Name: protocols for analyzing the role of paneth cells in regenerating the murine intestine using conditional cre-lox mouse models
Uploaded: 2015-11-21T14:00:00
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Description: 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

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.

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The authors have nothing to disclose.

Brug betingede CRE-lox transgene mus til at dissekere funktionen af gener og celler er et almindeligt anvendt metode. Disse modeller har været anvendt med stor succes i tarmen til at identificere og karakterisere de stamceller 2,4-6 og forstå deres rolle i sygdom 25. For fuldt ud at udnytte disse modeller kræver en omfattende karakteristik af systemet for at gøre det muligt for data, der skal fortolkes korrekt. En fuldstændig forståelse af disse systemer er vanskeligt at opnå på grund af gener sjældent er specifik for en enkelt celletype eller placering, manglende biologiske viden og ineffektivitet i de systemer, der anvendes til at inducere Cre-ekspression. De her beskrevne metoder vise, hvordan vi overvinde disse spørgsmål gennem eksperimentelle design og anvendelse af eksisterende viden. Selvom vi brugt disse metoder til at besvare et bestemt forskningsspørgsmål de teknikker præsenteres her er generiske og kan bruges til nogen forskning undersøger Murine tarmen. Fremstilling af tarmvæv Det afgørende skridt for at sikre robuste resultater er høstning og forarbejdning af vævet, som skal behandles i tide og fiksering protokoller nøje. Som næsten alle væsentlige spørgsmål nedstrøms kan tilskrives artefakter forbundet med vævet udtørring og / eller ufuldstændig fiksering. Timing er afgørende for at forhindre nedbrydning af væv arkitektur og / eller nukleinsyrer og proteiner. Ufuldstændige eller nidkære fiksering kan resultere i tab af histokemisk opløsning. Ufuldstændig fiksering på grund af utilstrækkelig tid eller sektioner for tykke til at tillade fiksativ indtrængning kan resultere i tab af opløsning inden for de intestinale krypter, der kan iagttages som et &quot;tidevand mark&quot; på IHC-analyse. Endvidere er det afgørende, at fiksering ikke strækker sig for længe, ​​som det nukleare β-catenin kan diffundere ud af kernen, medmindre omgående probejdet og voks indlejret efter formalin fiksering. Rolle Paneth celler i ISC Niche De data, der præsenteres her, viser effektivt betydningen af ​​Paneth celler i krypt regenerering i den voksne tarmen efter ISC tab. Men der var fortsat den mulighed, at Ah-CRE skåner en befolkning på individuelle servicekontrakter, at will-Cre-ER T2 mål. Tian et al. 26 elegant påvist, at Lgr5 hi individuelle servicekontrakter er erstattet af en befolkning på Lgr5 lo reserve individuelle servicekontrakter. Det virker nu sandsynligt, at disse individuelle servicekontrakter er sparet i Ah-CRE systemet på grund af reserven befolkningen er blevet identificeret som sekretorisk celle forstadier 6,7. Betydningen af ​​den modne Paneth celle i støtte disse sekretoriske celleforstadier når det er nødvendigt for at vende tilbage til en ISC staten mangler at blive besvaret. Som Paneth celler udgør ISC niche 8 og spille roller i reguleringen af ISC reaktioner på kalorieindtag 27 og inflammation 28 er det stadig sandsynligt, at deres sygepleje funktioner vil strække sig til deres egne forstadier. Nye metoder og teknologier til effektivt Model Menneskelig kolorektal cancer Opdagelsen af ISC førte til identifikation af gener, som nu bliver brugt til at generere nye musemodeller for at undersøge roller gener og celler i tarm biologi og sygdom, gennemgået af Clarke et al 9. De eneste begrænsninger for denne teknik er identifikationen af ​​gener at udtrykke Cre protein. I øjeblikket ISC rutinemæssigt undersøgt under anvendelse betingede transgene mus baseret på Lgr5 genekspression mønster. Mus, der udtrykker Cre fra Lgr5 promotoren er blevet anvendt til at slette APC genet hyppigst muteret i colorektal cancer (CRC), Hvilket viser ISC som celle af 25. Selektivt at slette andre CRC gener i disse celler er at give indsigt i sygdomsprogression og spredes f.eks. PTEN 29. Yderligere indsigt i ISC-funktionen bliver hentet ved specifikt ablation Lgr5 udtrykkende celler i mus ved hjælp af en menneskelig difteri toxin-receptor (DTR) genet bankede ind i Lgr5 locus 26. Andre strategier bruge Tet-O, ​​som muliggør løbende reversibel ekspression af mutantproteiner 30. Ved hjælp af disse værktøjer til at ændre genet (r) i forskellige celler 31 og steder 32,33 bruges til at forstå, hvordan kræft initiere, fremskridt og metastaserer 34. Alternativt mutagenese ved hjælp af sovende skønhed transposonet systemet identificere nye førere af CRC. Den fortsatte udvikling af mus, teknikker og genetiske ændringer strategier fortsætter med at udvikle mere patient relevante modeller. Ny metoder er blevet udviklet til karakterisering af tarmens epitel og individuelle servicekontrakter. Karakterisering af forholdet mellem epitelcelletyper kan opnås ved anvendelse af flowcytometri baseret på differentiel ekspression af lectin og CD24 35. Potentielt den største fremgang i forståelsen ISC biologi og deres rolle i sygdommen vil blive foretaget ved hjælp af ex vivo organoide kultur-system 36. Dette system gør det muligt for normale og maligne individuelle servicekontrakter til kultur i 3D, hvor de replikere og differentiere i en mere fysiologisk relevant måde. Det er håbet, at disse vil gøre det muligt direkte test af lægemidler på patientprøver in vitro, hvilket banede vejen for personlig medicin 37.

