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>
	<li>Cheng, H., Origin Leblond, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=differentiation+and+renewal+of+the+four+main+epithelial+cell+types+in+the+mouse+small+intestine.+V.+Unitarian+Theory+of+the+origin+of+the+four+epithelial+cell+types.">differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian Theory of the origin of the four epithelial cell types.</a> Am J Anat. 141 (4), 537-561 (1974).</li><li>Barker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+stem+cells+in+small+intestine+and+colon+by+marker+gene+Lgr5.">Identification of stem cells in small intestine and colon by marker gene Lgr5.</a> Nature. 449 (7165), 1003-1007 (2007).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The gut, a clonal conveyor belt. , (2015).</li><li>Snippert, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+crypt+homeostasis+results+from+neutral+competition+between+symmetrically+dividing+Lgr5+stem+cells.">Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells.</a> Cell. 143 (1), 134-144 (2010).</li><li>Lopez-Garcia, C., Klein, A. M., Simons, B. D., Winton, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+stem+cell+replacement+follows+a+pattern+of+neutral+drift.">Intestinal stem cell replacement follows a pattern of neutral drift.</a> Science. 330 (6005), 822-825 (2010).</li><li>Buczacki, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+label-retaining+cells+are+secretory+precursors+expressing+Lgr5.">Intestinal label-retaining cells are secretory precursors expressing Lgr5.</a> Nature. 495 (7439), 65-69 (2013).</li><li>Basak, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+early+fate+determination+in+Lgr5++crypt+stem+cells+using+a+novel+Ki67-RFP+allele.">Mapping early fate determination in Lgr5+ crypt stem cells using a novel Ki67-RFP allele.</a> EMBO J. 33 (18), 2057-2068 (2014).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paneth+cells+constitute+the+niche+for+Lgr5+stem+cells+in+intestinal+crypts.">Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts.</a> Nature. 469 (7330), 415-418 (2011).</li><li>Young, M., Ordonez, L., Clarke, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+are+the+best+routes+to+effectively+model+human+colorectal+cancer.">What are the best routes to effectively model human colorectal cancer.</a> Molecular oncology. 7 (2), 178-189 (2013).</li><li>Sauer, B., Henderson, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+DNA+recombination+in+mammalian+cells+by+the+Cre+recombinase+of+bacteriophage+P1.">Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1.</a> Proc Natl Acad Sci U.S.A. 85 (14), 5166-5170 (1988).</li><li>Brault, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+beta-catenin+gene+by+Wnt1-Cre-mediated+deletion+results+in+dramatic+brain+malformation+and+failure+of+craniofacial+development.">Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development.</a> Development. 128 (8), 1253-1264 (2001).</li><li>Fevr, T., Robine, S., Louvard, D., Huelsken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/&#946;-catenin+is+essential+for+intestinal+homeostasis+and+maintenance+of+intestinal+stem+cells.">Wnt/&#946;-catenin is essential for intestinal homeostasis and maintenance of intestinal stem cells.</a> Molecular and Cellular Biology. 27 (21), 7551-7559 (2007).</li><li>Ireland, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+Cre-mediated+control+of+gene+expression+in+the+murine+gastrointestinal+tract:+effect+of+loss+of+beta-catenin.">Inducible Cre-mediated control of gene expression in the murine gastrointestinal tract: effect of loss of beta-catenin.</a> Gastroenterology. 126 (5), 1236-1246 (2004).</li><li>Kemp, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elimination+of+background+recombination:+somatic+induction+of+Cre+by+combined+transcriptional+regulation+and+hormone+binding+affinity.">Elimination of background recombination: somatic induction of Cre by combined transcriptional regulation and hormone binding affinity.</a> Nucleic Acids Res. 32 (11), e92 (2004).</li><li>Madison, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cis+elements+of+the+villin+gene+control+expression+in+restricted+domains+of+the+vertical+(crypt)+and+horizontal+(duodenum,+cecum)+axes+of+the+intestine.">Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine.</a> J Biol Chem. 277 (36), 33275-33283 (2002).</li><li>Parry, L., Young, M., El Marjou, F., Clarke, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+crucial+role+of+paneth+cells+in+mediating+the+intestinal+response+to+injury.">Evidence for a crucial role of paneth cells in mediating the intestinal response to injury.</a> Stem Cells. 31 (4), 776-785 (2013).</li><li>Soriano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+lacZ+expression+with+the+ROSA26+Cre+reporter+strain.">Generalized lacZ expression with the ROSA26 Cre reporter strain.</a> Nat Genet. 21 (1), 70-71 (1999).</li><li>el Marjou, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+and+inducible+Cre-mediated+recombination+in+the+gut+epithelium.">Tissue-specific and inducible Cre-mediated recombination in the gut epithelium.</a> Genesis. 39 (3), 186-193 (2004).</li><li>Guillemot, F., Nagy, A., Auerbach, A., Rossant, J., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+Mash-2+in+extraembryonic+development.">Essential role of Mash-2 in extraembryonic development.</a> Nature. 371 (6495), 333-336 (1994).</li><li>Gregorieff, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+pattern+of+Wnt+signaling+components+in+the+adult+intestine.">Expression pattern of Wnt signaling components in the adult intestine.</a> Gastroenterology. 129 (2), 626-638 (2005).</li><li>van der Flier, L. G., Haegebarth, A., Stange, D. E., van de Wetering, M., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OLFM4+is+a+robust+marker+for+stem+cells+in+human+intestine+and+marks+a+subset+of+colorectal+cancer+cells.">OLFM4 is a robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells.</a> Gastroenterology. 137 (1), 15-17 (2009).</li><li>Merritt, A., Allen, T., Potten, C., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+small+intestinal+epithelial+from+p53-null+mice:+evidence+for+a+delayed,+p53-independent+G2/M-associated+cell+death+after+gamma-irradiation.">Apoptosis in small intestinal epithelial from p53-null mice: evidence for a delayed, p53-independent G2/M-associated cell death after gamma-irradiation.</a> Oncogene. 14 (23), 2759-2766 (1997).</li><li>Marshman, E., Ottewell, P., Potten, C., Watson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+activation+during+spontaneous+and+radiation-induced+apoptosis+in+the+murine+intestine.">Caspase activation during spontaneous and radiation-induced apoptosis in the murine intestine.</a> J Pathol. 195 (3), 285-292 (2001).</li><li>Fevr, T., Robine, S., Louvard, D., Huelsken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+is+essential+for+intestinal+homeostasis+and+maintenance+of+intestinal+stem+cells.">Wnt/beta-catenin is essential for intestinal homeostasis and maintenance of intestinal stem cells.</a> Mol Cell Biol. 27 (21), 7551-7559 (2007).</li><li>Barker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crypt+stem+cells+as+the+cells-of-origin+of+intestinal+cancer.">Crypt stem cells as the cells-of-origin of intestinal cancer.</a> Nature. 457 (7229), 608-611 (2009).</li><li>Tian, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reserve+stem+cell+population+in+small+intestine+renders+Lgr5-positive+cells+dispensable.">A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable.</a> Nature. 478 (7368), 255-259 (2011).</li><li>Yilmaz, O. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTORC1+in+the+Paneth+cell+niche+couples+intestinal+stem-cell+function+to+calorie+intake.">mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake.</a> Nature. 486 (7404), 490-495 (2012).</li><li>Adolph, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paneth+cells+as+a+site+of+origin+for+intestinal+inflammation.">Paneth cells as a site of origin for intestinal inflammation.</a> Nature. 503 (7475), 272-276 (2013).</li><li>Marsh, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+Pten+is+dispensable+for+intestinal+homeostasis+but+suppresses+adenoma+development+and+progression+after+Apc+mutation.">Epithelial Pten is dispensable for intestinal homeostasis but suppresses adenoma development and progression after Apc mutation.</a> Nat Genet. 40 (12), 1436-1444 (2008).</li><li>Jard&#233;, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+models+for+the+therapeutic+targeting+of+Wnt+signaling+using+a+Tet-O&#916;N89&#946;-catenin+system.">In vivo and in vitro models for the therapeutic targeting of Wnt signaling using a Tet-O&#916;N89&#946;-catenin system.</a> Oncogene. 32 (7), 883-893 (2013).</li><li>Hung, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+mouse+model+for+sporadic+and+metastatic+colon+tumors+and+its+use+in+assessing+drug+treatment.">Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment.</a> Proc Natl Acad Sci U S A. 107 (4), 1565-1570 (2010).</li><li>Dacquin, R., Starbuck, M., Schinke, T., Karsenty, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+alpha1(I)-collagen+promoter+is+the+best+known+promoter+to+drive+efficient+Cre+recombinase+expression+in+osteoblast.">Mouse alpha1(I)-collagen promoter is the best known promoter to drive efficient Cre recombinase expression in osteoblast.</a> Dev Dyn. 224 (2), 245-251 (2002).</li><li>Hinoi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+colonic+adenoma-carcinoma+progression+based+on+somatic+Apc+inactivation.">Mouse model of colonic adenoma-carcinoma progression based on somatic Apc inactivation.</a> Cancer Res. 67 (20), 9721-9730 (2007).</li><li>Sansom, O. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+Apc+allows+phenotypic+manifestation+of+the+transforming+properties+of+an+endogenous+K-ras+oncogene+in+vivo.">Loss of Apc allows phenotypic manifestation of the transforming properties of an endogenous K-ras oncogene in vivo.</a> Proc Natl Acad Sci U.S.A. 103 (38), 14122-14127 (2006).</li><li>Wong, V. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lrig1+controls+intestinal+stem-cell+homeostasis+by+negative+regulation+of+ErbB+signalling.">Lrig1 controls intestinal stem-cell homeostasis by negative regulation of ErbB signalling.</a> Nat Cell Biol. 14 (4), 401-408 (2012).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>van de Wetering, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+Derivation+of+a+Living+Organoid+Biobank+of+Colorectal+Cancer+Patients.">Prospective Derivation of a Living Organoid Biobank of Colorectal Cancer Patients.</a> Cell. 161 (4), 933-945 (2015).</li></ol>

The authors have nothing to disclose.

Verwendung bedingter cre-lox-transgenen Mäusen, die Funktion der Gene und Zellen zu zerlegen ist ein häufig verwendeter Ansatz. Diese Modelle sind mit großem Erfolg im Darm verwendet worden, um zu identifizieren und zu charakterisieren, die Stammzellen 2,4-6 und zu verstehen, ihre Rolle bei der Krankheit 25. Diese Modelle voll auszuschöpfen erfordert eine umfassende Charakterisierung des Systems zu ermöglichen, dass Daten richtig interpretiert werden. Ein vollständiges Verständnis dieser Systeme ist schwierig zu erreichen, aufgrund von Genen nur selten spezifisch für eine Einzelzelle Typ oder Ort, einer geringen biologischen Erkenntnisse und Ineffizienz der verwendet wird, um Cre Expression zu induzieren Systeme. Die hier beschriebenen Methoden zeigen, wie wir diese Probleme durch experimentelle Gestaltung und Anwendung des vorhandenen Wissens zu überwinden. Obwohl wir diese Methoden verwendet werden, um eine spezifische Fragestellung der hier vorgestellten Techniken sind generisch und kann für jede Forschung Untersuchung der Muri ausgenutzt werden, zu beantwortenne Darm. Vorbereitung der Darmgewebe Der entscheidende Schritt für die Gewährleistung robustere Ergebnisse ist die Ernte und Verarbeitung des Gewebes, die zeitnah und Fixierung Protokolle genau eingehalten verarbeitet werden muss. Da fast alle wichtigen Fragen können stromabwärts zu Artefakten mit dem Gewebe Austrocknen und / oder unvollständige Fixierung zugeordnet zurückzuführen. Timing ist entscheidend, um eine Verschlechterung der Gewebearchitektur und / oder Nukleinsäuren und Proteinen zu verhindern. Unvollständige oder übereifrigen Fixierung kann zum Verlust der histochemischen Auflösung führen. Unvollständige Fixierung aufgrund unzureichender Zeit oder Abschnitte zu dick, um zu ermöglichen Fixiermittel Penetration kann zu einem Verlust der Auflösung innerhalb der Darmkrypten, die als &quot;Wassermarke&quot; auf IHC-Analyse beobachtet werden kann, führen. Weiterhin ist es wichtig, dass Fixierung nicht zu lange zu verlängern, wie kern β-Catenin aus dem Zellkern, sofern nicht sofort pro diffundierenbeitet und Wachs eingebettet folgenden Formalinfixierung. Rolle der Paneth-Zellen in der ISC-Nischen- Die hier vorgestellten Daten effektiv zu zeigen, wie wichtig die Paneth-Zellen in Krypta Regeneration im adulten Darm folgenden ISC Verlust. Allerdings blieb die Möglichkeit, dass Ah-cre erspart eine Bevölkerung von ISCs dass vil-Cre-ER T2 Ziele. Tian et al. 26 elegant gezeigt, dass die Lgr5 hallo ISCs werden von einer Bevölkerung von Lgr5 lo Reserve ISCs ersetzt. Es scheint nun wahrscheinlich, dass diese ISCs in der Ah-cre-System aufgrund der Reservepopulation, die als sekretorischen Zellvorläufer 6,7 identifiziert verschont. Die Bedeutung des reifen Paneth Zelle bei der Unterstützung dieser sekretorischen Zellvorläufer bei Bedarf zu einem ISC Zustand zurück bleibt unbeantwortet. Wie Paneth-Zellen bilden die ISC Nischen 8 und spielen eine Rolle bei der Regulierung der ISC-Antworten, die Kalorienaufnahme 27 und Entzündung 28 bleibt es wahrscheinlich, dass ihre Pflegefunktionen, um ihre eigenen Vorprodukte zu verlängern. Neue Konzepte und Technologien zur effektiven Modell Menschen Darmkrebs Die Entdeckung des ISC führte zur Identifizierung von Genen, die nun verwendet werden, um neue Mausmodelle für die Untersuchung der Rolle der Gene und Zellen in Darm-Biologie und Erkrankung durch Clarke et al 9 Bewertung zu erzeugen. Die einzigen Einschränkungen dieser Technik ist die Identifizierung von Genen, die Cre-Protein zu exprimieren. Derzeit ISCs werden routinemäßig untersucht mit Hilfe von bedingten transgenen Mäusen auf der Grundlage der Lgr5 Genexpressionsmuster. Mäuse, die Cre vom Lgr5 Promotors exprimieren, wurden verwendet, um APC, das Gen am häufigsten bei Darmkrebs mutiert (CRC zu löschen), Was die ISC als Ursprungszelle 25. Selektiven Löschen anderer CRC-Gene in diesen Zellen wird einen Einblick in den Krankheitsverlauf und die Ausbreitung z. PTEN 29. Weitere Einblicke in ISC-Funktion wird von speziell Abtragen Lgr5 exprimierenden Zellen in Mäusen mit einem menschlichen Diphtherie-Toxin-Rezeptor abgerufen (DTR) Gen klopfte in die Lgr5 Locus 26. Andere Strategien nutzen die Tet-A-System, das laufende reversible Expression der mutierten Proteine ​​30 ermöglicht. Mit Hilfe dieser Werkzeuge, um Gen (en) in verschiedenen Zellen 31 und Standorte 32,33 ändern wird verwendet, um zu verstehen, wie Krebs auslösen, Fortschritt und metastasieren 34. Alternativ Mutagenese mit dem Transposon Sleeping Beauty System ist die Identifizierung neuer Fahrer von CRC. Die Weiterentwicklung von Mäusen, Techniken und genetische Veränderung Strategien weiterhin mehr Patienten relevanten Modelle zu entwickeln. New methoden wurden zur Charakterisierung der Darmepithelien und ISCs entwickelt. Charakterisierung des Verhältnisses der epithelialen Zelltypen können mit Strömung erreicht werden Zytometrie auf die differentielle Expression von Lectin und CD24 35 basierend. Möglicherweise der größte Fortschritt in der ISC Verständnis der Biologie und ihre Rolle bei der Krankheit mit der ex vivo organoiden Kultursystem 36 durchgeführt werden. Dieses System erlaubt es normalen und malignen ISCs zu Kultur in 3D, wo sie zu replizieren und zu differenzieren in einem physiologisch relevanten Art und Weise. Es ist zu hoffen, dass diese direkte Prüfung von Arzneimitteln am Patientenproben in vitro zu ermöglichen und damit den Weg für die personalisierte Medizin 37.

Die luminale Oberfläche der Säugerdarm Merkmale Repetiereinheiten der Krypten und fingerartigen Vorsprüngen, bezeichnet Zotten, die in das Lumen ragen. Diese Oberfläche ist eine kontinuierliche Bahn von Epithelien, die komplette Selbsterneuerung etwa alle 3 unterzogen - 4 Tage 1. Diese dynamische Gewebe wird durch eine Population rasch proliferierender Stammzellen unterstützt (ISCs; auch als Krypta Basis Cylinderzelle bekannt), die zunächst durch ihre Expression des Gens Lgr5 2,3 identifiziert wurden. Diese Zellen existieren in einer spezialisierten Nischen am Boden der Krypten der Lieberkühn. Zunächst wird die Feststellung, daß ISCs wurden rasch proliferierender war diskordanten mit der herrschenden Vorstellung, daß eine Stammzelle war Ruhe Natur. Vor der Identifizierung des Lgr5 + ISC wurde postuliert, dass eine Population von Ruheetikettenhaltende Zellen auf der + 4-Position, relativ zu der Basis der Krypta waren die ISCs 1. Neuere Forschungen hwie jetzt durch den Nachweis, dass in erster Linie gibt es einen Pool von äquipotent Radfahren ISCs in jeder Krypta, deren Schicksal durch seine Nachbarn 4,5 reguliert versöhnt diese Beobachtungen. Für den Fall, sie verloren sind diese durch ruhende Zellen, die normalerweise an den sekretorischen Linie verpflichtet, sondern kann auf ISCs zurück, wenn das ISC Bevölkerung beschädigt 6 ersetzt werden. ISC Nachbarn können entweder ISCs oder deren Tochterzellen. Die ISCs produzieren naiv Tochterzellen, die sich vermehren und differenzieren sich in den spezialisierten Zelltypen, die die epithelialen Blatt, Linien Darmlumen 1 umfassen. Der Kelch, enteroendokrinen, Enterozyten, Büschel und M Zellen wandern nach oben, um die luminale Oberfläche, wo sie bieten verschiedene Absorptions- und Regulierungsfunktionen bleiben jedoch die Paneth Zellen am Boden der Krypta, wo sie existieren, mit der ISCs vermischt. In den letzten Jahren wurde gezeigt, dass ein Anteil des naiven daughtER-Zellen für einen sekretorischen Linie dazu bestimmt sind Ruheetiketthalte Lgr5 lo Zellen, die Rückkehr zu einer ISC auf Verletzungen 6,7. Aufgrund ihrer Bedeutung in Krypta Regeneration eine Priorität auf das Verständnis der Wechselwirkungen zwischen der ISCs und seinen Nachbarn, insbesondere der Panethzellen platziert. Die Paneth-Zellen spielen eine entscheidende Rolle in der Nische, die die ISCs 8 unterstützt. Neben bakterizide Produkte die Paneth Zellen Signalmoleküle, die Wege, die ISC Erneuerung oder Differenzierung regieren zu aktivieren. Frühere Studien zeigten, dass die Lgr5 + ISCs könnte nur dann verbindlich, wenn sie für wesentliche Nische Signale durch ihre Tochter Panethzellen 8 vorgesehen konkurrieren könnte. Diese Studien untersuchten die Rolle von Paneth-Zellen an normalen Lgr5 + ISCs und nicht in einer Situation, wo sie beschädigt sind und Nachschub von einer Lgr5 lo Bevölkerung. <pclass = &quot;jove_content&quot;&gt; Um zu verstehen, Darm Biologie und Modellerkrankung untersuchen wir die funktionelle Rolle der Zellen und / oder Gene mit transgenen Mausmodellen 9,10. Häufig sind diese Modelle verwenden cre-lox-Technologie, um Gen (e) 9,10 bedingt zu ändern. Cre (Ursachen Rekombination) Rekombinase ist eine ortsspezifische Rekombinase des Integrase-Familie, isoliert aus dem Bakteriophagen P1. Cre katalysiert ortsspezifische Rekombination zwischen definierten 34 bp &#39; Lox P &#39;(Locus χ der Crossover-P1) Websites. Mäuse gentechnisch so verändert LoxP Websites, die interessierenden Bereiche, die bei der Expression der Cre-Rekombinase ausgeschnitten flankieren, enthalten. Verknüpfung der Expression der Cre-Gens in eine Zelle oder entwicklungsspezifischen Promotors ermöglicht die Änderung in einer räumlichen Mode 9,10 hergestellt werden, ist dies besonders nützlich bei der Überwindung embryonale letale Mutationen. Weitere Verknüpfung des Cre-Expression zu einem Rezeptor-Weg,daß künstlich aktiviert werden, erlaubt zeitlichen Veränderungen. Mit dieser Technologie haben wir inaktiviert die CatnB Gen 11 in den Darmepithelien. β-Catenin, das CatnB Genprodukt ist ein wichtiger Regulator des Wnt-Signalwegs, der ISC-Homöostase regelt. Zwei frühere Studien mit dieser Strategie erzeugt widersprüchlichen Ergebnissen 12,13. Die Studie von FEVR et al. 12 gezeigt Verlust von Stammzellen und Darm Homöostase. Während die Ireland et al. 14 Studie berichtet, dass im Anschluss an eine Verringerung der Lebensfähigkeit der Zellen die Krypta-villus Achse wurde aus Wildtyp-Zellen, die CatnB neu besiedelt. Der Hauptunterschied in diesen Studien war der Promotor verwendet werden, um Cre in Darmepithelien auszudrücken. Die FEVR et al., Verwendet Studie die Villin-Gen-Promotor an den Östrogenrezeptor verbunden, die durch die Verabreichung von t aktiviert werden kannamoxifen (vil-Cre-ER T2) 15,16. Im Gegensatz Ireland et al., Verwendet das Promotorelement des Ratten-Cytochrom P450A1 (CYP1A1) Gens Cre-Expression in Reaktion auf die xenobiotischen β-Naphthoflavon (Ah-cre) anzutreiben. Die Eigenschaften dieser verschiedenen Systemen erzeugten zwei Hypothesen für diese verschiedenen Beobachtungen erklären. Das erste, das CatnB effizienter im ISC mit der vil-Cre-ER T2 System im Vergleich zu den Ah-cre gelöscht wird, wodurch die Anzahl der ISCs, um Unter Repopulation Niveaus verringert wird. Alternativ war es aufgrund CatnB Deletion Differenz in der differenzierten Zellpopulation. Die vil-Cre-ER T2-System richtet sich an alle Epithelzellen der Krypta und Zotten während die Ah-cre-System zielt nur die nicht-Panethzellen des ISC-Nische und Krypta. Diese Systeme bereitgestellt ideale Werkzeuge für die Prüfung des Behavior der ISCs und ihrer Wechselwirkung mit den Paneth-Zellen. Hier stellen wir verschiedene ausführliche Protokolle auf, wie wir früher, diese Systeme zu bestimmen, dass Panethzellen eine entscheidende Rolle bei der Vermittlung der intestinalen Reaktion auf eine Verletzung 17 zu spielen.

<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

Die epitheliale Oberfläche der Säugerdarm ein dynamisches Gewebe, das alle 3 erneuert - 7 Tagen. Das Verständnis dieser Erneuerungsprozess identifizierten eine Population rasch proliferierender Darm-Stammzellen (ISCs), gekennzeichnet durch ihre Expression des Gens Lgr5. Diese werden von einem ruhenden Stammzellpopulation, von der BMI-1-Expression gekennzeichnet ist, in der Lage, im Falle einer Verletzung ersetzen unterstützt. Die Untersuchung der Wechselwirkungen zwischen diesen Populationen ist von entscheidender Bedeutung für das Verständnis ihrer Rolle in Erkrankungen und Krebs. Die ISCs existieren innerhalb Krypten auf der Darmoberfläche, diese Nischen unterstützen die ISC in Auffüllen der Epithelien. Die Wechselwirkung zwischen den aktiven und ruhenden ISCs wahrscheinlich mit anderen differenzierten Zellen innerhalb der Nische, wie es zuvor gezeigt worden ist, dass die &#39;&#39; stemness &#39;&#39; der Lgr5 ISC ist eng mit der Gegenwart ihrer benachbarten Panethzellen gebunden. Bedingte cre-lox-MausModelle, die wir untersucht den Effekt des Löschens der Mehrzahl der aktiven ISCs in Gegenwart oder Abwesenheit von Paneth-Zellen. Hier beschreiben wir die Techniken und Analysen durchgeführt, um den Darm zu charakterisieren und zu zeigen, dass die Paneth-Zellen spielen eine entscheidende Rolle in der ISC Nische bei der Unterstützung Erholung nach erheblichen Beleidigung.

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

Darmepithel-Stammzellen (ISCs) mit Paneth-Zellen vermischt. Diese Zellen sind differenziert Nachkommen des ISC, die den ISCs unterstützen und antibakteriellen Schutz. Hier zeigen wir, wie wir verwendet transgene bedingte Mausmodellen, um festzustellen, dass Paneth-Zellen eine entscheidende Rolle bei der Aufrechterhaltung der Darmepithelien zu spielen.

Protokolle für die Analyse der Rolle der Paneth-Zellen bei der Regeneration des Murine Darm mit Conditional<em&gt; Cre-lox</em&gt; Mausmodelle

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

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Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

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

17.6K Aufrufe.  Cardiff University.  Darmepithel-Stammzellen (ISCs) mit Paneth-Zellen vermischt. Diese Zellen sind differenziert Nachkommen des ISC, die den ISCs unterstützen und antibakteriellen Schutz. Hier zeigen wir, wie wir verwendet transgene bedingte Mausmodellen, um festzustellen, dass Paneth-Zellen eine entscheidende Rolle bei der Aufrechterhaltung der Darmepithelien zu spielen. 

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

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Alginat Encapsulation pluripotenter Stammzellen Mit einer Co-Axial Düsen

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Forschung

Entwicklungsbiologie

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- Wachstum der Maus Preantral Follikel in simulierter Schwerelosigkeit

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.

Live Imaging der Maus Sekundärer Gaumen 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.

Ein RANKL-basierte Osteoklasten Kultur Assay der Maus Knochenmark zu untersuchen, die Rolle von mTORC1 in Osteoklasten Bildung

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.

Anwendung von RNAi und Hitze-Schock-induzierte Transkription Faktor Ausdruck zu programmieren Keimzellen Neuronen 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)

Beurteilung der lysosomalen Alkalisierung in den Darm von Leben 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.

Computergestützte Analyse der Keimbahn Caenorhabditis Elegans , die Verteilung der Kerne, Proteine und das Zytoskelett zu studieren

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).

Visualisierung der Knoten und Notochordal Platte In Gastrulating Maus-Embryonen mittels Rasterelektronenmikroskopie und ganze Mount Immunfluoreszenz

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.

Erwachsenen Zebrafisch Verletzungen Modelle zur Untersuchung der Auswirkungen von Prednisolon bei der Regeneration von Knochengewebe

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.

Bewertung von oxidativen Schäden in den primären Maus-Augen-Oberflächenzellen/Stammzellen als Reaktion auf Uv-C-Schäden (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 ...

Protokolle für die Analyse der Rolle der Paneth-Zellen bei der Regeneration des Murine Darm mit Conditional

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