Mutant worm strains were kindly provided by the Caenorhabditis Genetics Center. This research was supported by a grant of the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (grant number: HI14C1277).

1. Sonde Etichettatura con digossigenina-dUTP mediante PCR <ol><li> Eseguire etichettatura PCR con 10x dNTP mix contenente digossigenina-dUTP come descritto in precedenza 13. </li><li> Purificare prodotto di PCR con la purificazione spin-colonna secondo le istruzioni del produttore. <ol><li> Se la sonda è più breve di 200 bp, rimuovere libera digossigenina-dUTP con cromatografia spin-colonna dalla miscela di reazione piuttosto che depurazione spin-colonna. </li></ol></li></ol> 2. Preparare diapositive polilisina rivestiti Nota: L&#39;intera procedura richiede circa 2 ore. La maggior parte dei passaggi sono eseguiti a temperatura ambiente tranne per la fase di essiccazione. <ol><li> Pulizia delle diapositive <ol><li> Porre i vetrini in un contenitore di plastica e lavare rapidamente i vetrini con acqua distillata (DW). Rimuovere l&#39;acqua e riempire il contenitore con DW contenente detergente per vetri 1%. </li><li> Agitare i vetrini per 15 min a 50 rpm a temperatura ambiente (RT). Lavare le diapositive con DW 3volte per 5 minuti ciascuno a temperatura ambiente. </li><li> Lavare i vetrini con il 70% di etanolo per 15 minuti con agitazione. Scartare 70% di etanolo e posizionare i vetrini su un blocco secca 65 ° C e asciugare per 15 min. </li></ol></li><li> Rivestimento polilisina di diapositive di vetro multi-well Nota: il rivestimento polilisina di vetro scorrevole è un passo importante, in quanto fornisce l&#39;adesione del campione durante la procedura di colorazione. Scarsamente scorrevole rivestito comporterà la perdita di campione. <ol><li> Diluire la soluzione polilisina magazzino al 0,01% (w / v) in acqua distillata. Aggiungere 20 ml di diluito 0,01% (w / v) polilisina ai pozzetti di un vetrino pulito. </li><li> Incubare il vetrino per 5 minuti a temperatura ambiente. </li><li> Porre i vetrini su un blocco di 65 ° C asciutto e asciugare per 1 ora. </li><li> Conservare le diapositive polilisina nella scatola senza polvere. </li></ol></li></ol> 3. Fissazione di Worms sul vetrino (figura 1) <ol><li> embrioni Preparazione per FISH Nota: i vermi Harvest prima di tutto tegli cibo batterica è consumato guardando i mezzi di crescita sotto il microscopio. La fame riduce la produzione di uova di vermi adulti e aumenta la schiusa delle uova. Metodi dettagliati sono descritti in 14,15. <ol><li> Crescere i vermi in 50 millimetri Petri-piatto secondo i metodi standard di 14. </li><li> Dopo tutto il cibo batterica è consumato, tagliare il supporto agar nel quartiere con la spatola. Sterilizzare la spatola prima del taglio per evitare contaminazioni. </li><li> Mettere tutto il pezzo di agar sul (NGM) piatto 100 millimetri terreni di crescita nematode. Girare il pezzo agar a testa in giù per i vermi per raggiungere il cibo batterica fresca. </li><li> Dopo 48-72 ore, raccogliere i vermi con tampone M9. Aggiungere 3 - 5 ml di tampone M9 sulla piastra NGM. Pipetta M9 tampone sulla superficie della NGM per lavare i vermi. </li><li> Raccogliere il liquido con i vermi e aggiungere in una provetta da 15 ml. Nota: Se c&#39;è detriti agar dopo il raccolto, centrifuga i vermi in una soluzione di saccarosio al 30%. Mentre i detriti sono pellettato, vermi galleggiano sula superficie. </li><li> Aggiungere tampone M9 per compensare il volume a 15 ml. Pellet i vermi mediante centrifugazione a 300 xg per 3 min e rimuovere la maggior parte del buffer M9. Ripetere questa operazione altre 2 volte. Nota: se i vermi sono ancora a galla dopo la centrifugazione, impostare il livello del freno della centrifuga al Valore = 1. </li><li> Aspirare tampone M9. Aggiungere la soluzione sbiancante per i vermi. Per 0,5 ml di vermi, aggiungere 6,5 ml di DW, 2 ml di ipoclorito, 1 ml di 5 M KOH. </li><li> Incubare i vermi con dondolo a temperatura ambiente per 3 minuti a 50 giri al minuto. vermi Vortex per 15 secondi per tosare meccanicamente i vermi ed esporre le uova. </li><li> Osservare il tubo sotto un microscopio di dissezione durante il candeggio. Assicurarsi che i vermi sono tagliati a metà e le uova vengono rilasciati. Quando