The authors would like to thank Mark Bishop, Mathew Zverev, Victoria Marsh-Durban, Adam Blackwood and Sylvie Robine. This work was funded by a programme grant from Cancer Research UK.

<ol>
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The authors have nothing to disclose.

Utilizzando topi transgenici condizionali Cre-lox di sezionare la funzione dei geni e cellule è un approccio comunemente utilizzato. Questi modelli sono stati utilizzati con grande successo a livello intestinale per identificare e caratterizzare le cellule staminali 2,4-6 e capire il loro ruolo nella malattia 25. Per sfruttare appieno questi modelli richiede una caratterizzazione completa del sistema per consentire i dati devono essere interpretati correttamente. Una completa comprensione di questi sistemi è difficile da raggiungere a causa di geni raramente essere specifico per un tipo di cella d&#39;isolamento o la posizione, una mancanza di conoscenza biologica e inefficienza dei sistemi utilizzati per indurre l&#39;espressione Cre. I metodi qui descritti dimostrano come abbiamo superato questi problemi attraverso il design sperimentale e l&#39;applicazione delle conoscenze esistenti. Anche se abbiamo usato questi metodi per rispondere a una domanda di ricerca specifica le tecniche presentate qui sono generiche e può essere sfruttata per qualsiasi ricerca indagando la muriintestino ne. Preparazione del tessuto intestinale Il passo fondamentale per garantire risultati affidabili è la raccolta e la lavorazione dei tessuti, che deve essere elaborato in modo tempestivo protocolli maniera e fissaggio rigorosamente rispettati. Come quasi tutti i problemi significativi a valle può essere attribuito ad artefatti associati con l&#39;essiccamento del tessuto e / o di fissaggio incompleta. Sincronizzazione è cruciale per prevenire la degradazione di architettura del tessuto e / o di acidi nucleici e proteine. Fissazione incomplete o troppo zelanti può causare perdita di risoluzione istochimica. Fissazione incompleta a causa del tempo o le sezioni troppo spessi per consentire la penetrazione fissativo può causare la perdita di risoluzione entro cripte intestinali che possono essere osservati come un &quot;segno di marea&quot; all&#39;analisi IHC insufficiente. Inoltre è importante che la fissazione non si estende troppo a lungo, come nucleare β-catenina può diffondere fuori dal nucleo meno immediatamente pronon trasformati e cera incorporati dopo fissazione in formalina. Ruolo delle cellule Paneth nella nicchia ISC I dati qui presentati mostrano efficacemente l&#39;importanza delle cellule Paneth nella rigenerazione cripta nell&#39;intestino adulto seguente perdita ISC. Tuttavia, vi è rimasta la possibilità che Ah-cre risparmia una popolazione di ISC che gli obiettivi T2 vil-Cre-ER. Tian et al. 26 elegantemente dimostrato che le ISC hi Lgr5 sono sostituiti da una popolazione di Lgr5 Lo ISC di riserva. Ora sembra probabile che queste ISC sono risparmiati nel sistema di Ah-cre a causa della popolazione di riserva essendo stato identificato come precursori delle cellule secretoria 6,7. L&#39;importanza della cella Paneth matura nel sostenere questi precursori delle cellule di secrezione, se necessario, per ripristinare uno stato di ISC resta da risolvere. Come le cellule Paneth costituiscono l&#39;hoSC nicchia 8 e svolgere ruoli nel regolare le risposte ISC di apporto calorico 27 e infiammazione 28 rimane probabile che le loro funzioni di cura si estenderanno alle loro precursori. Nuovi approcci e tecnologie in modo efficace modello umano cancro colorettale La scoperta del CSI ha portato alla identificazione di geni che ora vengono utilizzati per generare nuovi modelli di topo per studiare il ruolo dei geni e cellule in biologia e le malattie intestinali, recensito da Clarke et al 9. Le uniche limitazioni di questa tecnica è l&#39;identificazione di geni per esprimere la proteina Cre. Attualmente ISC sono regolarmente esaminati utilizzando topi transgenici condizionali in base al pattern di espressione genica Lgr5. I topi che esprimono Cre dal promotore Lgr5 sono stati utilizzati per eliminare Apc, il gene più frequentemente mutato nel tumore del colon-retto (CRC), Dimostrando l&#39;ISC come cellula di origine 25. Selettivamente eliminare altri geni CRC in queste cellule si permettono di approfondire la progressione della malattia e la diffusione ad es. PTEN 29. Una visione più completa funzione ISC è stato ripreso da specificamente ablazione Lgr5 esprimente cellule nei topi utilizzando un recettore della tossina difterica umano (DTR) gene buttato nel Lgr5 locus 26. Altre strategie utilizzano il sistema Tet-O che consente corso espressione reversibile delle proteine ​​mutanti 30. L&#39;utilizzo di questi strumenti per modificare gene (s) in diverse cellule 31 e le posizioni 32,33 viene utilizzato per capire come avviare il cancro, il progresso e metastasi 34. In alternativa, mutagenesi utilizzando il sistema di bellezza trasposoni sonno è identificare nuovi driver di CRC. Il continuo sviluppo di topi, tecniche e strategie di alterazione genetica continua a sviluppare modelli provvisti di più pazienti. Nuovo metodi sono stati sviluppati per la caratterizzazione dei epiteli intestinali e ISC. Caratterizzazione del rapporto di tipi di cellule epiteliali può essere raggiunto mediante citometria a flusso basato sulla espressione differenziale delle lectina e CD24 35. Potenzialmente il più grande progresso nella comprensione ISC biologia e il loro ruolo nella malattia saranno effettuati utilizzando la ex vivo organoide sistema di coltura 36. Questo sistema permette ISC normali e maligne alla cultura in 3D, dove si replicano e si differenziano in modo più fisiologicamente rilevanti. Si spera che questi permetteranno la verifica diretta delle droghe su campioni di pazienti in vitro, aprendo la strada per la medicina personalizzata 37.

