Name: rapid analysis of chromosome aberrations in mouse b lymphocytes by pna-fish
Uploaded: 2014-08-19T16:00:00.000+00:00
Duration: 7 min 54 s
Description: Watch this Scientific Journal Video about Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH at JoVE.com

Questo lavoro è sostenuto da NIH concedere R00 CA160574 (SFB) e da una borsa di studio del programma di formazione Biotecnologie Rutgers (a SMM).

Questa procedura è stata approvata dal Comitato di cura e l&#39;uso degli animali Istituzionale presso la Rutgers, l&#39;Università statale del New Jersey. I topi sono stati trattati in conformità con la guida NIH per la cura e l&#39;uso di animali da laboratorio. Gli scienziati dovrebbero consultare i loro organizzazioni nazionali ed istituzionali animali per linee guida stabilite e approvate. Prima di iniziare, preparare le soluzioni elencate nella tabella 1. 1 B Isolamento cellulare e attivazione Euthanize un mouse utilizzando CO 2 seguita da dislocazione cervicale. Posizionare il mouse in modo che il lato sinistro del corpo è alto e spruzzare il mouse con etanolo al 70% fino a quando il pelo è umido. Sezionare la milza e posto in un piatto di coltura tissutale 35 mm con tampone di lavaggio 2 ml. In una cappa a flusso laminare, delicatamente rompere la milza nel piatto di coltura di tessuti utilizzando la parte piatta di una siringa da 5 ml. <ol><li> Inserire una rete di nylon di 70 micron in un 50Tubo ml e trasferire il campione milza in 2 ml tampone di lavaggio nella rete di nylon. Interrompere delicatamente la milza nella rete utilizzando la parte piatta della siringa. </li><li> Risciacquare retro della siringa e la placca 35 mm, con 8 ml di tampone di lavaggio e filtrata attraverso la maglia di nylon 70 micron. Centrifugare a 300 xg per 10 min a 4 ° C. </li><li> Aspirare e scartare il surnatante e risospendere il pellet in 3 ml di tampone di lisi ACK. Pipettare su e giù brevemente per interrompere grumi e incubare per 5 min. Aggiungere 10 ml di tampone di lavaggio e centrifugare a 300 xg per 10 min a 4 ° C. </li><li> Aspirare e scartare il surnatante e risospendere il pellet toccando il fondo della provetta poi pipettando in 1 ml di tampone di lavaggio. Aggiungere 50 ml di anti-CD43 MACS micro-perline e incubare in ghiaccio per 30 min. Aggiungere 10 ml di tampone di lavaggio, mescolare delicatamente e centrifugare a 300 xg per 10 min a 4 ° C. Aggiungere 10 ml di tampone di lavaggio, mescolare delicatamente e centrifugare a 300 xg per 10 min a 4 ° C. </li><li> Impostare unMini-MACS colonna posizionando una colonna sul magnete MACS. Posizionare un marcato tubo da 15 ml di sotto della colonna. Aggiungere 1 ml di tampone di lavaggio alla colonna e lasciar correre attraverso il tubo da 15 ml. </li><li> Aspirare il surnatante e risospendere in 1 ml di tampone di lavaggio. Caricare il campione nella colonna dopo che il tampone di lavaggio è eseguito attraverso. </li><li> Dopo che il campione è percorrere la colonna, aggiungere 1 ml di tampone di lavaggio per lavare la colonna. Aggiungere 7 ml di tampone di lavaggio direttamente al campione prelevato nella provetta da 15 ml. </li><li> Contare le cellule utilizzando un emocitometro. Trasferire il volume richiesto di cellule necessarie per 5.000.000 cellule / pozzetto in una provetta da 50 ml. Centrifugare a 300 xg per 10 min a 4 ° C. Supplemento medio delle cellule B con LPS 1: 500 e IL4 1: 1.000. </li><li> Aspirare il surnatante e