la maggior parte del corpo adulto è sciolto, aggiungere tampone M9 per compensare il volume a 15 ml. </li><li> Centrifugare a 300 xg per 3 min. Aspirare la maggior parte della M9 e aggiungere tampone M9 fresco per compensare il volume a 15 ml. fase di lavaggio ripetere 3 volte. Nota:Evitare di utilizzare quantità eccessiva di vermi, in quanto ostacolano il processo di sbiancamento. Mantenere il tempo di reazione complessivo inferiore a 8 minuti fino a quando il lavaggio. Over-sbiancato uova producono forti autofluorescenza. </li><li> Aggiungere tampone fosfato con polisorbato-20 (PBST) fino a 200 ml e 200 ml di 4% paraformaldeide (PFA) per rendere il 2% PFA. Attenzione: Dal momento che PFA è cancerogeno, indossare indumenti protettivi, guanti e protezione per gli occhi prima di utilizzare PFA. </li><li> Aggiungere 40 ml di uova a 2% PFA sul pozzo di rivestito polilisina diapositiva. </li><li> Porre i vetrini in una camera umida e incubare per 15 minuti a RT. Chiudere la camera umida subito dopo le diapositive sono collocati all&#39;interno. Nota: Le uova si depositano sul fondo della slitta pur essendo fisso. </li></ol></li><li> Preparazione gonadi sezionati per i pesci <ol><li> vermi Harvest adulti coltivati ​​sul 50 mm Piastra NGM pipettando 1 ml di tampone M9. vermi Harvest prima di alimentare batterica è esaurito. </li><li> Lavare i vermi da qualsiasi batteri ingegnoh tampone M9, 2 volte. Nota: i batteri residui possono interferire con le gonadi sezionati si attacchino alle diapositive rivestito polilisina. </li><li> Pellet i vermi per centrifugazione a 300 x g. Rimuovere M9 e trasferire i vermi alla piastra NGM vuota micropipetta. </li><li> Aggiungere 30 ml di tampone M9 contenente 2 mM levamisolo su un pozzo di polilisina trattata diapositiva. </li><li> Aggiungere 500 microlitri di tampone M9 ad una provetta 1 ml. Utilizzare questo buffer per trasferire i vermi per bocca pipetta. </li><li> Riempire la punta della pipetta bocca con tampone M9 mettendo un capillare nel tubo 1 ml contenente il tampone M9. </li><li> Sotto il microscopio dissezione, inserire la punta della pipetta bocca di fronte alla testa di adulto verme e trascinare bocca pipetta in modo che la testa di vermi entra pipetta bocca. Una volta che la testa di vermi entra la punta, l&#39;intero corpo di verme sarà disegnato nella punta. </li><li> Trasferire i vermi alla diapositiva rivestito polilisina usando la bocca pipetta. </li><li> Usando un rasoio, taglio dif la testa o la punta della coda di vermi sul vetrino. Quando il worm viene tagliato, le gonadi uscirà fuori. Gonadi si attaccano alle diapositive. <ol><li> Preparare almeno 30 vermi in un pozzetto. Più pozzetti possono essere utilizzati per altri 30 vermi. </li></ol></li><li> Mettere il vetrino nella camera umida e aspirare via il buffer M9 con la bocca pipetta. </li><li> Fissare il campione aggiungendo 20 ml di 2% PFA a temperatura ambiente per 15 minuti in camera umida. </li></ol></li></ol> 4. Fissazione e permeabilizzazione <ol><li> Posizionare un blocco di alluminio in ghiaccio secco e riporla in un congelatore (-80 ° C). Conservare metanolo e acetone -20 ° C. </li><li> Dopo la fissazione PFA passo 3.1.15 o 3.2.11, rimuovere fissativo utilizzando micropipetta lasciando ~ 5 ml di fissativo. </li><li> Mettere un altro vetrino rivestito polilisina sulla diapositiva campione. Rimuovere il fissativo con tovagliolo di carta se la soluzione è eccessiva. Non spostare le diapositive una volta che sono attaccati insieme. </li><li> Congelare le diapositivesul blocco di alluminio per almeno 15 min. Nota: I campioni possono essere conservati per almeno 2 - 3 giorni. </li><li> Mentre le diapositive vengono congelati, mettere i vasi contenenti metanolo freddo e acetone su ghiaccio. </li><li> Prendere le diapositive fuori e torcere loro di congelare-crack del campione. Eliminare la slitta superiore. immergere immediatamente il vetrino in metanolo ghiacciato per 5 min. </li><li> Trasferire i vetrini in acetone ghiacciata per 5 min. </li><li> Lavare i vetrini 3 volte con PBST per 5 minuti per rimuovere fissativo residuo. Procedere alla fase successiva o conservare le diapositive in 100% di etanolo a 4 ° C. Nota: I campioni possono essere conservati per almeno 2 - 3 