Den luminale overflade af pattedyr-tyndtarm funktioner gentagelsesenheder af krypter og finger fremspring, betegnet villi, som rager ind i hulrummet. Denne overflade er en kontinuerlig plade af epitel, der undergår fuldstændig selvfornyelse ca. hver 3 - 4 dage 1. Denne dynamiske væv understøttes af en population af kort cyklustid stamceller (HTT; også kendt som krypt basen søjleformede celler), som oprindeligt blev identificeret ved deres ekspression af genet Lgr5 2,3. Eksisterer disse celler i en niche i bunden af ​​krypterne af Lieberkuhn. I første omgang, den opdagelse, at indgå individuelle servicekontrakter blev hurtigt cykling var uharmonisk med den fremherskende idé om, at en stamcelle var inaktiv i naturen. Forud for identifikation af Lgr5 + ISC det blev postuleret, at en population af hvilende etiket fastholde cellerne på fire stilling i forhold til bunden af krypten, var ISC 1. Nyere forskning hsom nu forenes disse observationer ved at demonstrere, at primært er der en pulje af ækvipotent cykling individuelle servicekontrakter i hvert krypten hvis skæbne er reguleret af sine naboer 4,5. I tilfælde af, de er tabt disse kan erstattes af hvilende celler, der normalt er forpligtet til det sekretoriske afstamning, men kan gå tilbage til individuelle servicekontrakter, hvis ISC befolkning er beskadiget 6. ISC naboer kan enten være individuelle servicekontrakter eller deres datterceller. De individuelle servicekontrakter producerer naive datterceller, som formerer sig og differentierer i de specialiserede celletyper, der omfatter epithelial ark, som linjer tarmlumen 1. Pokalen, enteroendokrin, enterocytter, tot og M celler migrerer opad til den luminale overflade, hvor de giver forskellige absorberende og regulerende funktioner, men forbliver Paneth celler i bunden af ​​krypten, hvor de findes blandet med individuelle servicekontrakter. I de senere år er det blevet vist, at en del af det naive daughtER celler bestemt til en sekretorisk afstamning er hvilende etiket fastholde Lgr5 lo celler er i stand til at vende tilbage til en ISC efter skade 6,7. På grund af sin betydning i krypt regenerering prioriteres blev placeret på at forstå samspillet mellem individuelle servicekontrakter og dets naboer, især Paneth cellerne. De Paneth celler spiller en afgørende rolle i den niche, der understøtter ISC 8. Udover bakteriedræbende produkter de Paneth celler producerer signalmolekyler, der aktiverer de veje, der styrer ISC fornyelse eller differentiering. Tidligere undersøgelser viste, at Lgr5 + individuelle servicekontrakter kunne kun eksistere, når de kunne konkurrere om vigtige niche signaler, som deres datter Paneth celler 8. Disse studier undersøgte rolle Paneth celler på normal Lgr5 + individuelle servicekontrakter og ikke i en situation, hvor de er beskadiget og kræver genopfyldning fra en Lgr5 lo befolkning. <pclass = &quot;jove_content&quot;&gt; For at forstå intestinal biologi og model sygdommen vi undersøge den funktionelle rolle af celler og / eller gener ved anvendelse af transgene musemodeller 9,10. Ofte disse modeller anvender cre-lox-teknologi til betinget ændre gen (er) 9,10. Cre (Årsager rekombination) rekombinase er et site specifik rekombinase af integrase familien, isoleret fra bakteriofag P1. Cre katalyserer stedspecifik rekombination mellem definerede 34 bp &#39; Lox P &#39;(locus χ af crossover P1) sites. Mus er genetisk manipuleret til at indeholde loxP-sites, som flankerer områder af interesse, der ved ekspression af Cre-rekombinase udskæres. Sammenkædning ekspressionen af Cre-gen til en celle eller udviklingsmæssig specifik promotor muliggør ændringer der skal foretages i en rumlig mode 9,10, dette er især nyttig i at overvinde embryonale letale mutationer. At knytte Cre-ekspression til en receptor pathway,der kan aktiveres kunstigt, tillader tidsmæssige ændringer. Ved hjælp af denne teknologi, vi inaktiverede CatnB genet 11 i den intestinale epitel. β-catenin, at CatnB genproduktet, er en vigtig regulator af den kanoniske Wnt signalvej, som regulerer ISC homeostase. To tidligere undersøgelser ved hjælp af denne strategi modstridende resultater 12,13. Undersøgelsen fra Fevr et al. 12, viste tab af stamceller og intestinal homeostase. Henviser til, at Irland et al. 14 undersøgelse rapporterede, at efter en reduktion i cellernes levedygtighed krypten-villus aksen blev genopbyggede fra vild type celler, der udtrykker CatnB. Den største forskel i disse studier var initiativtageren anvendes til at udtrykke Cre i tarmens epitel. Den Fevr et al., Undersøgelse benyttede villin genpromotor forbundet med østrogenreceptoren, som kan aktiveres ved at administrere tamoxifen (will-Cre-ER T2) 15,16. I modsætning Irland et al., Anvendt promotorelementet af rotte cytochrome P450A1 (CYP1A1) genet til at drive Cre-ekspression som reaktion på de xenobiotiske β-naphthoflavon (Ah-CRE). Kendetegnene for disse forskellige systemer genereret to hypoteser for at tage højde for disse forskellige observationer. Det første, at CatnB mere effektivt slettes i ISC hjælp af vIL-Cre-ER T2 systemet i forhold til Ah-cre, hvorved antallet af ISC til sub-genudsættelse niveauer. Alternativt var det på grund af forskelle CatnB deletion i differentieret cellepopulation. De vIL-Cre-ER T2 systemets mål alle epitelceller i krypten og villus henviser Ah-cre systemet er kun rettet mod de ikke-Paneth celler af ISC niche og krypt. Disse systemer forudsat ideelle værktøjer til at undersøge Behavior af individuelle servicekontrakter og deres samspil med Paneth cellerne. Her præsenterer vi flere detaljerede protokoller baseret på, hvor vi anvendte disse systemer at fastslå, at Paneth celler spiller en afgørende rolle ved mediering af den intestinale respons på skade 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