aggiungere il volume appropriato di supporto integrato delle cellule B necessaria per la concentrazione finale di 1.000.000 cellule / ml. </li><li> Cellule piastra in piastre da 6 pozzetti con 5 ml di cellule a 1.000.000 cellule / ml e posto in un umidifiEd coltura cellulare incubatore a 37 ° C, 5% di CO 2. </li></ol> 2 B Cella di fissaggio <ol><li> Aggiungere 50 ml di Colcemid al pozzo contenente 5 ml di cellule B (per una concentrazione finale di 100 ng / ml) e incubare 1 ora a 37 ° C. Preriscaldare soluzione KCl 75 mM in un bagno di 37 ° C Acque. Trasferire le cellule B ad una etichetta tubo 15 ml. Coprire l&#39;etichetta con nastro e centrifugare a 300 xg per 10 min a 4 ° C. </li><li> Aspirare il surnatante lasciando 1 ml nella provetta e risospendere il pellet pipettando. Aggiungere 5 ml della preriscaldata KCl goccia a goccia, mentre toccando o pulsare in un vortice. </li><li> Aggiungere 10 ml di KCl e mescolare capovolgendo. Incubare in un bagno d&#39;acqua a 37 ° per 15 min. Aggiungere 5 gocce di fissativo fresco e mescolare capovolgendo. Centrifugare a 300 xg per 10 min a 4 ° C. Aspirare il surnatante lasciando 1 ml nella provetta e risospendere pellet pipettando su e giù. </li><li> Aggiungere 5 ml di fissativo fresco goccia a goccia, mentre toccando o pulsante su un VORtex. Aggiungere altri 10 ml di fissativo e incubare per 30 minuti a temperatura ambiente. </li><li> Centrifugare a 300 xg per 10 min a 4 ° C. Aspirare il surnatante lasciando 1 ml in provetta e risospendere da pipettaggio. Aggiungere 14 ml di fissativo fresco e centrifugare a 300 xg per 10 min a 4 ° C. </li><li> Ripetere il punto 2.7 altre due volte. </li><li> Aggiungere 14 ml di fissativo fresco. Sigillare i tappi con Parafilm e conservare una notte a -20 ° C. </li></ol> 3 Preparazione di metafase spread cromosomi <ol><li> Impostare una camera ambientale regolata a 22,9 ° C e 52% di umidità. </li><li> Centrifugare le cellule B fissato a 300 xg per 10 min a 4 ° C. Aspirare fissativo lasciando 70 microlitri nel tubo e risospendere da pipettaggio. </li><li> Posizionare i vetrini etichettati in camera umida con un angolo di 35 °. Goccia 35 ml di cellule B risospese su un vetrino e rilasciare due scivoli per campione. Lasciare i vetrini si asciughino in camera umida per 30 minuti poi memorizzare le diapositivein una scatola di diapositive a 37 ° C. </li></ol> 4. telomeri PNA FISH <ol><li> Preparazione della sonda <ol><li> Pre-caldo formammide deionizzata a 37 ° C. Mescolare 2 ml di sonda PNA telomeri con 7 microlitri formammide deionizzata preriscaldata e incubare a 37 ° C con agitazione per 1 ora. </li><li> Pre-caldo mix FISH maestro a 37 ° C. Aggiungere 7 ml di pre-riscaldato mix FISH maestro alla miscela dal punto 4.1.1 e incubare a 37 ° C per 1-3 ore. </li><li> Denaturare la miscela sonda per 8 minuti a 80 ° C. Pre-temprare la sonda per 1 ora a 37 ° C. </li></ol></li><li> Pretrattamento dei vetrini cromosomi <ol><li> Pre-riscaldare un barattolo di vetro scorrevole colorazione contenente 50 ml di HCl 0,01 M a 37 ° C in un bagno d&#39;acqua. </li><li> Collocare i vetrini in un altro vaso di vetro scorrevole-colorazione contenente 2x SSC per 5 minuti a temperatura ambiente. </li><li> Aggiungere 2 ml di pepsina magazzino al vetro pre-riscaldato slide-colorazione barattolo e mescolare capovolgendo. Trasferire i vetrini di questo vetro sljar ide-colorazione e incubare 90 sec a 37 ° C. </li><li> Lavare i vetrini 2x