giorni. </li></ol> 5. L&#39;ibridazione di cellule fissate <ol><li> Aggiungere 20 ml di soluzione di RNasi (PBST contenente 10 ug / ml RNasi A). Incubare il vetrino nella camera umida a 37 ° C per 1 ora. </li><li> Lavare il vetrino due volte in soluzione salina 2x e citrato di sodio polisorbato-20 (2x SSCT) per 15 minuti ciascuno. </li><li> Aggiungere 20ml di soluzione di ibridazione e di mettere la camera umida nel 37 ° C incubatore. Dopo 1 ora, rimuovere la soluzione di ibridazione pipettando. </li><li> Prima di rimuovere soluzione di ibridazione, di preparare la sonda. Se la sonda è DNA a doppia elica, denaturare le sonde mediante riscaldamento a 95 ° C per 5 min su un blocco secca. Dopo il riscaldamento, raffreddare la sonda brevemente ghiaccio. </li><li> Aggiungere 10 ml di soluzione di ibridazione contenente sonde al campione. Per sonda PNA, concentrazione uso con un rapporto di 1: 2.000 e per sonda dig-marcato, la concentrazione uso con un rapporto di 1: 200. Coprire il campione con vetro di copertura. </li><li> Mettere un tovagliolo di carta imbevuto di acqua sul blocco di calore (80 ° C). Mettere un coperchio di plastica sul blocco di calore per preservare l&#39;umidità e la temperatura. </li><li> Dopo che la temperatura del blocco termico è stabilizzata (a 80 ° C), collocare il vetrino campione sul tovagliolo di carta riscaldato e coprire con il coperchio di plastica. Denaturare il campione per 3 min. </li><li> IncUbate i vetrini in camera umida notte a 37 ° C. </li></ol> 6. Lava ed immunofluorescenza <ol><li> Riscaldare la soluzione di lavaggio di ibridazione (2x SSC, il 50% formammide) a 37 ° C. </li><li> Lavare il campione in PBST due volte a temperatura ambiente per 5 min. Rimuovere il vetro di copertura. </li><li> Lavare il campione in soluzione di lavaggio ibridazione a 37 ° C per 30 min. </li><li> Lavare il vetrino del campione in PBST 3 volte a temperatura ambiente. Nota: eseguire tutti i passaggi successivi nella camera umida a temperatura ambiente. </li><li> Aggiungere 20 ml di soluzione di blocco e incubare per 1 ora a RT nella camera umida. </li><li> Rimuovere soluzione bloccante e aggiungere FITC coniugato soluzione di anticorpo anti-digossigenina (1: 200) per 3 ore a temperatura ambiente o per una notte a 4 ° C. </li></ol> 7. Montaggio e di osservazione <ol><li> Lavare il vetrino campione con PBST 2 volte per 15 minuti ciascuno. </li><li> Aggiungere 10 ml di soluzione di montaggio con DAPI. Mettere delicatamente il vetro di copertura e premere. Rimuovere la soluzione in eccessocon un tovagliolo di carta. </li><li> Per evitare l&#39;evaporazione della soluzione di montaggio, sigillare i bordi del vetro di copertura con smalto. </li><li> Osservare al microscopio confocale. Esclusione di embrioni con fondo elevato. Focus su un campo con 4 - 20 nuclei. </li><li> Prendere le immagini in base alle istruzioni del produttore con lente obiettivo 100X. Nota: Excite campione con 405 nm laser per DAPI, con 555 nm laser per Cy3, con 488 nm laser per FITC. </li></ol> 8. Quantificazione dei telomeri Signal Nota: quantificazione è stata effettuata come descritto in precedenza 16. Tutte le immagini che devono essere confrontate vengono presi con stessa impostazione compreso il tempo di esposizione e la sorgente luminosa. <ol><li> Esportare l&#39;immagine in formato tif. </li><li> Scaricare e installare il software di analisi delle immagini. </li><li> Eseguire il software di analisi di immagine e fare clic sul pulsante d&#39;accordo. </li><li> Fare clic sul pulsante aperta. Aprire le immagini con telomeri FISH facendo doppio clic sul file di immagine. </li><li> Clic[Modifica] - [seleziona regione di elaborazione], selezionare regione di interesse da parte di sinistra del mouse e trascinamento. Escludere tutte le colorazione aspecifica. </li><li> Fare clic su [misura] - [Spot densità ottiche], selezionare il canale con il segnale dei telomeri e immettere il nome del file per salvare i risultati in file txt. Nota: La colonna di risultati sono nel seguente ordine: fluorescenza di spot, intensità di spot e area di spot background. </li><li> Copiare i valori e sottrarre l&#39;intensità di spot sfondo da fluorescenza della macchia. I valori possono essere analizzati statisticamente. </li></ol>

<ol>
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The authors have nothing to disclose.

Il vantaggio principale del nostro protocollo è la semplicità della procedura senza danni evidenti alla morfologia della struttura cellulare. Diversi passaggi sono stati ottimizzati per C. elegans FISH in questo protocollo. I passaggi critici per i pesci di successo includono l&#39;etichettatura di sonde, la fissazione di embrioni e la penetrazione. metodo di etichettatura digossigenina-dUTP fornisce un metodo di etichettatura di facile utilizzo mediante PCR o nick-translation. Per etichettare lunga sequenza ...

Telomeri protegge le estremità cromosomiche dalla fusione aberranti e degrado. telomeri mammiferi è composta da G-ricchi ripete esamerica, TTAGGG, e complessi shelterin. La sequenza di ripetizione telomerica del nematode è simile a quelle dei mammiferi (TTAGGC). La maggior parte degli eucarioti utilizzano telomerasi per aggiungere le ripetizioni dei telomeri alle loro estremità cromosomiche. Tuttavia, il 10 - 15% delle cellule tumorali utilizzano telomerasi meccanismo indipendente, conosciuto come allungamento dei telomeri alternativo (ALT) 3. In precedenza, abbiamo riportato che ripete telomeri e le sue sequenze associate, come il nome TALT, sono stati amplificati nei telomeri di linee mutanti telomerasi che sono sopravvissuti la sterilità critica 2. La lunghezza dei telomeri è stata misurata mediante PCR quantitativa o Southern blot, che fornisce la lunghezza media dei telomeri totale 4,5,6,7. Leggi conteggio di ripetizione dei telomeri in tutto il sequenziamento del genoma dei dati è anche un indicatore del totale contenuti telomeri 8. Sebbene SinGLE Telomere Lunghezza Analysis (STELA) potrebbe fornire la lunghezza di un singolo telomero, essa non può fornire informazioni spaziali dei telomeri 9. Mentre POT-1 :: mCherry proteina reporter fornisce le informazioni spaziali dei telomeri in vivo, non può rappresentare lunghezze dei telomeri a doppio filamento, come POT-1 è un telomero singolo filamento proteico il 10 vincolante. Mentre i metodi di cui sopra prevedono l&#39;informazione media delle sequenze ripetitive, ibridazione in situ fluorescente (FISH) permette di osservare la quantità e la distribuzione spaziale delle singole sequenze di interesse su scala cromosomica. Invece di purificazione del DNA, tessuti o cellule vengono fissate per mantenere le informazioni nativo spaziale FISH. Così, il pesce è uno strumento sia quantitativa e qualitativa per l&#39;osservazione delle singole sequenze ripetute, come ripete telomeri. Questo protocollo fornisce un metodo efficiente per la rilevazione simultanea di entrambi telomeri e altre repliche basate su miglioramenti da metodi descritti in precedenza 11,12. C. elegans larve o adulti sono organismo multicellulare con cellule altamente differenziate. L&#39;eterogeneità delle cellule impedisce sull&#39;analisi quantitativa di un gran numero di macchie telomeri. Per massimizzare il numero di cellule analizzate, gli embrioni sono isolati e diffondere sugli scivoli polilisina rivestita per i pesci. Inoltre, questo protocollo può anche essere combinato con immunofluorescenza. Come prova che il protocollo funziona, mostriamo che è possibile osservare e quantificare due differenti sequenze ripetitive. sonda di DNA contro TALT1 è stata generata con semplice PCR che incorpora digossigenina-dUTP. Poi questa sonda TALT1 e sonda PNA telomeri fluorescenza marcati sono stati ibridati simultaneamente. Successivamente, digossigenina stato rilevato con metodi di immunofluorescenza canonici. Vi presentiamo qui le immagini rappresentative dove TALT1 colocalizzava con il telomero in TRT-1 </eM&gt; i sopravvissuti.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PNA probe</td>
			<td>PANAGENE</td>
			<td>custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin-Fluorescein, Fab fragments</td>
			<td>Roche</td>
			<td>11207741910</td>
			<td>use 1:200 diluted in PBST</td>
		</tr>
		<tr>
			<td>Digoxigenin-dUTP</td>
			<td>Roche</td>
			<td>11573152910</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>SIGMA-ALDRICH</td>
			<td>A-7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-6148</td>
