Yazarlar Benjamin J Weigler, D.V.M., doktora teşekkür etmek istiyorum ve Walt Mandeville, D.V.M. (hayvan kaynakları ve kampüs katılıyor veteriner, University of Nevada, Reno) mükemmel hayvan hizmet için onların avukatı yanı sıra fareler sağlanan cerrahi işlemler.

Aşağıdaki iletişim kuralı kurumsal hayvan bakım ve kullanmak Komitesi (IACUC) University of Nevada-Reno (UNR) hayvan kaynakları tarafından onaylandı ve araştırma hayvanlar kullanımı ile ilgili tüm kurumsal etik kuralları ile uyumludur.
1. hayvanlar.
<ol><li>Olgun (4-6 hafta eski) C57BL/6 fareler arasında 20-30 g. ev laboratuvar fareler UNR hayvan kaynakları merkezi bir hayvan tesisi kolonisi Tartım elde edilir.</li>
</ol>2. kısmi tıkanıklık cerrahi
Not: Ameliyatları cerrahi işlemler için adanmış bir odada gerçekleştirilir. Tüm cerrahi aletler ameliyat öncesi autoclaved. Steril Cerrahi önlük ve eldiven cerrahi odasında tüm personeli tarafından her zaman giyilmelidir.
<ol><li>Cerrahi fare için hazırlık<ol>
<li>Oksijen kaynağı sağlamak için anestezik teslim sistemi kontrol edin ve isoflurane yordamı için yeterlidir. Tedarik oksijen açın. Gaz akış ölçer üzerinde açmak ve indüksiyon odasında hayvan 500-1000 mL/dk. yer ayarlamak ve üst mühür.</li>
		<li>%5 için isoflurane Buharlaştırıcı açmak ve yaslanmış şeklini alıncaya kadar hayvan izlemek. Anestezik iletim sistemi için burun konisi geçin.</li>
		<li>Herhangi bir kalıntı gaz oksijen ile indüksiyon TMMOB floş, sonra indüksiyon odası hattan burun konisi satır tutarken açık dönün.</li>
		<li>Hayvan odasından kaldırın ve dikkatle oftalmik merhem hayvan gözler yerleştirin.</li>
		<li>Gaz akışı devam ederken bir Önceden ısıtılmış sıcak yastık üzerinde burun konisi yerleştirin.</li>
		<li>100-200 mL/dk, % 2-3 isoflurane ile oksijen akışını değiştirmek. Hayvan hareket başlarsa, gentlyrestrain üzerinde burun konisi kadar hayvanla tam olarak tekrar anestezi.</li>
		<li>Solunum ve stimülasyon işlemi sırasında yanıt izlemek ve isoflurane (% 2-5) yüzdesini gerektiği gibi ayarlayın. Cerrahi yapılmadan önce hayvan anesthetization düzey ayak çimdik refleks eksikliği tarafından izlenir.</li>
		<li>Ağrı kesici (buprenorfin, vücut ağırlığının 1 µg/g) intraperitoneally kesi sitesinden ayrıldığınızda enjekte et.</li>
		<li>Saç Temizleme losyonu temiz pamuk bez kullanarak karnına uygulayın. Gazlı bez ve pamuk temizleme bezi kullanarak saç kaldırmak belgili tanımlık losyon oturmak için fare, 3-5 dk sonra izin. Tüm saç fare karından kaldırılıncaya kadar bu adımı yineleyin.</li>
		<li>Cilt gazlı bez ve pamuk temizleme bezi kullanarak % 70 etanol ile temizleyin. Karın temizlemek için swabsticks veya povidone-iyot çözümü uygulayın.</li>
	</ol>
</li>
	<li>Kısmi tıkanıklık cerrahi<ol>
<li>Cerrahi alan orta açma 2.5 x 2, 5 cm ile 25 x 50 cm steril kağıt kullanarak cerrahi sitesi asmak. Örtüsü hayvan için açılış ve cilt sınırlarını steril şeritler yerleştirerek güvenli.</li>
		<li>Bir ~3.0 cm karın kesi boyuna No. 15 bıçak neşter sadece cilt basamağında sağlanması ve şu anda musculoperitoneal katman içine kesme kaçınarak kullanarak olun.</li>