in un altro vaso di vetro scorrevole-colorazione contenente 1x PBS per 5 minuti ciascuno a temperatura ambiente, agitando. Quindi lavare i vetrini per 5 min in PBS 1x / MgCl 2 a temperatura ambiente, agitando. </li><li> Trasferire i vetrini in un vaso di vetro scorrevole-colorazione contenente appena preparato 1% di formaldeide / 1x PBS / MgCl 2 per 10 minuti a temperatura ambiente. </li><li> Lavare i vetrini per 5 min in un altro vaso di vetro scorrevole-colorazione contenente 1x PBS a temperatura ambiente, agitando. </li><li> Preparare una serie di disidratazione di etanolo avendo in vetro separata vasetti di slide-colorazione contenenti il ​​70%, 90% e il 100% di etanolo. </li><li> Trasferire i vetrini per ogni barattolo per 3 min, iniziando con etanolo al 70%, quindi il 90% di etanolo, e poi il 100% di etanolo. Posizionare vasetti etanolo a -20 ° C una volta terminato. </li><li> Lasciare i vetrini all&#39;aria secca toccando eccesso di etanolo su un tovagliolo di carta e puntellare le diapositive con un angolo di 35 °. Una volta asciutto, mark un&#39;area 18 x 18 mm per ibridazione sul retro delle diapositive utilizzando una penna diamante. </li></ol></li><li> Denaturazione delle diapositive per FISH <ol><li> Applicare 120 microlitri formammide 70% deionizzata / 2X SSC a un coprioggetto 24 x 60 mm. Toccare la diapositiva per il coprioggetto. Denaturare il vetrino a 80 ° C su una piastra calda per 90 sec. </li><li> Far scorrere velocemente e con cura il vetrino e porre il vetrino in ghiaccio freddo etanolo al 70% per 3 minuti, seguito da 90% di etanolo, e il 100% di etanolo ciascuno per 3 min. Lasciare i vetrini all&#39;aria secca. </li><li> Preparare una camera umida allineando una scatola di plastica con un coperchio stretto e carta velina bagnata. La carta deve essere umido, ma non saturo d&#39;acqua. </li><li> In una camera umida, applicare la miscela pre-ricotto sonda dal passo 4.1.3 per l&#39;area contrassegnata sul vetrino, coprire con un coperchio 18 x 18 mm, antiscivolo e incubare a 37 ° C per 1 ora. </li></ol></li><li> Lavaggi post-incubazione <ol><li> Preriscaldare il 50% formammide / 2x SSC, SSC 1x, 4x e SSC / 0,1% Tween-20 in un 45 °; C bagnomaria. </li><li> Rimuovere con attenzione il coprioggetto e lavare i vetrini in pre-riscaldato 50% formammide / 2x SSC 3xfor 5 minuti ciascuna nel buio e agitando. Lavare i vetrini in pre-riscaldato 1x SSC 3xfor 5 minuti ciascuno, tremante. Infine, lavare i vetrini in pre-riscaldato 4x SSC / 0,1% Tween-20 3xfor 5 minuti ciascuno. </li><li> Colorare i vetrini per 3 min in DAPI in una luce protetta vetrino-colorazione vaso. </li><li> Lavare i vetrini per 5 min in 2x SSC, tremante. Applicare 35 ml di mezzo di montaggio Mowiol, coprire con 24 x 60 mm coprioggetto e conservare le diapositive a 4 ° C. </li></ol></li></ol> 5 Analisi microscopica di cromosomi <ol><li> Analizzare le diapositive metafase utilizzando un microscopio a fluorescenza standard. Utilizzare una piattaforma di imaging con una fase automatizzata, ad esempio la piattaforma METASYSTEMS Metafer per ottenere migliori risultati. Per ogni spread metafase, raccogliere un&#39;immagine per DAPI fluorescenza di visualizzare cromosomi, e un&#39;immagine per il segnale fluorescente dal telomero probe. (Ad esempio, Cy3- altri fluors sono disponibili e funzionano altrettanto bene.) </li><li> Sovrapponete le immagini con un programma di analisi di immagine e analizzare. </li></ol>