			<td>prepare 4% paraformaldehyde by heating in DW with few drops of NaOH. add 0.1 volume of 10x PBS.</td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridizaiton solution</td>
			<td></td>
			<td></td>
			<td>3X SSC, 50% formamide, 10% (w/v) dextran sulfate, 50 ug/ml heparin, 100 ug/ml yeast tRNA , 100ug/ml sonicated salmon sperm DNA</td>
		</tr>
		<tr>
			<td>Hybridizaiton wash solution</td>
			<td></td>
			<td></td>
			<td>2X SSC, 50% formamide</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>BIONEER</td>
			<td>C-9012</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carlo Erba</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Carlo Erba</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>SIGMA-ALDRICH</td>
			<td>H3393</td>
			<td>make 10 mg/ml for stock solution</td>
		</tr>
		<tr>
			<td>Dextran sulfate</td>
			<td>SIGMA-ALDRICH</td>
			<td>67578</td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td></td>
			<td></td>
			<td>For 1 Liter DW : 80 g NaCl, 2.0 g KCl, 27 g Na2HPO4:7H2O, 2.4 g KH2PO</td>
		</tr>
		<tr>
			<td>PBST</td>
			<td></td>
			<td></td>
			<td>1X PBS, 0.1% tween-20</td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-2287</td>
			<td>Commercial name is Tween-20</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine solution (0.1 % w/v)</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-8920</td>
			<td>prepare fresh 0.01 % w/v solution before use</td>
		</tr>
		<tr>
			<td>M9</td>
			<td></td>
			<td></td>
			<td>3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 L</td>
		</tr>
		<tr>
			<td>Bleaching solution</td>
			<td></td>
			<td></td>
			<td>20% sodium hypochlorite, 0.5 M KOH</td>
		</tr>
		<tr>
			<td>Antibody buffer</td>
			<td></td>
			<td></td>
			<td>1X PBST, 1mM EDTA, 0.1% BSA, 0.05% Sodium azide (toxic)</td>
		</tr>
		<tr>
			<td>Blocking solution</td>
			<td></td>
			<td></td>
			<td>Antibody buffer with 5% bovine serum albumin (BSA)</td>
		</tr>
		<tr>
			<td>illustra Microspin G-50</td>
			<td>GE healthcare</td>
			<td>27-53310-01</td>
			<td></td>
		</tr>
		<tr>
			<td>20X SSC</td>
			<td></td>
			<td></td>
			<td>To make 1L, 175.3 g of NaCl, 88.2 g of sodium citrate, H2O to 1 L, adjust pH to 7.0</td>
		</tr>
		<tr>
			<td>2X SSCT</td>
			<td></td>
			<td></td>
			<td>2X SSC, 0.1 % tween-20</td>
		</tr>
		<tr>
			<td>10x digoxigenin-dUTP mix</td>
			<td></td>
			<td></td>
			<td>1 mM dATP, 1 mM dGTP, 1 mM dCTP, 0.65mM dTTP, 0.35mM DIG-11-dUTP</td>
		</tr>
		<tr>
			<td>PCR purification columns</td>
			<td>Cosmo genetech</td>
			<td>CMR0112</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cleaner / ULTRA CLEAN</td>
			<td>Dukssan pure chemicals</td>
			<td>8AV721</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-well glass slide</td>
			<td>MP biomedicals</td>
			<td>96041205</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode growth media</td>
			<td></td>
			<td></td>
			<td>to make 1 L, 3 g of NaCl, 17 g of agar, 2.5 g of peptone, H2O to 974 mL. Autoclave and cool the flask. Add 1 mL of 1M CaCl2, 1 ml of 4 mg/mL cholesterol in ethanol, 1 ml of 1 M MgSO4, 25 mL of 1 M KPO4.</td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>SIGMA-ALDRICH</td>
			<td>196142</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor</td>
			<td>Feather</td>
			<td>blade No. 11</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Enzynomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>SIGMA-ALDRICH</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microsope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td>EC Plan-Neofluar 100x was used as objective lens.</td>
		</tr>
		<tr>
			<td>Dry block / aluminum block</td>
			<td>Labtech</td>
			<td>LBH-T03</td>
			<td>Set temperature to 80&#8451;</td>
		</tr>
		<tr>
			<td>Humid chamber</td>
			<td></td>
			<td></td>
			<td>Plastic box filled with paper towel soaked in DW</td>
		</tr>
		<tr>
			<td>Image Analysis Software&#160;</td>
			<td>Dr. Peter Landsdorp</td>
			<td>TFL-telo</td>
			<td><a href="http://www.flintbox.com/public/project/502">http://www.flintbox.com/public/project/502</a></td>
		</tr>
	</tbody>
</table>