La superficie luminale dell&#39;intestino dei mammiferi caratteristiche ripetendo unità di cripte e dito come proiezioni, chiamato villi, che sporgono nel lume. Questa superficie è un foglio continuo di epiteli che subisce completa auto-rinnovo circa ogni 3 - 4 giorni 1. Questo tessuto dinamico è supportato da una popolazione in rapida ciclismo cellule staminali (ISC, conosciuta anche come la base cripta cellule colonnari), che inizialmente sono stati identificati per la loro espressione del gene Lgr5 2,3. Queste cellule esistono in una nicchia specializzata in fondo le cripte di Lieberkuhn. Inizialmente, la scoperta che ISC stavano rapidamente il ciclismo era discordante con l&#39;idea prevalente che una cellula staminale era quiescente in natura. Precedente all&#39;identificazione del Lgr5 + ISC è stato postulato che una popolazione di un&#39;etichetta quiescente trattenere cellule alla posizione 4, rispetto alla base della cripta, sono stati i ISC 1. Recenti ricerche hcome ora riconciliati queste osservazioni dimostrando che in primo luogo vi è una piscina di ciclismo equipotenti ISC in ogni cripta cui destino sono regolati dai suoi vicini di 4,5. Nel caso in cui si perdono questi possono essere sostituiti da cellule quiescenti che normalmente si impegnano a lignaggio secretoria ma possibile ripristinare ISCs se la popolazione ISC è danneggiato 6. Vicini ISC possono essere sia ISC o le loro cellule figlie. Le ISC producono cellule figlie naif che si moltiplicano e si differenziano in tipi di cellule specializzate che compongono il foglio epiteliale che riveste lume intestinale 1. La coppa, enteroendocrine, enterociti, ciuffo e cellule M migrano verso la superficie luminale dove forniscono varie funzioni di assorbimento e regolamentari, tuttavia, le cellule Paneth rimangono sul fondo della cripta dove esistono inframmezzati al CSI. Negli ultimi anni è stato dimostrato che una parte del daught naiveer cellule destinate a un lignaggio di secrezione sono etichetta riposo mantenendo cellule Lgr5 Lo capaci di un ritorno ad un ISC su 6,7 infortunio. Per la sua importanza nella rigenerazione cripta una priorità fu posta sulla comprensione delle interazioni tra l&#39;ISC ei suoi vicini, in particolare le cellule Paneth. Le cellule Paneth svolgono un ruolo cruciale nella nicchia che sostiene la ISC 8. Oltre ai prodotti battericidi cellule Paneth producono molecole di segnalazione che attivano le vie che regolano il rinnovo ISC o differenziazione. Precedenti studi hanno dimostrato che il Lgr5 + ISC può esistere solo quando potevano competere per i segnali di nicchia essenziali forniti dalla figlia cellule Paneth 8. Questi studi hanno esaminato il ruolo delle cellule Paneth sul normale Lgr5 + ISC e non in una situazione in cui sono danneggiati e richiedono la ricarica da una popolazione Lgr5 lo. <pclass = &quot;jove_content&quot;&gt; Per capire intestinale biologia e modello di malattia si esamina il ruolo funzionale delle cellule e / o geni che utilizzano modelli di topi transgenici 9,10. Spesso questi modelli utilizzano la tecnologia cre-lox modificare condizionale gene (s) 9,10. Cre (Cause ricombinazione) ricombinasi è un ricombinasi site specific della famiglia dell&#39;integrasi, isolato dal batteriofago P1. Cre catalizza ricombinazione sito specifico tra definita 34 bp &#39; Lox P &#39;(locus χ di P1 di crossover) siti. I topi sono geneticamente per contenere siti loxP che fiancheggiano le regioni di interesse che al momento espressione della Cre ricombinasi sono rimossi. Collegando l&#39;espressione del gene Cre a una cella o di sviluppo promotore specifico permette di modifica da effettuare in modo spaziale 9,10, questo è particolarmente utile nel superare mutazioni letali embrionali. Inoltre collegando l&#39;espressione Cre di un percorso del recettore,attivabile artificialmente, permette alterazioni temporali. Grazie a questa tecnologia abbiamo inattivato il gene CatnB 11 nei epiteli intestinali. β-catenina, il prodotto del gene CatnB, è un regolatore chiave della via di segnale Wnt canonica che regola ISC omeostasi. Due studi precedenti utilizzando questa strategia hanno prodotto risultati contrastanti 12,13. Lo studio di févr et al. 12, hanno dimostrato la perdita di cellule staminali e omeostasi intestinale. Considerando che l&#39;Irlanda et al. 14 studio ha riportato che a seguito di una riduzione della vitalità cellulare sull&#39;asse cripta-villo fu ripopolata da cellule wild type che esprimono CatnB. La principale differenza in questi studi è stato il promotore usata per esprimere Cre nei epiteli intestinali. La FEVR et al., Studio ha utilizzato il promotore del gene villin legato al recettore degli estrogeni che può essere attivato somministrando tamoxifen (vil-Cre-ER T2) 15,16. In contrasto con l&#39;Irlanda et al., Utilizzato l&#39;elemento promotore del gene del ratto del citocromo P450A1 (CYP1A1) per guidare l&#39;espressione Cre in risposta alla xenobiotici β-naftoflavone (Ah-cre). Le caratteristiche di questi diversi sistemi generati due ipotesi per spiegare queste diverse osservazioni. Il primo che CatnB è più efficiente cancellata ISC utilizzando il sistema T2 vil-Cre-ER rispetto al Ah-cre, riducendo così il numero di ISC a livelli sub-ripopolamento. In alternativa era causata dalla differenza CatnB delezione nella popolazione di cellule differenziate. Le vil-Cre-ER obiettivi del sistema T2 tutte le cellule epiteliali della cripta e villi, mentre il sistema di Ah-cre si riferisce solo alle cellule non Paneth della nicchia ISC e la cripta. Questi sistemi forniti strumenti ideali per l&#39;esame della behavior della ISC e la loro interazione con le cellule Paneth. Qui vi presentiamo diversi protocolli dettagliati in base a come abbiamo usato questi sistemi per determinare che le cellule Paneth svolgono un ruolo cruciale nel mediare la risposta al danno intestinale 17.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Acetate buffer</td>
			<td>*</td>
			<td>*</td>
			<td>To make 100ml: 4.8ml 0.2M Acetic acid, 45.2ml 0.2M Sodium acetate &#38; 50ml distilled water</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Fisher Scientific</td>
			<td>C/0400/PB17</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic anhydride</td>
			<td>Sigma</td>
			<td>A6404</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic anhydride solution</td>
			<td>*</td>
			<td>*</td>
			<td>2 M Acetic anhydride in 0.1 M triethanolamine hydrochloride</td>
		</tr>
		<tr>
			<td>Alcian Blue</td>
			<td>Sigma</td>
			<td>A5268</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue ph2.5</td>
			<td>*</td>
			<td>*</td>
			<td>To make 500ml: 15ml acetic acid, 5g Alcian Blue &#38; 485ml distilled water</td>
		</tr>
		<tr>
			<td>anti-digoxigenin alkaline phosphatase conjugated antibody</td>
			<td>Abcam</td>
			<td>ab119345</td>
			<td></td>
		</tr>
		<tr>
			<td>B(beta)-Naphthoflavone</td>
			<td>Sigma</td>
			<td>N3633</td>
			<td>BNF, inject without allowing the solution to cool too much as compound will drop out of solution. Solution can be re-used &#8211; store at -20&#176;C between uses, do not reheat more than twice.</td>
		</tr>
		<tr>
			<td>Bloxall</td>
			<td>Vector Labs</td>
			<td>SP-6000</td>
			<td></td>
		</tr>
		<tr>
			<td>BM purple</td>
			<td>Roche</td>
			<td>11442074001</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A4503</td>
			<td>Bovine serum albumin</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C/4920/17</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate Buffer/Antigen Unmasking Solution</td>
			<td>Vector Labs</td>
			<td>H-3300</td>
			<td></td>
		</tr>
		<tr>
			<td>Corn oil</td>
			<td>Sigma</td>
			<td>C8627</td>
			<td></td>
		</tr>
		<tr>
			<td>Demucifiying solution</td>
			<td>*</td>
			<td>*</td>
			<td>For 500ml: 50ml glycerol, 50ml Tris 0.1M pH8.8, 100ml EtOH, 300ml saline (0.9% NaCl in water), DTT 1.7g.&#160; Demucifying solution can be made in advance and stored, but DTT sholud be added just before incubation (340mg/100ml).</td>
		</tr>
		<tr>
			<td>DEPC treated water</td>
			<td>Life Technologies</td>
			<td>750023</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>101509944</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>O3690</td>
			<td>0.5M</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>E/0650DF/17</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>3000917</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin&#160;</td>
			<td>Sigma</td>
			<td>SLBL11382V</td>
			<td>Neutral buffered formalin</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma</td>
			<td>F5786</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehye</td>
			<td>Sigma</td>
			<td>G6257</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma</td>
			<td>216763</td>
			<td></td>
		</tr>
		<tr>
			<td>Haematoxylin</td>
			<td>Raymond A Lamb</td>
			<td>12698616</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14175-053</td>
			<td>HBSS (-MgCl2+; -CaCl2)</td>
		</tr>
		<tr>
			<td>Hybridisation buffer</td>
			<td>*</td>
			<td>*</td>
			<td>&#160;5&#215; SSC, 50% formamide, 5% SDS, 1 mg/ml heparin, 1 mg/ml calf liver tRNA</td>
		</tr>
		<tr>
			<td>Hydroquinone</td>
			<td>Sigma</td>
			<td>H9003</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmPACT DAB Peroxidase</td>
			<td>Vector Labs</td>
			<td>SK-4105</td>
			<td></td>
		</tr>
		<tr>
			<td>Immpress HRP Anti-Mouse IgG Kit</td>
			<td>Vector Labs</td>
			<td>MP-7402</td>
			<td></td>
		</tr>
		<tr>
			<td>Immpress HRP Anti-Rabbit IgG Kit</td>
			<td>Vector Labs</td>
			<td>MP-7401</td>
			<td></td>
		</tr>
		<tr>
			<td>Intestinal tissue powder</td>
			<td>*</td>
			<td>*</td>