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N., Reijo Pera, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaphase+spreads+and+spectral+karyotyping+of+human+embryonic+stem+cells.">Metaphase spreads and spectral karyotyping of human embryonic stem cells.</a> Cold Spring Harb Protoc. 2008, (2008).</li><li>Schreck, R. R., Disteche, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+4.">Chapter 4.</a> Chromosome banding techniques. Unit 2, (2001).</li><li>Gilley, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-PKcs+is+critical+for+telomere+capping.">DNA-PKcs is critical for telomere capping.</a> Proc Natl Acad Sci USA. 98 (26), 15084-15088 (2001).</li><li>Bolzan, A. 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Considerando che i linfociti B attivati ​​sono particolarmente adatti alla preparazione degli spread cromosomi mitotici, possono essere utilizzati anche altri tipi di cellule. Linfociti T condividere molti dei vantaggi delle cellule B, in quanto possono essere purificati dai nodi milza o linfonodi, e hanno un elevato indice mitotico, quando stimolato dalla crescita in terreno contenente mitogeni appropriate 8. Le cellule staminali embrionali (cellule ES) sono adatti anche per l&#39;analisi della metafase ...

A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH.&#160;Table 1 (the list of solutions to prepare) was omitted from the Protocol section. The contents of Table 1 are as follows:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Reagents</td>
			<td>Comments</td>
			<td>Storage</td>
		</tr>
		<tr>
			<td rowspan="3">B Cell Wash Buffer</td>
			<td>500 ml 1x Hanks Balanced Salt Solution</td>
			<td rowspan="3">Filter through 0.22 &#956;m stericup filtration unit</td>
			<td rowspan="3">4 &#176;C</td>
		</tr>
		<tr>
			<td>5 ml 50 &#176;C heat inactivated FCS</td>
		</tr>
		<tr>
			<td>5 ml 100x Pen/Strep</td>
		</tr>
		<tr>
			<td rowspan="8">B Cell Medium</td>
			<td>418.5 ml RPMI-1640</td>
			<td rowspan="8">Filter through 0.22 &#956;m stericup filtration unit</td>
			<td rowspan="8">4 &#176;C</td>
		</tr>
		<tr>
			<td>50 ml 50 &#176;C heat inactivated FCS</td>
		</tr>
		<tr>
			<td>5 ml 100x Pen/Strep</td>
		</tr>
		<tr>
			<td>5 ml glutamine</td>
		</tr>
		<tr>
			<td>5 ml nonessential amino acids</td>
		</tr>
		<tr>
			<td>5 ml sodium pyruvate</td>
		</tr>
		<tr>
			<td>1.8 ml 14.2 M 2-mercaptoethanol</td>
		</tr>
		<tr>
			<td>5 ml 1M HEPES</td>
		</tr>
		<tr>
			<td>Fixative</td>
			<td>3:1 v/v methanol/glacial acetic acid</td>
			<td></td>
			<td>Make fresh</td>
		</tr>
		<tr>
			<td rowspan="2">0.075 M KCl</td>
			<td>1.395 g KCl</td>
			<td rowspan="2"></td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>250 ml distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="3">FISH Master Mix</td>
			<td>40 ml 50% Dextran sulfate</td>
			<td rowspan="3">Vortex the solution and leave in a shaking platform overnight</td>
			<td rowspan="3">Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>20 ml 20x SSC</td>
		</tr>
		<tr>
			<td>40 ml sterile distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="3">70% Formamide/2x SSC</td>
			<td>10 ml 20x SSC</td>
			<td rowspan="3">Adjust to pH 7 with 1M HCl</td>
			<td rowspan="3">Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>20 ml distilled H2O</td>
		</tr>
		<tr>
			<td>70 ml deionized formamide</td>
		</tr>
		<tr>
			<td rowspan="2">0.01 M HCl</td>
			<td>1 ml 1M HCl</td>
			<td rowspan="2">Adjust to pH 2.0</td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>99 ml distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="2">1x PBS/MgCl2</td>
			<td>50 ml 1M MgCl2</td>
			<td rowspan="2"></td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>950 ml 1x PBS</td>
		</tr>
		<tr>
			<td rowspan="3">50% Formamide/2x SSC</td>
			<td>20 ml 20x SSC</td>
			<td rowspan="3">Adjust to pH 7.25 
			Formamide used in this step does not have to be deionized.</td>
			<td rowspan="3">Make fresh</td>
		</tr>
		<tr>
			<td>80 ml distilled H2O</td>
		</tr>
		<tr>
			<td>100 ml formamide</td>
		</tr>
		<tr>
			<td rowspan="3">4x SSC/0.1% Tween20</td>
			<td>50 ml 20x SSC</td>
			<td rowspan="3"></td>
			<td rowspan="3">Room Temperature</td>
		</tr>
		<tr>
			<td>200 ml distilled H2O</td>
		</tr>
		<tr>
			<td>250 &#956;l Tween20</td>
		</tr>
		<tr>
			<td rowspan="2">DAPI staining solution (80 ng/ml)</td>
			<td>40 &#956;l 0.2mg/ml DAPI stock</td>
			<td rowspan="2"></td>
			<td rowspan="2">4 &#176;C in light protected staining jar</td>
		</tr>
		<tr>
			<td>100 ml 2x SSC</td>
		</tr>
		<tr>
			<td rowspan="5">Mowiol Mounting Medium</td>
			<td>6 g glycerol</td>
			<td rowspan="5">Incubate at 50 &#176;C with stirring for 2hrs</td>
			<td rowspan="5">Aliquot and store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>2.4 g Mowiol 4-88</td>
		</tr>
		<tr>
			<td>6 ml distilled H2O</td>
		</tr>
		<tr>
			<td>12 ml 0.2 M TrisCl pH 8.5</td>
		</tr>
		<tr>
			<td>230 &#956;l 1% Thimerosal (w/v in water)</td>
		</tr>
	</tbody>
</table>
Table 1. List of solutions.&#160;

A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH.

Erratum: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Il cancro è causato da un accumulo di mutazioni che interessano i geni che regolano la crescita delle cellule normali. La mutazione è una conseguenza di modifiche alla struttura e la sequenza del genoma causato da danni al DNA. Danno al DNA può avvenire attraverso una varietà di processi, compresi gli agenti esogeni come radiazioni ionizzanti, e come un sottoprodotto del normale metabolismo cellulare, come deaminazione spontanea di basi nucleotidiche o danni che si verificano per contatto con le specie reattive dell&#39;ossigeno 1. Anche se le cellule di mammifero in possesso di una serie di attività di riparazione che può invertire il danno del DNA o ripristinare la sequenza in siti rottura, le mutazioni si accumulano comunque per tutta la durata di una cella. Danno al DNA può contribuire inoltre alla senescenza e la perdita di efficacia delle cellule staminali, due processi che sono associati con la malattia associata all&#39;invecchiamento 2. Comprendere la riparazione dei danni al DNA è quindi di fondamentale importanza per affrontare due segnoquestioni ificant nella salute pubblica. Una crescente evidenza suggerisce che mammiferi percorsi di riparazione del DNA possono contribuire all&#39;evoluzione del genoma delle cellule di cancro 3,4, il che rende ancora più indispensabile per capire i processi coinvolti nella soppressione mutazione a livello molecolare. Visualizzazione diretta di aberrazioni cromosomiche è un potente e quantitativi mezzi per determinare il grado di instabilità genomica in un particolare tipo di cellula. Cromosomi abbreviato da cellule in metafase possono essere isolate e ispezionati utilizzando la luce o la microscopia a fluorescenza. Tali approcci citogenetiche sono stati in pratica per diversi decenni e può essere utilizzato per dimostrare l&#39;aspetto di traslocazioni o specifici tipi di aberrazioni cromosomiche associate alla perdita di attività di riparazione del DNA. Il protocollo si presta a diversi potenziali estensioni: i cromosomi possono essere etichettati con sonde per cariotipo spettrale (SKY) o multicolor fluorescente in situ ibridazione(MFISH) per identificare traslocazioni 5,6. Queste tecniche permettono anche la frequenza di traslocazioni cromosomiche e la struttura di traslocazioni cromosomiche complesse da determinare, che fornisce informazioni aggiuntive di là di quanto è possibile con questo protocollo. In alternativa, sonde sequenza-specifiche possono essere generati e utilizzati per testare la frequenza di rottura del DNA in siti genomici selezionati 7. In questo protocollo, si descrive la preparazione del cromosoma metafase diffonde da linfociti B. Un peptide acido nucleico (PNA) sonda fluorescenza marcata per le ripetizioni telomeriche viene utilizzato, che segna in modo efficiente telomeri in metafase cromosoma si diffonde Questo protocollo ha diversi vantaggi. Cellule B possono essere indotte a crescere ad alto indice mitotico in modo che gli spread di alta qualità possono essere prodotti in modo coerente. Cellule B da topi geneticamente modificati sono anche molto meno probabile che contengono mutazioni genetiche secondarie che possono confondere l&#39;analisi del contributodi geni specifici per l&#39;integrità genomica. L&#39;approccio PNA-FISH può essere completato in un giorno, e permette il punteggio più accurata delle rotture cromosomiche. Usando questo approccio, in particolare in combinazione con attrezzature specifiche descritte in questo protocollo, è possibile produrre molto consistenti, spread alta qualità e analizzare rapidamente la velocità e il tipo di instabilità genomica.

<table><tbody><tr><td>50% Dextran sulfate</td><td>Intergen</td><td>S4030</td><td></td></tr><tr><td>Deionized formamide</td><td>Ambion</td><td>9342</td><td></td></tr><tr><td>Pepsin (5 g)</td><td>Sigma</td><td>P 6887&amp;nbsp;</td><td></td></tr><tr><td>FCS</td><td>Gemini</td><td>100-106</td><td></td></tr><tr><td>LPS (100 mg)</td><td>Sigma</td><td>L2630</td><td></td></tr><tr><td>IL-4 (5 &amp;mu;g)</td><td>Sigma</td><td>I1020</td><td></td></tr><tr><td>PNA Telomere Probe</td><td>PNA Bio Inc</td><td>F1002</td><td>(CCCTAACCCTAACCCTAA)</td></tr><tr><td>Colcemid</td><td>Roche</td><td>295892</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Sigma</td><td>81381-50g</td><td></td></tr><tr><td>CD43 (ly-48_) micro-beads</td><td>Miltenyl Biotec.</td><td>130-049-801</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>32670-5MG</td><td></td></tr><tr><td>Eclipse E800</td><td>Nikon</td><td></td><td></td></tr><tr><td>AxioImager.Z2</td><td>Zeiss</td><td></td><td></td></tr><tr><td>CDS-5 Cytogenetic Drying Chamber</td><td>Thermotron</td><td></td><td></td></tr></tbody></table>

rapid analysis of chromosome aberrations in mouse b lymphocytes by pna-fish

Riparazione del DNA difettoso porta ad una maggiore instabilità genomica, che è la causa principale delle mutazioni che portano alla tumorigenesi. Analisi della frequenza e il tipo di aberrazioni cromosomiche in diversi tipi cellulari permette difetti nei percorsi di riparazione del DNA da chiarire. Comprensione mammiferi riparazione del DNA biologia è stato molto aiutato dalla produzione di topi con ko in geni specifici. L&#39;obiettivo di questo protocollo è quello di quantificare l&#39;instabilità genomica nei linfociti B del mouse. Etichettatura dei telomeri con sonde PNA-FISH (peptide di acido nucleico - ibridazione fluorescente in situ) facilita l&#39;analisi rapida di instabilità genomica degli spread metafase cromosomiche. Cellule B hanno vantaggi specifici relativi ai fibroblasti, perché hanno ploidia normale e un indice mitotico superiore. Cultura a breve termine di cellule B consente quindi la misurazione precisa di instabilità genomica in una popolazione di cellule primarie, che rischia di avere mutazione genetica secondaria menos di quello che è tipicamente presenti nei fibroblasti trasformati o linee cellulari di pazienti.

Cromosomi in metafase derivati ​​da una cella dovrebbe formare un cluster discreto contenente 40 cromosomi (se si utilizza cellule di topo) (Figura 1A). Ogni cromosoma ha un centromero ad una estremità, che è visibile come una sfera azzurro. In cromosomi normali, due segnali telomeri sono visti alla fine dei cromatidi e due segnali di ciascun centromero. Nuclei in interfase saranno presenti sul vetrino pure. Questi saranno visibili come sfere blu, con segnali telomeri rosse, ma non cromosomi conde...

Watch this Scientific Journal Video about Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH at JoVE.com

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Percorsi di riparazione del DNA sono essenziali per il mantenimento dell&#39;integrità genomica e prevenire la mutazione e il cancro. L&#39;obiettivo di questo protocollo è quello di quantificare l&#39;instabilità genomica mediante l&#39;osservazione diretta di aberrazioni cromosomiche in metafase si diffonde dalle cellule B del mouse utilizzando ibridazione in situ fluorescente (FISH) per ripetizioni telomeriche del DNA.

Analisi rapida di aberrazioni cromosomiche in linfociti B mouse da PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

rapid-analysis-chromosome-aberrations-mouse-b-lymphocytes-pna

Research

JoVE Journal

Immunology and Infection

16.9K Views.  Rutgers, the State University of New Jersey.  Percorsi di riparazione del DNA sono essenziali per il mantenimento dell'integrità genomica e prevenire la mutazione e il cancro. L'obiettivo di questo protocollo è quello di quantificare l'instabilità genomica mediante l'osservazione diretta di aberrazioni cromosomiche in metafase si diffonde dalle cellule B del mouse utilizzando ibridazione in situ fluorescente (FISH) per ripetizioni telomeriche del DNA. 

