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.

En utilisant des souris transgéniques de Cre-lox conditionnelles à disséquer la fonction des gènes et des cellules est une approche couramment utilisée. Ces modèles ont été utilisés avec succès dans l&#39;intestin pour identifier et caractériser les cellules souches 2,4-6 et de comprendre leur rôle dans la maladie 25. Pour exploiter pleinement ces modèles requiert une caractérisation complète du système pour permettre aux données d&#39;être interprété correctement. Une compréhension complète de ces systèmes est difficile à réaliser en raison de gènes étant rarement spécifiques à un type de cellule d&#39;isolement ou de l&#39;emplacement, un manque de connaissances biologiques et l&#39;inefficacité des systèmes utilisés pour induire l&#39;expression Cre. Les méthodes décrites ici montrent comment nous surmontons ces questions par le biais de la conception expérimentale et l&#39;application des connaissances existantes. Bien que nous avons utilisé ces méthodes pour répondre à une question spécifique de recherche les techniques présentées ici sont génériques et peuvent être exploitées pour toute recherche portant sur la muriintestin ne. Préparation du tissu intestinal L&#39;étape cruciale pour assurer des résultats robustes est la récolte et la transformation du tissu, qui doit être traité en temps opportun des protocoles de modalités et la fixation strictement respectés. Comme la quasi-totalité des questions importantes peuvent être attribués à l&#39;aval des artefacts liés à l&#39;assèchement des tissus sur et / ou la fixation incomplète. Le timing est crucial pour empêcher la dégradation de l&#39;architecture tissulaire et / ou des acides nucléiques et des protéines. Fixation incomplète ou trop zélé peut entraîner la perte de la résolution histochimique. Fixation incomplète en raison de temps ou sections trop épaisse pour permettre la pénétration du fixateur peut entraîner la perte de la résolution dans les cryptes intestinales qui peuvent être observés comme une «marque de marée» sur l&#39;analyse IHC insuffisante. En outre, il est crucial que la fixation ne se prolonge pas trop longtemps, comme β-caténine nucléaire peut diffuser hors du noyau à moins immédiatement proétat brut et la cire incorporé après la fixation du formol. Rôle des cellules de Paneth dans la niche ISC Les données présentées ici montrent effectivement l&#39;importance des cellules de Paneth dans la crypte de régénération dans l&#39;intestin de l&#39;adulte en cas de perte ISC. Cependant, il restait la possibilité que Ah-cre épargne une population de CSI que les objectifs de T2 vil-Cre-ER. Tian et al. 26 élégamment démontré que les CSI hi LGR5 sont remplacés par une population de LGR5 lo CSI de réserve. Il semble maintenant probable que ces entrepreneurs indépendants sont épargnés dans le système Ah-cre en raison de la population de réserve ayant été identifié comme précurseurs de cellules de sécrétion 6,7. L&#39;importance de la cellule mûre Paneth au soutien de ces précurseurs de cellules sécrétoires en cas de besoin de revenir à un état ISC reste à répondre. Comme les cellules de Paneth constituent le jeSC niche 8 et jouer un rôle dans la régulation des réponses Icc à l&#39;apport calorique et l&#39;inflammation 28 27 il reste probable que leurs fonctions infirmières vont étendre à leurs propres précurseurs. Nouvelles approches et technologies pour modéliser efficacement du cancer colorectal humain La découverte de l&#39;ISC a conduit à l&#39;identification des gènes qui sont actuellement utilisés pour générer de nouveaux modèles de souris pour étudier les rôles des gènes et des cellules en biologie et les maladies intestinales, examiné par Clarke et al 9. Les seules limitations de cette technique est l&#39;identification des gènes à exprimer la protéine Cre. Actuellement CSI sont systématiquement étudiés en utilisant des souris transgéniques conditionnels basés sur le profil d&#39;expression génique LGR5. Les souris qui expriment Cre à partir du promoteur LGR5 ont été utilisées pour supprimer APC, le gène le plus fréquemment muté dans le cancer colorectal (CRC), Ce qui démontre l&#39;ISC que la cellule 25 d&#39;origine. Suppression sélective d&#39;autres gènes dans ces cellules CRC fournit un aperçu de progression de la maladie et la propagation par exemple. PTEN 29. En outre aperçu ISC fonction est extraite par l&#39;ablation de cellules exprimant spécifiquement LGR5 chez la souris en utilisant un récepteur de la toxine diphtérique humain (DTR) dans le gène frappé LGR5 26 locus. D&#39;autres stratégies utilisent le système Tet-O qui permet l&#39;expression réversible cours des 30 protéines mutantes. En utilisant ces outils pour modifier gène (s) dans différentes cellules 31 et 32,33 emplacements est utilisée pour comprendre comment le cancer initier, de progrès et métastaser 34. Alternativement mutagenèse en utilisant le système de transposon beauté de couchage est d&#39;identifier de nouveaux pilotes de CRC. La poursuite du développement des souris, des techniques et des stratégies de modification génétique continue de développer des modèles plus pertinents patients. Nouveau modes ont été développés pour la caractérisation des épithéliums et CSI intestinaux. Caractérisation du ratio de types de cellules epitheliales peuvent être obtenus en utilisant la cytométrie de flux basé sur l&#39;expression différentielle de 35 lectine et CD24. Potentiellement, le plus grand progrès dans la compréhension de la biologie ISC et leur rôle dans la maladie seront effectués en utilisant l&#39;ex vivo organoïde système de culture 36. Ce système permet CSI normales et malignes à la culture en 3D, où ils se répliquent et se différencient d&#39;une manière plus physiologiquement pertinent. Il est à espérer que ces tests permettront directe des médicaments sur des échantillons de patients in vitro, ouvrant la voie à la médecine personnalisée 37.

La surface luminale de l&#39;intestin des mammifères caractéristiques des unités de cryptes et de répéter doigt comme des projections, appelées villosités, qui font saillie dans la lumière. Cette surface est une feuille continue de l&#39;épithélium qui subit une auto-renouvellement complet environ tous les 3 - 4 jours 1. Ce tissu dynamique est supporté par une population de vélo rapidement les cellules souches (CSI; également connu en tant que base de crypte des cellules cylindriques), qui ont été initialement identifiés par leur expression du gène LGR5 2,3. Ces cellules existent dans un créneau spécialisé au fond des cryptes de Lieberkuhn. Initialement, la découverte que les CSI sont rapidement cyclisme est discordante avec l&#39;idée répandue selon laquelle une cellule souche est au repos dans la nature. Avant l&#39;identification de la LGR5 + ISC il a été postulé qu&#39;une population de cellules retenant l&#39;étiquette de repos à la position 4, par rapport à la base de la crypte, ont été les CSI 1. Des recherches récentes hcomme maintenant réconcilié ces observations en démontrant que surtout il ya une piscine du cyclisme équipotente ISC dans chaque crypte dont le sort sont régis par ses voisins de 4,5. Dans le cas où ils sont perdus ceux-ci peuvent être remplacés par des cellules au repos qui habituellement se sont engagés à la lignée de sécrétion, mais peut revenir à CSI si la population ISC est endommagé 6. Voisins ISC peuvent être soit des entrepreneurs indépendants ou leurs cellules filles. Les entrepreneurs indépendants produisent des cellules filles naïves qui se multiplient et se différencient en types de cellules spécialisées qui composent la feuille épithéliale qui tapisse les lumière intestinale 1. Le gobelet, entéroendocrine, entérocytes, touffe et les cellules M migrent vers le haut pour la surface luminale où ils fournissent diverses fonctions d&#39;absorption et réglementaires, cependant, les cellules de Paneth restent au fond de la crypte où ils existent entremêlées avec l&#39;ISC. Ces dernières années, il a été démontré qu&#39;une proportion de la daught naïfcellules er destinés à une lignée de sécrétion sont étiquette de repos en conservant les cellules LGR5 lo capables de revenir à un entrepreneur indépendant sur ​​blessure 6,7. En raison de son importance dans la régénération crypte une priorité a été placée sur la compréhension des interactions entre la CSI et ses voisins, en particulier les cellules de Paneth. Les cellules de Paneth jouent un rôle crucial dans la niche qui soutient la CSI 8. En plus des produits bactéricides les cellules de Paneth produisent des molécules qui activent les voies qui régissent le renouvellement ISC ou la différenciation de signalisation. Des études antérieures ont montré que le LGR5 + CSI ne pouvait exister quand ils pourraient concurrencer pour les signaux de niche essentiels fournis par leur fille cellules de Paneth 8. Ces études ont examiné le rôle des cellules de Paneth sur LGR5 normale + CSI et non pas dans une situation où ils sont endommagés et nécessitent reconstitution d&#39;une population LGR5 lo. <pclass = &quot;jove_content&quot;&gt; Pour comprendre la biologie et le modèle maladie intestinale nous examinons le rôle fonctionnel de cellules et / ou des gènes en utilisant des modèles de souris transgéniques 9,10. Souvent, ces modèles utilisent la technologie cre-lox de modifier conditionnellement gène (s) 9,10. Cre (causes recombinaison) recombinase est une recombinase spécifique du site de la famille de l&#39;intégrase, isolé de bactériophage P1. Cre catalyse recombinaison spécifique de site entre défini 34 pb &#39; Lox P &#39;(locus χ de CrossOver P1) sites. Les souris sont génétiquement modifiées pour contenir des sites LoxP qui flanquent les régions d&#39;intérêt qui lors de l&#39;expression de la recombinase Cre sont excisés. Lier l&#39;expression du gène Cre à une cellule ou promoteur spécifique de développement permet de changement qui pourra être fait de façon spatiale 9,10, ce qui est particulièrement utile pour surmonter les mutations létales embryonnaires. Relierons encore davantage l&#39;expression Cre à une voie du récepteur,qui peuvent être activés artificiellement, permet des modifications temporelles. Grâce à cette technologie, nous inactivé le gène CatnB 11 dans l&#39;épithélium intestinal. β-caténine, le produit du gène CatnB, est un régulateur clé de la voie de signalisation Wnt canonique qui régit l&#39;homéostasie ISC. Deux études précédentes en utilisant cette stratégie a produit des résultats contradictoires 12,13. L&#39;étude par févr et al. 12, a montré une perte de cellules souches et de l&#39;homéostasie intestinale. Alors que l&#39;Irlande et al. 14 étude a signalé que suite à une réduction de la viabilité cellulaire de l&#39;axe crypte-villosité a été repeuplé à partir de cellules de type sauvage exprimant CatnB. La principale différence dans ces études était le promoteur utilisé pour exprimer la Cre dans l&#39;épithélium intestinal. La févr et al., Étude a utilisé le promoteur du gène de la villine lié au récepteur d&#39;œstrogène qui peut être activé par administration de tamoxifen (vil-Cre-ER T2) 15,16. En revanche Ireland et al., Utilise l&#39;élément promoteur du gène du cytochrome rat P450A1 (CYP1A1) pour diriger l&#39;expression Cre en réponse à la β-naphtoflavone xénobiotique (Ah-cre). Les caractéristiques de ces différents systèmes ont généré deux hypothèses pour expliquer ces différentes observations. La première est que CatnB plus efficacement supprimée dans la CAI en utilisant le système de T2 vil-Cre-ER par rapport à la Ah-cre, réduisant ainsi le nombre d&#39;entrepreneurs indépendants à des niveaux sous-repeuplement. Alternativement, il est dû à une différence de CatnB suppression dans la population de cellules différenciées. Le système T2 vil-Cre-ER cibles toutes les cellules épithéliales de la crypte et de villosités alors que le système Ah-cre ne cible les cellules non-Paneth de la niche ISC et la crypte. Ces systèmes constituent des outils idéaux pour examiner le comporIOR de l&#39;ISC et leur interaction avec les cellules de Paneth. Ici, nous présentons plusieurs protocoles détaillés basés sur la façon dont nous avons utilisé ces systèmes pour déterminer que les cellules de Paneth jouent un rôle crucial dans la médiation de la réponse à une lésion 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 surface de l&#39;épithélium de l&#39;intestin des mammifères est un tissu dynamique qui renouvelle toutes les 3 - 7 jours. Comprendre ce processus de renouvellement identifié une population de vélo rapidement les cellules souches intestinales (CSI) caractérisées par leur expression du gène LGR5. Ceux-ci sont prises en charge par une population de cellules souches quiescentes, marqué par Bmi-1 expression, capable de les remplacer en cas de dommage. Enquêter sur les interactions entre ces populations est cruciale pour comprendre leurs rôles dans la maladie et le cancer. Les entrepreneurs indépendants existent dans des cryptes sur la surface intestinale, ces niches soutiennent l&#39;ISC à reconstituer l&#39;épithélium. L&#39;interaction entre les ISC actives et inactives implique probablement d&#39;autres cellules différenciées dans le créneau, comme il a déjà été démontré que le &#39;&#39; stemness &#39;&#39; de l&#39;LGR5 ISC est étroitement liée à la présence de leurs cellules de Paneth voisins. Utilisation conditionnelle souris cre-loxon modèles testés pour effet de supprimer la majorité des CSI actifs dans la présence ou l&#39;absence des cellules de Paneth. Nous décrivons ici les techniques et les analyses menées pour caractériser l&#39;intestin et démontrent que les cellules de Paneth jouent un rôle crucial dans le créneau ISC en aidant la récupération après l&#39;insulte substantielle.