Epiteloverfladen pattedyr-tyndtarm er et dynamisk væv, der fornyer hver 3 - 7 dage. Forståelse denne fornyelsesproces identificeret en befolkning på hurtigt at cykle tarm stamceller (HTT) er kendetegnet ved deres udtryk af Lgr5 genet. Disse er understøttet af en hvilende stamceller population, kendetegnet ved Bmi-1-ekspression, er i stand til at erstatte dem i tilfælde af skade. Undersøgelse samspillet mellem disse populationer er afgørende for at forstå deres roller i sygdomme og kræft. De individuelle servicekontrakter eksistere inden krypter på tarmens overflade, disse nicher understøtter ISC i påfyldning af epitel. Samspillet mellem aktive og rolige individuelle servicekontrakter sandsynligvis involverer andre differentierede celler i den niche, som det tidligere er blevet påvist, at &#39;&#39; stemness &#39;&#39; af Lgr5 ISC er tæt knyttet til tilstedeværelsen af deres nabolande Paneth celler. Brug af betinget cre-lox musmodeller testede vi virkningen af ​​at slette de fleste aktive ISC i nærvær eller fravær af Paneth celler. Her beskriver vi de teknikker og analyser gennemført for at karakterisere tarmen og vise, at de Paneth celler spiller en afgørende rolle i ISC niche i at hjælpe inddrivelse efter betydelig fornærmelse.

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

Tarmepitel stamceller (HTT) er blandet med Paneth celler. Disse celler er differentieret afkom af ISC, som støtter individuelle og fælles give antibakteriel beskyttelse. Her viser vi, hvordan vi anvendte transgene betingede musemodeller at fastslå, at Paneth celler spiller en afgørende rolle i at opretholde tarmens epitel.

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

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

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

Anvendelse af RNAi og Heat-shock-induceret transskription faktor udtryk til at omprogrammere kønsceller til neuroner i 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

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Vurdering af lysosomale basisk i tarmen af 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

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

Beregningsmæssige analyse af Caenorhabditis elegans kønscelleoverførsel at studere Distribution kerner, proteiner og cytoskelettet

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

Visualisere Node og Notochordal plade i Gastrulating mus embryoner ved hjælp af Scanning elektronmikroskopi og hele Mount immunfluorescens

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

Voksen zebrafisk skade modeller at studere virkningerne af Prednisolon i regenererende knoglevæv

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

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

Vurdering af oxidative skader i den primære mus okulære overfladeceller / stamceller som reaktion på Ultraviolet-C (UV-C) Skader

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