		<li>Forseps ve Cerrahi makas kullanarak, dikkatle cilt musculoperitoneal katmanından herhangi bir kesi musculoperitoneal katmana neden olmadan ayırın. Katmanları yeterince (yaklaşık 1 cm x 4 cm) ayrılmıştır sonra linea tanımlamak alba musculoperitoneal katman ve kesme ~ 2 cm boyunca linea alba mikro-forseps ve makas kullanarak mayi boşluğu ortaya çıkarmak için.</li>
		<li>Dikkatle bulun ve çekum tanımlamak. Yavaş ve nazikçe çekum mikro-forseps, proksimal kolon ve steril örtü üzerine dışında çekum ile ileum getirerek ile mayi boşluğu kaldırın. Hemen bağırsak dokusu % 0,9 steril serum fizyolojik batırılmış gazlı bez ile nemlendirin ve karın boşluğu dışında oldukları sırada her zaman nemlendirilmiş maruz doku tutun.</li>
		<li>Bulmanıza ve tanımlamanıza ileum ve proksimal kolon arasında bıçaklanmasından. Bir kesik (~ 1 cm) içinde bıçaklanmasından için ve hemen altında ileum paralel yapmak ve herhangi bir damarlara kesme önlemek.</li>
		<li>Bir autoclaved silikon yüzüğü (uzunluk, 4 mm dış çapı, 3.5 mm iç çapı 6 mm) al. Tüp açın ve mikro-Forseps ile halka açmak için boyuna kesilmiş.</li>
		<li>Açık halka kesi aracılığıyla bir ucunu bıçaklanmasından dokusunda bağlayın. Yüzüğü yüzük tarafından çevrili ileum ile temas başka bir son veriyorum tarafından tamamlanan yüzük şekle geri.</li>
		<li>Silikon halka tamamen ileum çevreleyen emin olun, dikiş yüzükle kapatın ve dikkatle bağırsak mayi boşluğunda yer.</li>
	</ol>
</li>
	<li>Cerrahi kapatma<ol>
<li>Basit bir gerçekleştirmek sürekli dikiş boyunca linea musculoperitoneal katmandaki alba musculoperitoneal kapatmak için yara ile absorbe bir dikiş. Dikişi tamamlandıktan sonra kanama % 0,9 steril serum fizyolojik batırılmış gazlı bez ile temizleyin.</li>
		<li>Tamamen ayrı naylon sütür ile yara kapatmak için basit sürekli dikiş cilt üzerinde gerçekleştirin.</li>
		<li>Her iki dikiş tamamladıktan sonra yeni swabstick veya povidone-iyot yarayla temiz.</li>
		<li>İntraperitoneally antibiyotik (gentamisin, 20-30 g vücut ağırlığı üzerinde temel fare başına 150 μL) enjekte et.</li>
		<li>Yordamı, tamamlandıktan açtıktan sonra isoflurane Buharlaştırıcı kapalı ve hayvan o başlamak kadar bilinç kazanıyor tek akan oksijen nefes sağlar.</li>
		<li>Hayvan uyanık olduğunda, hayvan tamamen iyileşti kadar termal destekli bir ayrı kurtarma alana yerleştirin.</li>
	</ol>
</li>
</ol>3. sonrası operasyon gözlem.
<ol><li>Cerrahi tamamlanmasından sonra hayvanlar için bir kurtarma odada bir kuluçka nerede sıcaklık ve nem düzenlenmiştir hareket ettirin. Hayvanlar da kuluçka makinesine iken ikinci saat için ilk saat sonra her 30 dakikada hayvanlar postoperatively her 15dk izlemek.</li>
	<li>Reçete gözlem tam olduğu gibi hayvanlar kendi bireysel kafese taşımak ve klinik belirtiler ağrı18ve cerrahi yara düzgün komplikasyonlar () herhangi bir belirti iyileşme sağlamak için günlük izlemek yırtılması) mevcut.</li>
</ol>

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Yazarlar ifşa gerek yok.