Video: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

L&#39;analisi del particolato ambientale indotto aberrazioni cromosomiche L&#39;utilizzo di un<em&gt; In Vitro</em&gt; sistema

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Ricerca

Genetica

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

Rilevazione di Inter-aberrazioni cromosomiche stabili per fluorescenza multipla<em&gt; In Situ</em&gt; Ibridazione (mFISH) e spettrale cariotipo (SKY) in topi irradiati

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

FISH for Pre-implantation Genetic Diagnosis 

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

FISH for Pre-implantation Genetic Diagnosis

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

FISH per preimpianto Diagnosi Genetica

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

Medicina

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

Preparazione delle scime cromosomiche meiotiche da Spermatociti di pesce zebra

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

Zebrafish Modello di Metastasi del Neuroblastoma

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

Ricerca sul cancro

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Chromosomics: Rilevamento delle alterazioni numeriche e strutturali in tutti i 24 cromosomi umani simultaneamente utilizzando un nuovo metodo di analisi FISH OctoChrome

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Biologia

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Immunofluorescenza combinato e FISH DNA 3D conservato nuclei in interfase di studiare i cambiamenti in 3D Organizzazione nucleare

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the variable coding exons of the Ig heavy and light chain gene loci. This process of somatic hypermutation (SHM) requires the enzyme activation-induced cytidine deaminase (AID), which converts deoxycytidines (C), into deoxyuridines (U). Processing the AID-generated U:G mismatches into mutations by the base excision and mismatch repair pathways introduces new Ig coding sequences that may produce a higher affinity Ig. Mutations in AID or DNA repair genes can block or significantly alter the types of mutations observed in the Ig loci. We describe a protocol to quantify JH4 intron mutations that uses fluorescence activated cell sorting (FACS), PCR, and Sanger sequencing. Although this assay does not directly measure Ig affinity maturation, it is indicative of mutations in Ig variable coding sequences. Additionally, these methods utilize common molecular biology techniques which&#160;analyze&#160;mutations in Ig sequences of multiple B cell clones. Thus, this assay is an invaluable tool in the study of SHM and Ig diversification.

Presented here is an assay to quantify somatic hypermutation within the immunoglobulin heavy chain gene locus using germinal center B cells from mouse Peyer&#8217;s patches.

Analisi dell'ipermutazione somatica nell'introne JH4 delle cellule del Centro Germinale B dalle patch di Mouse Peyer

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the ...

Immunologia e infezioni

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 ...

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and T lymphocytes. Studies of zebrafish lymphopoiesis are difficult because antibodies recognizing D. rerio cell surface markers are generally not available, complicating isolation and characterization of different lymphocyte populations, including B-lineage cells. Transgenic lines with lineage-specific fluorophore expression are often used to circumvent this challenge. The transgenic lck:eGFP line has been used to study D. rerio T cell development, and has also been utilized to model T cell development and acute lymphoblastic leukemia (T-ALL). Although lck:eGFP fish have been widely used to analyze the T-lineage, they have not been used to study B cells. Recently, we discovered that many zebrafish B cells also express lck, albeit at lower levels. Consequently, lck:eGFP B cells likewise express low levels of GFP. Based on this finding, we developed a protocol to purify B-lineage cells from lck:eGFP zebrafish, which we report here. Our method describes how to utilize a fluorescent-activated cell sorter (FACS) to purify B cells from lck:eGFP fish or related lines, such as double-transgenic rag2:hMYC; lck:eGFP fish. In these lines, B cells, particularly immature B cells, express GFP at low but detectable levels, allowing them to be distinguished from T cells, which express GFP highly. B cells can be isolated from marrow, thymus, spleen, blood, or other tissues. This protocol provides a new method to purify D. rerio B cells, enabling studies focused on topics like B cell development and B lymphocyte malignancies.

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Transgenic lck:eGFP zebrafish express GFP highly in T lymphocytes, and have been used to study T cell development and acute lymphoblastic leukemia. This line can be used to study B cells, which express lck at lower levels. This protocol describes purification of malignant and non-malignant B cells from lck:eGFP zebrafish.

Isolando il maligno e le cellule di B Non-maligna da lck:eGFP Zebrafish

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and ...

Analisi rapida di aberrazioni cromosomiche in linfociti B mouse da PNA-FISH

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L'analisi del particolato ambientale indotto aberrazioni cromosomiche L'utilizzo di un

Rilevazione di Inter-aberrazioni cromosomiche stabili per fluorescenza multipla