2 P0 4 (Sigma): dH 2 O a volume 0,1 M NaH 2 P0 4 1 L </strong&gt; 14,2 g di NaH 2 P0 4 (Sigma) dH 2 O a volume. PN: Regolare il pH di 0,1 M Na 2 HP0 4 a pH 8,0 con 0,1 M NaH 2 P0 4. Filtro sterilizzare e aggiungere 1 ml di NP-40. PNM 50 ml 1.25 g non grassi del latte secco (Sigma) 25 ml tampone PN (ricetta sopra) Mescolare per 15-20 minuti mescolando costantemente. Spin 2 volte a 400 xg per 10 min. Utilizzare il surnatante, e fare attenzione a non disturbare proteine ​​del latte precipitate. </td></tr></tbody></table>

chromosome replicating timing combined with fluorescent in situ hybridization

Replicazione del DNA di mammifero avvia in siti multipli lungo cromosomi in momenti diversi durante la fase S, seguendo un programma di replica temporale. La specifica dei tempi di replica è pensato per essere un processo dinamico regolato da stimoli tessuto-specifiche e di sviluppo che sono sensibili alle modificazioni epigenetiche. Tuttavia, i meccanismi che regolano dove e quando la replicazione del DNA inizia lungo cromosomi rimane poco chiaro. Cromosomi omologhi di solito replicare in modo sincrono, ma ci sono eccezioni a questa regola. Per esempio, in cellule di mammifero femmina uno dei due cromosomi X diventa tardiva replica attraverso un processo noto come inattivazione X 1. Insieme a questo ritardo nella temporizzazione replica, stimato a 2-3 ore, la maggior parte dei geni diventano trascrizionalmente tacere su un cromosoma X. In aggiunta, una discreta azione cis locus, noto come il centro inattivazione X, regola questo processo di inattivazione X, compresi thinduzione e dei tempi di replica ritardata sul cromosoma X inattivo intero. Inoltre, alcuni riarrangiamenti trovano nelle cellule tumorali e cellule esposte a radiazioni ionizzanti visualizzare un significativo ritardo nella temporizzazione replica di&gt; 3 ore che interessa l&#39;intero cromosoma 2,3. Un recente lavoro del nostro laboratorio indicano che l&#39;alterazione di discreta azione cis autosomica risultato loci in un fenotipo estremamente tardiva replica che interessa l&#39;intero cromosoma 4. Ulteriori studi &#39;di ingegneria cromosomica&#39; indicano che alcuni riarrangiamenti cromosomici che interessano molti risultati diversi cromosomi in questa anomala replica-timing fenotipo, suggerendo che tutti i cromosomi di mammifero contengono discrete cis-che agiscono loci che controllano tempistica corretta replicazione dei cromosomi individuali 5. Qui, presentiamo un metodo per l&#39;analisi quantitativa di temporizzazione cromosoma replica combinato con ibridazione in situ fluorescente. Questometodo consente un confronto diretto di temporizzazione replica tra cromosomi omologhi all&#39;interno della stessa cella, ed è stato adattato da 6. Inoltre, questo metodo consente l&#39;identificazione univoca di riarrangiamenti cromosomici che correlano con modifiche nei tempi di replica che interessano l&#39;intero cromosoma. Questo metodo ha dei vantaggi rispetto recentemente sviluppato un elevato throughput di micro-array o sequenza protocolli che non è possibile distinguere tra alleli omologhi presenti sul riarrangiati e poco risistemato cromosomi. Inoltre, poiché il metodo descritto qui valuta singole cellule, si può rilevare variazioni di temporizzazione sulla replicazione cromosomica riarrangiamenti cromosomici che sono presenti in una frazione di cellule in una popolazione.

Un esempio di analisi replica temporizzazione per cromosoma umano 6 è mostrato in Figura 2. Cellule contenenti una delezione del gene ASAR6 4, situato in 6q16.1, sono stati esposti a BrdU per 5 ore, le cellule raccolte per mitotici e trattati per FISH con sonda cromosoma 6 vernice (Vysis) e per incorporazione di BrdU. Notare che vi è una differenza significativa nella configurazione BrdU bande tra i due 6, che è coerente con un ritardo nella temporizzazione replica di&gt; 2 ore per uno dei...

Watch this Scientific Journal Video about Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization at JoVE.com

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Un metodo per l&#39;analisi quantitativa di temporizzazione cromosoma replica è descritto. Il metodo utilizza incorporazione di BrdU in combinazione con fluorescente<em&gt; In situ</em&gt; Ibridazione (FISH) per valutare la tempistica replicazione dei cromosomi di mammifero. Questa tecnica permette il confronto diretto dei cromosomi riarrangiati e un-riarrangiato all&#39;interno della stessa cella.

Cromosoma Timing Replica combinazione con fluorescente<em&gt; In situ</em&gt; Ibridazione

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

chromosome-replicating-timing-combined-with-fluorescent-situ

Research

JoVE Journal

Biology

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

13.9K Views.  Oregon Health & Science University. Un metodo per l'analisi quantitativa di temporizzazione cromosoma replica è descritto. Il metodo utilizza incorporazione di BrdU in combinazione con fluorescente In situ Ibridazione (FISH) per valutare la tempistica replicazione dei cromosomi di mammifero. Questa tecnica permette il confronto diretto dei cromosomi riarrangiati e un-riarrangiato all'interno della stessa cella.

Video: Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Doppia Monte intero ibridazione in situ di embrioni di pollo primi

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Ricerca

Biologia

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebrafish intero monte ad alta risoluzione doppia fluorescente In Situ Ibridazione

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Fluorescenza In situ</emIbridazione> (FISH) Protocollo di sperma umano

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Tutto il monte In Situ</emIbridazione> di E8.5 a E11.5 embrioni di topo

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

Ibridazione in situ per la localizzazione precisa delle Trascrizioni nelle piante

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Rilevamento di RNA virale per fluorescenza<em&gt; In situ</em&gt; Hybridization (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

High-throughput mappatura fisica dei cromosomi con modalità automatizzate<em&gt; In situ</em&gt; Ibridazione

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Analisi di Single-cell trascrizione del gene per l&#39;RNA fluorescente<em&gt; In Situ</em&gt; Ibridazione (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Tutta la Monte RNA fluorescente<em&gt; In situ</em&gt; L&#39;ibridazione di<em&gt; Drosophila</em&gt; Embrioni

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualizzazione e l&#39;analisi di molecole di mRNA mediante fluorescenza<em&gt; In Situ</em&gt; Ibridazione in<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...