Biz sürekli olarak ve tekrarlanarak bağırsak PO cerrahi alan fareler insan bağırsak tıkanıklığı hareketini taklit eden nazik bağırsak düz kas hipertrofisi geliştirmek gösterdi. Bağırsak tıkanıklığı ameliyatları fareler7, fareler10, eskiden şiling şimdi pigs11, köpek kedi ve12 13de dahil olmak üzere farklı hayvanlar için geliştirilmiştir. Fare modeli bağırsak tıkanıklı...

Bir kısmi veya tam tıkanma sindirilmiş gıda, sıvı ve gaz bağırsak1ile hareket etmelerini engeller küçük veya büyük bağırsak bağırsak engelleri vardır. Tıkanıklık nedeniyle, kalınlaşmış olmak için bağırsak duvarlar2Lümen daralması tıkanma neden olmaktadır. Bağırsak tıkanıklığı karın yapışma doku oluşmasına neden olur karın veya pelvik ameliyatlar sonucunda veya iltihabi bağırsak hastalıkları (Crohn hastalığı), divertikülit, Fıtıklar, volvulusu, yapışıklıkları gibi GI bozuklukları ortaya çıkabilir, İnvajinasyon, kabızlık, dışkı sıkışması, Psödo obstrüksiyonu, kanser ve tümör3,4,5. Bu durumda bağırsak engelleri genellikle barsak6tunica muscularis hipertrofisi yol.
Lümen PO bağırsak şişkinlik neden olmaktadır ve düz kas tabakası kalınlığı yanıt olarak fonksiyonel peristaltizm7,8,9,10devam etmek gerek tıkanıklığı çevresinde artırır, 11,12,13. Bağırsak po hayvan modelleri fareler7, fareler10, eskiden şiling şimdi pigs11, köpekler12ve sürekli geliştirmek içinde benzer hipertrofisi kediler13 düz kas hipertrofisi incelemek için geliştirilmiştir bağırsak kas tabakaları.
Bağırsak PO bir fare modeli oluşturmak ve bağırsak engelleri vivo içindeçalışma için en uygun maliyetli yöntemidir. İnce bağırsak engelleri farelerde ileum çevreleyen cerrahi olarak yerleştirilmiş bir silikon ring kullanarak gerçekleştirilir. PO fareler hücreleri (Hiperplazi) erken bir artış ve bir artış sonra PO cerrahi8,15kas tabakası kalınlığı (hipertrofisi) gösterdi. SMC Hipertrofik koşulları14yanıt olarak düz kas katmanlar içinde büyüyen birincil plastik hücreleri ama ICC gibi diğer hücreleri ve SMC ile yakından ilişkili olan, aynı zamanda yeniden doldurulur PDGFRα+ hücreleri vardır. Biz daha önce PO fareler ince bağırsak hipertrofisi geliştirmek bildirdin hangi SMC son derece proliferatif7,15,16PDGFRα+ hücrelere dediferansiye. Diğer taraftan, ICC dejenere ve düz kas Hipertrofik katmanlar içinde bağırsak obsruction7geliştirilmesi sırasında kaybetti. Başka bir önemli yararı PO modelinin enterik sinir sistemi ve sinir motor desen yayılıyor değişiklikleri ikna etmek için onun kapasitesidir. Büyük yayılıyor fare ince bağırsak Nörojenik motor desen Nörojenik ve does değil istemek ICC ya da elektrik yavaş dalgalar17geçirme motor karmaşık (MMC), olduğunu. PO modeli MMC ve enterik sinir tarafından kısmi tıkanıklık nasıl remodeled içine açık anlayışlar sağlayabilir.
Burada, bir fare protokol silikon ring kullanarak bağırsak PO ameliyat için teklif ediyorum. PO cerrahi güvenilir bir şekilde alma fareler bağırsağın tunica muscularis hipertrofisi üretmek. Hipertrofik kas içinde SMC, ICC, PDGFRα+ve nöronal hücre önemli ölçüde remodeled.