			<td>The small intestines of 5 adult mice were combined and homogenised in the minimum volume of ice cold PBS. 4 volumes of ice cold acetone were added to the homogenised intestine, which was mixed thoroughly and incubated on ice for 30 minutes. This was centrifuged and the pellet was washed using ice cold acetone. This was further centrifuged and the resulting pellet spread onto filter paper and allowed to dry. Once thoroughly dry the material was ground to a fine powder using a pestle and mortar.&#160;</td>
		</tr>
		<tr>
			<td>K-ferricyanide</td>
			<td>Sigma</td>
			<td>P-3667</td>
			<td></td>
		</tr>
		<tr>
			<td>K-ferrocyanide</td>
			<td>Sigma</td>
			<td>P3289</td>
			<td></td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>Sigma</td>
			<td>L0380000</td>
			<td></td>
		</tr>
		<tr>
			<td>Methacarn</td>
			<td>*</td>
			<td>*</td>
			<td>60% Methanol:30% Chloroform:10% Acetic acid</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>M/4000/17</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Vector Labs</td>
			<td>S-1012</td>
			<td>NGS</td>
		</tr>
		<tr>
			<td>Normal rabbit serum</td>
			<td>Dako</td>
			<td>X0902</td>
			<td>NRS</td>
		</tr>
		<tr>
			<td>NTMT</td>
			<td>*</td>
			<td>*</td>
			<td>100 mM NaCl, 100 mM Tris HCl, 50 mM MgCl2, 0.1% Tween20, 2 mM Levamisole</td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Vector</td>
			<td>H-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>PBT</td>
			<td>*</td>
			<td>*</td>
			<td>0.5M NaCl, 10mM TrisHCL pH7.5, 0.1% Tween 20</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100x solutiuon.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (10x)</td>
			<td>Fisher Scientific</td>
			<td>BP3994</td>
			<td>Dilluted 1:10 with distilled water to make 1x</td>
		</tr>
		<tr>
			<td>PLL slides</td>
			<td>Sigma</td>
			<td>P0425-72EA</td>
			<td>Poly-L-lysine microscope slides</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma</td>
			<td>P2308</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase k solution</td>
			<td>*</td>
			<td>*</td>
			<td>Dilute Proteinase K at 200 &#181;g/ml in 50 mM Tris, 5 mM EDTA.</td>
		</tr>
		<tr>
			<td>Ralwax</td>
			<td>BDH</td>
			<td>36154 7N</td>
			<td></td>
		</tr>
		<tr>
			<td>Reducer Solution</td>
			<td>*</td>
			<td>*</td>
			<td>To make 100ml: 1g Hydroquinone, 5g sodium sulphite &#38; 100ml distilled water</td>
		</tr>
		<tr>
			<td>RnaseA</td>
			<td>Sigma</td>
			<td>R6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>*</td>
			<td>*</td>
			<td>0.9% NaCl in distilled water</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma</td>
			<td>I3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep serum</td>
			<td>Sigma</td>
			<td>S3772</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Sigma</td>
			<td>S/1240/46</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver solution</td>
			<td>*</td>
			<td>*</td>
			<td>To make 100ml: 10ml Acetate buffer, 87ml distilled water, 3ml 1% silver nitrate</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Fisher Scientific</td>
			<td>S/2120/53</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S6753</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Sodium sulfite</td>
			<td>Sigma</td>
			<td>239321</td>
			<td></td>
		</tr>
		<tr>
			<td>SSC</td>
			<td>Sigma</td>
			<td>93017</td>
			<td>20x saline sodium citrate</td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td>Fisher Scientific</td>
			<td>12960495</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td>TAM, inject without allowing the solution to cool too much as compound will drop out of solution. Solution can be re-used &#8211; store at -20&#176;C between uses, do not reheat more than twice.</td>
		</tr>
		<tr>
			<td>TBS/T</td>
			<td>Cell Signalling</td>
			<td>#9997</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine hydrochloride</td>
			<td>Sigma</td>
			<td>T1502</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCL</td>
			<td>Invitrogen</td>
			<td>15567-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Sigma</td>
			<td>TP9416</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaMount</td>
			<td>Vector Labs</td>
			<td>H-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaShield Hardset mounting Medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain ABC Kit</td>
			<td>Vector Labs</td>
			<td>PK-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Promega</td>
			<td>V3941</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal fixative</td>
			<td>*</td>
			<td>*</td>
			<td>2% formaldehyde, 0.1% glutaraldehyde in 1xPBS</td>
		</tr>
		<tr>
			<td>X-gal stain</td>
			<td>*</td>
			<td>*</td>
			<td>X-gal stain; 200ul X-gal (A) in 50ml solution B (0.214g MgCl2, 0.48g K-ferricyanide, 0.734g K-ferrocyanide in 500ml PBS).&#160; Solution B can be made up oin advance and stored at 4&#176;C</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>X/0200/21</td>
			<td></td>
		</tr>
	</tbody>
</table>

protocols for analyzing the role of paneth cells in regenerating the murine intestine using conditional cre-lox mouse models

La superficie epiteliale intestinale di mammifero è un tessuto dinamico che rinnova ogni 3 - 7 giorni. La comprensione di questo processo di rinnovamento identificato una popolazione di cellule staminali in rapida ciclismo intestinali (ISC) caratterizzati dalla loro espressione del gene Lgr5. Questi sono supportati da una popolazione di cellule staminali quiescenti, caratterizzato da Bmi-1, in grado di sostituirle in caso di lesioni. Indagare le interazioni tra queste popolazioni è fondamentale per capire il loro ruolo nella malattia e il cancro. Le ISC esistono all&#39;interno cripte sulla superficie intestinale, queste nicchie supportano l&#39;ISC nel rifornire epiteli. L&#39;interazione tra ISC attive e quiescenti probabilmente coinvolge altre cellule differenziate all&#39;interno della nicchia, così come è già stato dimostrato che la &#39;&#39; staminalità &#39;&#39; del Lgr5 ISC è strettamente legata alla presenza dei loro vicini cellule Paneth. Utilizzando il mouse condizionale cre-loxmodelli abbiamo testato l&#39;effetto di eliminare la maggior parte delle ISC attivi in ​​presenza o assenza di cellule Paneth. Qui si descrivono le tecniche e le analisi svolte per caratterizzare l&#39;intestino e dimostrano che le cellule Paneth svolgono un ruolo fondamentale all&#39;interno della nicchia ISC in favoreggiamento recupero dopo sostanziale insulto.