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

Cellules souches épithéliales intestinales (CSI) sont entremêlées avec des cellules de Paneth. Ces cellules se différencient descendance de l&#39;ISC, qui soutiennent la CSI et de fournir une protection antibactérienne. Ici, nous démontrons comment nous avons utilisé des modèles de souris transgéniques conditionnelles pour établir que les cellules de Paneth jouent un rôle crucial dans le maintien de l&#39;épithélium intestinal.

Protocoles pour analyser le rôle des cellules de Paneth dans la régénération de l&#39;intestin murin utilisant conditionnelle<em&gt; Cre-lox</em&gt; Modèles de souris

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 vues.  Cardiff University.  Cellules souches épithéliales intestinales (CSI) sont entremêlées avec des cellules de Paneth. Ces cellules se différencient descendance de l'ISC, qui soutiennent la CSI et de fournir une protection antibactérienne. Ici, nous démontrons comment nous avons utilisé des modèles de souris transgéniques conditionnelles pour établir que les cellules de Paneth jouent un rôle crucial dans le maintien de l'épithélium intestinal. 

Vidéo: 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 production de cellules souches pluripotentes de souris amniotique Cellules Fluid Utilisation d&#39;un système de transposon

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

Recherche

Biologie du développement

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 Croissance des follicules Preantral souris sous microgravité simulée

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.

Imagerie en direct de la souris Fusion de palais secondaire

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.

Une base RANKL Osteoclast Culture dosage de moelle osseuse de souris pour enquêter sur le rôle de mTORC1 dans la Formation des ostéoclastes

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.

Application de RNAi et Expression du facteur de Transcription induite par la chaleur-choc pour reprogrammer les cellules germinales de neurones chez 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)

Évaluer l’alcalinisation Lysosomal dans l' intestin de vivre 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.

Analyse de calcul de la lignée germinale de Caenorhabditis elegans pour étudier la Distribution des noyaux, les protéines et le cytosquelette

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).

Visualiser le nœud et la notochorde plaque en Gastrulating des embryons de souris à l’aide de la microscopie électronique à balayage et toute monture Immunofluorescence

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.

Modèles de lésion du poisson-zèbre adulte pour étudier les effets de la Prednisolone dans la régénération de tissu osseux

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.

Évaluation des dommages oxydatifs dans les cellules de surface oculaires de la souris primaire/cellules souches en réponse aux dommages causés par les ultraviolets-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 ...