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		<tr>
			<td>Petrolatum ophthalmic ointment</td>
			<td>Puralube VET</td>
			<td></td>
			<td>3.5 g</td>
		</tr>
		<tr>
			<td>Fluriso (isoflurane)</td>
			<td>Vetone</td>
			<td>V1 502017</td>
			<td>250 ml</td>
		</tr>
		<tr>
			<td>Steri-strip reinforced skin closure</td>
			<td>3M</td>
			<td>R1547</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical gloves</td>
			<td>Medline</td>
			<td>MSG2270</td>
			<td></td>
		</tr>
		<tr>
			<td>Ear loop face mask</td>
			<td>The safety zone</td>
			<td>RS700</td>
			<td></td>
		</tr>
		<tr>
			<td>Avant gauze non-woven sponges</td>
			<td>Caring</td>
			<td>PRM25444</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical cup</td>
			<td>Admiral&#160; craft OYC-2</td>
			<td>725-A42</td>
			<td>2.5 oz</td>
		</tr>
		<tr>
			<td>Swabstick</td>
			<td>ChloraPrep</td>
			<td>260103</td>
			<td>2% w/v Chlorhexidine&#160; Gluconate (CHG) and 70% v/v Isopropyl Alcohol (IPA)</td>
		</tr>
		<tr>
			<td>Cotton tipped applicator</td>
			<td>Puritan</td>
			<td>806-WC</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Zoo pharm</td>
			<td>BZ8069317</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Gentamycin sulfate</td>
			<td>Vetone</td>
			<td>G-6336-04</td>
			<td>100 mg/ml</td>
		</tr>
		<tr>
			<td>Fast acting gel cream remover</td>
			<td>Veet</td>
			<td>8111002</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>AHS</td>
			<td>AH01T2516</td>
			<td>1 ml with needle</td>
		</tr>
		<tr>
			<td>Silicon ring</td>
			<td>VWR</td>
			<td>60985-720</td>
			<td>6 mm in length, 4 mm exterior diameter, 3.5 mm interior diameter</td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>The Jackson Laboratory</td>
			<td></td>
			<td>4-6 weeks old</td>
		</tr>
	</tbody>
</table>

a mouse model of intestinal partial obstruction

Engel veya Peristaltik hareket engellemek, bağırsak engelleri, karın yapışıklıklar ve tümör büyümeleri de dahil olmak üzere en gastrointestinal (GI) hastalıklar tarafından neden olabilir. Ancak, mekanizmaları remodeling cep dahil ve neden, bağırsak engelleri kötü anlaşılır. Bağırsak engelleri çeşitli hayvan modeller geliştirilmiştir, ama fare modeli en maliyet/saat etkili. Fare modeli doğru şekilde gerçekleştirilir, yüksek mortalite oranına sahip bir bağırsak kısmi tıkanıklık (PO) cerrahi implantasyon kullanır. Buna ek olarak, uygun bir abluka kullanılmaz veya düzgün bir şekilde yerleştirilmiş hipertrofisi geliştirmek fareler PO cerrahi alma başarısız. Burada, biz çok düşük mortalite ile güvenilir ve tekrarlanabilir bağırsak engelleri üreten PO ameliyat için detaylı bir protokol tanımlamak. Bu iletişim kuralı küçük bağırsakta sindirim hareketi kısmen blokları ileum çevreleyen bir ameliyatla yerleştirilen silikon yüzük kullanır. Kısmi tıkanıklık sindirim hareketi durdurmak nedeniyle büyümüş haline bağırsak yapar. Dilatasyon bağırsak düz kas hipertrofisi aşamalı olarak ölüm nedenleri kadar 2 hafta içinde geliştirir yüzüğü ağız tarafında neden olmaktadır. Cerrahi PO fare modeli Hipertrofik bağırsak dokusu patolojik değişiklikler bağırsak hücreleri düz kas hücreleri (SMC), interstisyel hücreleri ve Cajal (ICC), PDGFRα+, dahil olmak üzere, eğitim için yararlı bir vivo içinde modeli sunar ve nöronal hücreleri bağırsak tıkanıklığı geliştirilmesi sırasında.