Watch this Scientific Journal Video about Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models at JoVE.com

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Cellule intestinali staminali epiteliali (ISC) sono mescolati con le cellule Paneth. Queste cellule sono differenziate progenie della ISC, che supportano la ISC e fornire protezione antibatterica. Qui mostriamo come abbiamo utilizzato modelli transgenici condizionali mouse per stabilire che le cellule Paneth svolgono un ruolo cruciale nel mantenimento degli epiteli intestinali.

Protocolli per l&#39;analisi del ruolo delle cellule Paneth in Rigenerazione della murino Intestino con condizionale<em&gt; Cre-lox</em&gt; Modelli mouse

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

protocols-for-analyzing-role-paneth-cells-regenerating-murine

Research

JoVE Journal

Developmental Biology

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

17.5K Views.  Cardiff University.  Cellule intestinali staminali epiteliali (ISC) sono mescolati con le cellule Paneth. Queste cellule sono differenziate progenie della ISC, che supportano la ISC e fornire protezione antibatterica. Qui mostriamo come abbiamo utilizzato modelli transgenici condizionali mouse per stabilire che le cellule Paneth svolgono un ruolo cruciale nel mantenimento degli epiteli intestinali. 

Video: Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

La produzione di cellule staminali pluripotenti da mouse amniotico cellule del liquido Utilizzando un sistema di trasposoni

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Ricerca

Biologia dello sviluppo

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

In Vitro Crescita dei follicoli preantrali Mouse sotto condizioni di microgravità simulata

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Imaging Live di Mouse Seconda Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

Un base di RANKL Osteoclast cultura Assay di midollo osseo del topo per studiare il ruolo di mTORC1 nella formazione degli osteoclasti

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Applicazione di RNAi ed espressione di fattore di trascrizione indotta da calore-shock per riprogrammare le cellule germinali ai neuroni in c. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

Valutazione Lysosomal alcalinizzazione l' intestino di Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Analisi computazionale della linea germinale Caenorhabditis elegans di studiare la distribuzione dei Nuclei, le proteine ed il citoscheletro

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualizzare il nodo e la piastra Notochordal In embrioni di topo Gastrulating mediante microscopia elettronica a scansione e intero Monte immunofluorescenza

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Modelli di lesione di Zebrafish adulto per studiare gli effetti del Prednisolone nella rigenerazione di tessuto osseo

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

Valutazione dei danni ossidativi nelle cellule primarie della superficie oculare del topo/cellule staminali in risposta ai danni ultravioletti-C (UV-C)

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...