observation and quantification of telomere and repetitive sequences using fluorescence in situ hybridization (fish) with pna probes in caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

In precedenza era stato riferito che sopravvissuto ALT può emergere dal mutante telomerasi-deficienti, TRT-1 (ok410), in bassa frequenza replicando localizzato internamente &#39;Modello di ALT&#39; (TALT) sequenze per telomeri manutenzione 2. Utilizzando la sonda PNA, siamo stati in grado di visualizzare telomeri nelle gonadi sezionati (Figura 2A). Il segnale telomeri debole è stato rilevato sia in TRT-1 (ok410) e ALT superstite. Il segnale...

Watch this Scientific Journal Video about Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans at

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

Osservazione e quantificazione dei telomeri e sequenze ripetitive Usando fluorescenza<em&gt; In Situ</em&gt; Ibridazione (FISH) con sonde PNA in<em&gt; Caenorhabditis elegans</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

observation-quantification-telomere-repetitive-sequences-using

Research

JoVE Journal

Biology

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

10.3K Views.  Seoul National University. We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT). 

Video: Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

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 ...

Ricerca

Biologia

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Microscopia time-lapse di Embriogenesi All&#39;inizio del Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

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 ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Di base<em&gt; Caenorhabditis elegans</em&gt; Metodi: sincronizzazione e di osservazione

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

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 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

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 ...

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 ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Prostaglandina estrazione e l&#39;analisi in<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

Robusto FISH 3D DNA con sonde marcate direttamente

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.