Kısmi obstrüksiyonu (PO) cerrahi olarak silikon ringin etrafında ileum ileocecal sfinkter yakın yerleştirerek bir ay önce fareler akımıdır. Bir kısmi tıkanıklık ileum içinde oluşturulan bu yüzüğü. (Böylece aynı zamanda bir yüzük olmadan açık yaş/cinsiyet gerçekleştirilmiştir) sahte operasyonlar fareler eşleşen ve bu farelerde herhangi bir belirti PO farelerde bulunan belli etmedi. Fare hızlı PO cerrahiden birkaç saat içinde yeniden elde etmek. İlk hafta...

Watch this Scientific Journal Video about A Mouse Model of Intestinal Partial Obstruction at JoVE.com

A Mouse Model of Intestinal Partial Obstruction

Bir kısmi veya tam tıkanma Şiddetli karın ağrısı, bulantı, kusma ve dışkı geçiş engelleme neden olabilir bağırsak bağırsak engelleri vardır. Bağırsak kısmi obsructions farelerde oluşturmak için bu yordamı patolojik hücre büyümesi ve ölüm bağırsak temel mekanizmaları eğitim güvenilirdir.

Bağırsak kısmi tıkanıklık bir fare modeli

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

a-mouse-model-of-intestinal-partial-obstruction

Research

JoVE Journal

Biology

20.9K Görüntüleme.  University of Nevada, Reno School of Medicine. Bir kısmi veya tam tıkanma Şiddetli karın ağrısı, bulantı, kusma ve dışkı geçiş engelleme neden olabilir bağırsak bağırsak engelleri vardır. Bağırsak kısmi obsructions farelerde oluşturmak için bu yordamı patolojik hücre büyümesi ve ölüm bağırsak temel mekanizmaları eğitim güvenilirdir.

Video: A Mouse Model of Intestinal Partial Obstruction

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo Görüntüleme ve Yumurtalık Kanseri Ortotopik Fare Modeli Tedavi Tedaviler

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Biyoloji

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

İBH DSS-kaynaklı Modeli Bağırsak Enflamasyon incelenmesi

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

Metabolik Profil Gerçek Zamanlı Analizi<em&gt; Ex vivo</em&gt; Fare Bağırsak Crypt organoid Kültürleri

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Lentiviral İletim ve Bağırsak Organoids Mansabında Analizi için Protokol

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

Kısmi safra kanalı ligasyonu fare: kontrollü manken yerelleştirilmiş obstrüktif Histiositoz olgusu

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-iç hücre embriyonik kök hücrelerin kitle enjeksiyon daha yüksek Chimerism oranları için yol açar.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

EYFP etiketli CENP-A (EYFP-CENP-A) Ubiquitylation tanımlamak için Kütle Spektrometresi Analizi

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

Bel Omurgası Kararsızlığının Fare Modeli

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

Organoid Kaynaklı Epitel Tek Katmanlı: Bağırsak Bariyer Fonksiyonu için Klinik Olarak İlgili Bir In Vitro Model

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Partial Hepatectomy in Adult Zebrafish

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy livers are capable of regenerating from toxic damage, but in advanced liver disease, the natural ability of the liver to regenerate is impaired. Zebrafish have emerged as a powerful experimental system for studying regeneration. They are an ideal model for studying liver regeneration from partial hepatectomy, a procedure with direct clinical relevance in which part of the liver is surgically removed, leaving the rest intact. There is no standard protocol for partial hepatectomy; previous studies using this model have used slightly different protocols and reported disparate results. Described here is an efficient, reproducible protocol for performing a partial hepatectomy in adult zebrafish. We use this technique to demonstrate that zebrafish are capable of epimorphic regeneration of the resected lobe. This protocol can be used to further interrogate the mechanisms required for liver regeneration in zebrafish.

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

Yetişkin Zebra Balıklarında Kısmi Hepatektomi

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy ...