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.

Genler ve hücrelerin fonksiyonunu incelemek için koşullu CRE-LOX transgenik fareler kullanılarak yaygın olarak kullanılan bir yöntemdir. Bu modeller belirlemek ve tanımlamak kök hücreleri 2,4-6 ve hastalık 25 rollerini anlamak için bağırsaklarda büyük bir başarı ile kullanılmaktadır. Tam bu modeller yararlanmak için doğru yorumlanmalıdır verileri sağlamak için sistemin kapsamlı bir karakterizasyonu gerektirir. Bu sistemlerin tam anlaşılması nedeniyle genleri nadiren yalnız hücre tipi veya konumu, biyolojik bilgi ve Cre ifadesini uyarmak için kullanılan sistemlerin verimsizlik eksikliği özgü olmasının elde etmek zordur. Burada açıklanan yöntemler deneysel tasarım ve mevcut bilginin uygulanması yoluyla bu sorunları aşmak nasıl gösterilmektedir. Burada sunulan genel ve muri araştıran herhangi araştırma için istismar edilebilir teknikler belirli bir araştırma soruyu cevaplamak için bu yöntemleri kullanmasına rağmenne bağırsak. Bağırsak Doku Hazırlanması Sağlam sonuçlar sağlamaya yönelik önemli bir adım kesinlikle uyulması zamanında bir şekilde ve tespit protokollerinde işlenmesi gereken doku hasat ve işleme. Hemen hemen tüm önemli konular aşağı doku kuruma ve / veya eksik fiksasyonu ile ilişkili eserler isnat edilebilir. Zamanlama doku mimarisi ve / veya nükleik asitlerin ve proteinlerin bozulmasını önlemek için çok önemlidir. Eksik veya overzealous tespit histokimyasal çözünürlük kaybına neden olabilir. Yeterli zaman veya fiksatif penetrasyon İHK analizi üzerine bir &quot;gelgit işareti&quot; olarak görülebilir bağırsak kriptlerin içinde çözünürlük kaybına neden olabilir izin vermek için çok kalın bölümlere bağlı Eksik tespit. Bundan başka, nükleer β-katenin hemen yanlısı sürece çekirdeğin dışına dağılacaktır gibi tespit, çok uzun süre uzatmak etmediğini önemlidirulaşıldığı ve formalin fiksasyonu aşağıdaki gömülü balmumu. ISC Niche Paneth Hücrelerinin Rolü Burada sunulan veriler etkili bir ISC kaybını takiben yetişkin bağırsakta kript rejenerasyonu Paneth hücrelerinin önemini göstermektedir. Ancak orada Ah-CRE o vil-Cre-ER T2 hedefleri ISCs nüfusu yedek ihtimalini kalmıştır. Tian ve diğ. 26 zarif Lgr5 hi ISCs Lgr5 lo karşılık ISCs nüfusu ile ikame edildiğini göstermiştir. Şimdi bu ISCs nedeniyle rezerv nüfus salgı hücre öncüleri 6,7 olarak tespit edilerek için Ah-CRE sisteminde bağışladı olduğu muhtemel görünüyor. ISC duruma dönmek için gerektiğinde bu salgı hücre öncüleri destekleyen olgun Paneth hücrenin önemi cevaplanması gereken kalır. Paneth hücrelerinin I teşkil gibiSC niş 8 ve onların hemşirelik işlevleri kendi öncüleri uzatmak olasıdır kalır kalori alımı 27 ve inflamasyon 28 ISC yanıtlarını düzenlenmesinde rol oynamaktadır. Yeni Yaklaşımlar ve Teknoloji Etkili Model İnsan Kolorektal Kanser için ISC keşfi şimdi Clarke ve diğerleri tarafından gözden 9 barsak biyoloji ve hastalık, gen ve hücre rollerini araştırmak için yeni fare modelleri oluşturmak için kullanılan genlerin belirlenmesine yol açmıştır. Bu teknik için tek sınırlama, Cre proteinini ifade etmek için genlerin tanımlanmasıdır. Şu ISCs rutin Lgr5 gen ekspresyon modeline göre koşullu transgenik fareler kullanılarak incelenmiştir. Lgr5 promoterinden Cre ifade Fareler aPC, en yaygın olarak kolorektal kanserde mutasyona uğramış gen (CRC silmek için kullanılmıştır), Orijin 25 hücre olarak ISC gösteren. Seçerek silme bu hücrelerde diğer CRC genleri hastalığın ilerlemesi içgörü sağlanması ve yayılır örneğin. PTEN 29. ISC işlevi ayrıntılı fikir özellikle ablasyon Lgr5 insan difteri toksini reseptörü kullanılarak farelerde hücrelerin -expressing tarafından alındığını (DTR) geninin Lgr5 içine lokusu 26 çaldı. Diğer stratejiler mutant proteinlerin 30 devam eden geri dönüşümlü ifadesini sağlayan Tet-O sistemi kullanın. Farklı hücreler 31 ve yerlerde 32,33 yılında gen (ler) değiştirmek için bu araçları kullanarak kanser başlatmak nasıl anlamak, ilerleme ve 34 metastaz için kullanılır. Alternatif uyuyan güzellik transpozon sistemi kullanılarak mutajenez ÇHS yeni sürücüleri tespitidir. Farelerde, teknikleri ve genetik değişim stratejileri sürekli gelişimi daha sabırlı, ilgili modeller geliştirmeye devam ediyor. Yeni methods bağırsak epitel ve ISCs karakterizasyonu için geliştirilmiştir. Akış kullanılarak elde edilebilir epitel hücre tiplerinin oranı karakterizasyonu sitometri Lektin ve CD24 35 farklı ekspresyonu göre. Potansiyel büyük anlayış ISC biyolojide kaydedilen ilerleme ve hastalık rolleri ex vivo organoid kültür sistemi kullanılarak 36 yapılacaktır. Bu sistem, bu çoğaltma ve fizyolojik olarak daha uygun bir şekilde ayırt 3B, kültür normal ve habis ISCs sağlar. Bu kişiselleştirilmiş tıp 37 önünü, in vitro hasta örnekleri üzerinde ilaçların doğrudan test sağlayacak umulmaktadır.

Memeli bağırsak lümen yüzeyi projeksiyonları gibi kriptalarının ve parmak tekrar birimlerinden özellikleri, lümen içine çıkıntı villus olarak adlandırılan. 4 gün 1 - Bu yüzey, yaklaşık her 3 tam kendini yenileme uğrar epitel sürekli bir tabakadır. Bu dinamik Doku hızla kök hücreleri bisiklet nüfusu tarafından desteklenmektedir (ISCs, aynı zamanda kript baz kolon hücreleri olarak da bilinir) ilk olarak Lgr5 geni 2,3 kendi ifadesi ile tanımlandı. Bu hücreler Lieberkühn kript altındaki özel bir niş bulunmaktadır. Başlangıçta, ISCs hızla bisiklet vardı keşif kök hücre doğada sakin olduğunu hakim fikri ile uyumsuz oldu. O durgun etiket nüfus crypt tabanına göre 4 pozisyonunda hücreleri, istinat öne sürülmüştür Lgr5 + ISC tanımlanmasına Önceki, ISCs 1 idi. Son zamanlarda yapılan araştırmalar hşimdi öncelikle kader komşuları 4,5 düzenlenir her crypt ekipotent bisiklet ISCs bir havuz olduğunu göstererek bu gözlemleri barıştırdı. Halinde bunlar, bu normalde salgı soyu adadık ancak ISC nüfus 6 hasar görmüşse ISCs dönebilirsiniz durgun hücreleri tarafından değiştirilebilir kaybolur. ISC komşuları ISCs veya kızları hücreler olabilir ya. ISCs çarpma ve hangi hatlar bağırsak lümen 1 epitel tabaka oluşturan özelleşmiş hücre tiplerine ayırt naif kızı hücreleri üretirler. Goblet, enteroendokrin, enterositler, püskül ve M hücreleri çeşitli emici ve düzenleyici işlevleri sağlamak lümen yüzeyine yukarı göç, ancak, Paneth hücreleri ISCs ile iç içe bulunmamakta crypt dibinde kalır. Son yıllarda ortaya konmuştur, bu naif kız çocuğu bir kısmınınbir salgı soy için mukadder er hücreleri yaralanma 6,7 üzerine bir ISC geri döndürme yeteneğine Lgr5 lo hücrelerini koruyarak hareketsiz etiket vardır. Nedeniyle kript rejenerasyon önemi bir öncelik ISCs ve komşuları, özellikle Paneth hücreleri arasındaki etkileşimleri anlamaya yerleştirildi. Paneth hücrelerinin ISCs 8 destekleyen niş önemli bir rol oynamaktadır. Bakterisit ürünlerinin yanı sıra Paneth hücrelerinin ISC yenileme veya farklılaşmasını düzenleyen yolları aktive sinyal molekülleri üretirler. Önceki çalışmalar onların kızları Paneth hücreleri 8 tarafından sağlanan temel niş sinyalleri için rekabet edebilecek ne zaman Lgr5 + ISCs sadece var olduğunu gösterdi. Bu çalışmalar, normal Lgr5 + ISCs üzerinde değil, onlar zarar ve bir Lgr5 lo nüfus ikmal gerektiren bir durumda Paneth hücrelerinin rolü incelenmiştir. <pclass = &quot;jove_content&quot;&gt; Biz transgenik fare modelleri 9,10 kullanılarak hücrelerin ve / veya genlerin işlevsel rolünü incelemek bağırsak biyoloji ve model hastalığını anlamak için. Sıkça bu modeller şartlı gen (ler) 9,10 değiştirmek için CRE-lox teknolojisini kullanmaktadır. Cre rekombinaz bakteriyofaj P1 izole integraz ailesinin bir site özgü rekombinaz olduğunu (rekombinasyon Nedenleri). Cre tanımlanan 34 bp arasındaki alana özgü rekombinasyon katalize &#39; Lox P &#39;siteleri (geçit P1 odağı χ). Fareler genetik Cre rekombinazın ifadesi üzerine eksize ilgi alanları kuşatan LoxP siteleri içerecek şekilde tasarlanmıştır. Bir hücre ya da gelişimsel belirli bir promotere Cre geni ekspresyonunu bağlama işleminin tahrip edici bir mekansal biçimde 9,10 yapılabilmesi için, bu oluşum safhasındaki ölümcül mutasyonlar aşılmasında özellikle yararlıdır sağlar. Ayrıca, bir alıcı yolu Cre ifade bağlayanBu yapay aktive edilebilir, zamansal değişiklikler izin verir. Bu teknolojiyi kullanarak, biz bağırsak epitel CatnB genini 11 inaktive. β-katenin, CatnB gen ürünü, ISC homeostazı tabi doğal Wnt sinyal yolunun bir anahtar düzenleyicisidir. Bu stratejiyi kullanarak iki önceki çalışmalarda çelişkili sonuçlar 12,13 üretti. AEVR ve ark., 12 tarafından yapılan çalışmada, kök hücre ve bağırsak homeostazisinin kaybı gösterdi. İrlanda ve ark. Oysa 14 çalışma hücre canlılığında bir azalma, aşağıdaki kript-villus ekseni CatnB eksprese vahşi tip hücrelerden yeniden doldurulur olduğunu bildirdi. Bu çalışmalarda önemli fark bağırsak epitel Cre ifade etmek için kullanılan promotör oldu. AEVR ve diğ., Çalışma t tatbik edilmesiyle aktive edilebilir östrojen reseptörüne bağlanmış villin gen promotörü kullanılıramoxifen (vil-Cre-ER T2) 15,16. Buna karşılık Ireland ve arkadaşlarının., Ksenobiyotik β-naphthoflavone (Ah-CRE) yanıt olarak Cre sentezlenmesini tahrik etmek için, sıçan sitokrom P450A1 (CYP1A1) geni promoter dizisi kullanılmıştır. Bu farklı sistemlere dair özellikler bu farklı gözlemler için hesap, iki hipotez oluşturulur. CatnB daha verimli ve böylece alt repopulation seviyelerine ISCs sayısını azaltarak, Ah-CRE kıyasla vil-Cre-ER T2 sistemi kullanılarak ISC silinir ilk. Alternatif olarak farklılaşmış bir hücre popülasyonunda CatnB silme diferansiyel kaynaklanıyordu. Vil-Cre-ER T2 sistemi hedefleri Ah-CRE sistemi ise crypt ve villuslarının tüm epitel hücreleri yalnızca ISC niş ve crypt olmayan Paneth hücreleri hedefliyor. Bu sistemler Behav incelenmesi için ideal araçlar teminISCs ve ior ve Paneth hücreleri ile etkileşimi. İşte biz Paneth hücrelerinin yaralanma 17 bağırsak cevabı aracılık önemli bir rol oynadığını belirlemek için bu sistemlerin nasıl kullandığını dayalı birçok detaylı protokoller sunuyoruz.

<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

7 gün - memeli bağırsak epitel yüzeyi her 3 yenileyen dinamik bir dokudur. Hızla Lgr5 geninin kendi ifadesi ile karakterize bağırsak kök hücreleri (ISCs) bisiklet nüfusu bu yenilenme sürecini belirlenen anlama. Bunlar yaralanma durumunda bunların yerine yeteneğine Bmi-1 ifadesi ile işaretlenmiş bir sakin kök hücre popülasyonu, tarafından desteklenmektedir. Bu nüfus arasındaki etkileşimi araştıran hastalığı ve kanser rollerini anlamak için çok önemlidir. ISCs bağırsak yüzeyindeki crypts içinde var, bu nişler epitel Yenileyici de ISC destekliyoruz. Bir önceki Lgr5 ISC&#39;nin &#39;stemness&#39; yakından komşu Paneth hücreleri varlığına bağlı olduğunu göstermiştir edildiği gibi, aktif ve pasif ISCs arasındaki etkileşim olasılığı, kovan içinde diğer farklılaşmış hücreleri içerir. Koşullu CRE-lox Fareyi kullanarakmodeller Paneth hücreleri varlığında veya yokluğunda aktif ISCs çoğunluğu silme etkisi test edilmiştir. Burada bağırsak karakterize ve taahhüt teknikler ve analiz Paneth hücrelerinin önemli hakaret aşağıdaki kurtarma yardım ISC niş içinde çok önemli bir rol oynadığını göstermektedir açıklar.

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

Bağırsak epitel kök hücreler (ISCs) Paneth hücreleri ile iç içe. Bu hücreler ISCs destekleyen ve antibakteriyel koruma sağlamak ISC, soyunu ayırt edilirler. İşte biz Paneth hücrelerinin bağırsak epithelia sürdürmek çok önemli bir rol oynadığını kurmak için transgenik fare koşullu modellerini nasıl kullandıklarını göstermektedir.

Şartlı kullanarak Fare İnce Bağırsağında Yenileyici içinde Paneth Hücrelerinin Rolü analiz için protokoller<em&gt; Cre-lox</em&gt; Fare Modelleri

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 Görüntüleme.  Cardiff University.  Bağırsak epitel kök hücreler (ISCs) Paneth hücreleri ile iç içe. Bu hücreler ISCs destekleyen ve antibakteriyel koruma sağlamak ISC, soyunu ayırt edilirler. İşte biz Paneth hücrelerinin bağırsak epithelia sürdürmek çok önemli bir rol oynadığını kurmak için transgenik fare koşullu modellerini nasıl kullandıklarını göstermektedir. 

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

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

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

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

Bir Transpozon Sistemi Kullanılarak Fare Amniyotik Sıvı Hücreleri pluripotent kök hücrelerin üretimi

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

Gelişim Biyolojisi

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.

Vitro Simüle mikrogravite altında fare Preantral follikül gelişimi

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.

Fare İkincil Palin Füzyonunun Canlı Görüntüsü

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.

Bir RANKL tabanlı Osteoklast kültür tahlil, fare mTORC1 Osteoklast oluşumunda rol araştırmak için ilik

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.

Uygulama için yeniden programlamak Germ hücreleri için C. elegans nöronlarda RNAi ve transkripsiyon faktörü ifade ısı şok bağlı

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)

Bağırsak hayatın Caenorhabditis elegans lizozomal Alkalinization değerlendirilmesi

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.

Çekirdeklerin dağıtım, proteinler ve sitoiskeleti çalışmaya Caenorhabditis elegans Germline sayısal analizi

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).

Düğüm ve Notochordal plaka Gastrulating fare embriyo Tarama elektron mikroskobu ve bütün Dağı ayirt kullanarak görselleştirme

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.

Kemik doku Yenileyici prednizolon etkilerini incelemek için yetişkin zebra balığı yaralanma modelleri

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.

Ultraviyole-C (UV-C) Hasarına Yanıt Olarak Birincil Fare Oküler Yüzey Hücrelerinde/Kök Hücrelerinde Oksidatif Hasarın Değerlendirilmesi

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