Name: a genetically engineered mouse model of sporadic colorectal cancer
Uploaded: 2017-07-06T12:30:00
Description: Watch this Scientific Journal Video about A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer at JoVE.com

Bu eser Prof. Moritz Koch&#39;un anısına ithaf edilmiştir.

Burada sunulan hayvan deneyleri, kurumsal ve resmi bir Hayvan Bakımı ve Kullanım Komitesi tarafından bağımsız olarak gözden geçirildi ve onaylandı ve FELASA (Laboratuar Hayvan Bilimi Dernekleri Federasyonu) kurallarına göre gerçekleştirildi. Anestezi ve analjezi ya da gerekirse erken ötanazi gibi acıyı en aza indirmek için olası tüm önlemler alındı. 1. Cerrahi Adeno-cre Enfeksiyonu ile Lokal Tümör İndüksiyonu <ol><li> Ameliyat için hayvanların ...

<ol>
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L., 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=Analysis+of+lung+tumor+initiation+and+progression+using+conditional+expression+of+oncogenic+K-ras.">Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.</a> Genes Dev. 15 (24), 3243-3248 (2001).</li><li>Olive, K. 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Genellikle üretilmesi ve bakımı kolay olsa da, hücre çizgisi enjeksiyonuna dayanan klasik CRC fare modelleri yapaytır ve insan hastalığını tam olarak tekrar edememektedir. Sonuç olarak GEMM&#39;ler geliştirildi. İlk CRC GEMM, Apc geninde heterozigot boş bir mutasyona sahip Apc Min fare idi, bu nedenle insan kalıtsal hastalık ailevi adenomatöz polipozis (FAP) taklit eder. Bununla birlikte, Apc Min fare- lerinde her zaman kolond ile sınırlı olmayan çok sayıda bağırsak adenomu ...

Kolorektal kanser (ÇHS), batı ülkelerinde kansere bağlı ölümlerin başlıca nedenlerinden biri olmayı sürdürüyor. Erken evre hastalığı olan hastaların prognozu iyi olmakla birlikte, birçok tedavi seçeneği olmasına rağmen prognozu sınırlı olan sonraki aşamalarda birçok tümör tanısı konur. 2 , 3 , 4 , 5 Mevcut CRC fare modellerinin çoğunluğu, hücre hatlarından veya hasta tümörlerinden türetilen tümör hücrelerinin immün yetersiz farelere implante edilmesine dayanır. 6 , 7 , 8 Bu, lokal yol açar ve enjeksiyon yerine ve enjeksiyon için kullanılan tümör hücrelerine bağlı olarak, bazen metastatik tümörler oluşturur. Ancak, ortaya çıkan xenograft modelleri majo&#39;yduSınırlamaları. Bağışıklık eksikliği olan farelerde kurulmalılar, böylece tümör ve bağışıklık sistemi arasındaki karmaşık etkileşim ortadan kaldırılmalıdır. Buna ek olarak, tümör stroması konukçu hücrelerden türetildiğinde, insan tümörü parankimi ile mürin stroması arasındaki etkileşim kusurludur ve bu nedenle hastalığı temsil etmez. Bu eksiklikler, enjeksiyon için fare hücre dizilerinin kullanımı ile engellenebilir. Bununla birlikte, sadece birkaç fare CRC hücre çizgisi mevcuttur ve en çok bulunan insan CRC hücre çizgilerine benzer şekilde, monoklonal ve oldukça anaplastiktir. Özetle, halihazırda mevcut CRC fare modellerinin çoğu yapaytır ve insan hastalığını tam olarak temsil etmez. CRC&#39;nin genetik olarak tasarlanmış fare modelleri (GEMM&#39;ler), bu iki dezavantajdan kaçınılabildikleri için kolondaki CRC&#39;nin anahtar mutasyonlarının indüksiyonu yoluyla oluşturulan gerçek fare tümörleri özelliği taşırlar. 12 , 13 ,14 Bu, kolorektal mukozadaki kreatin recombinaz tarafından koşullu (ökelenmiş) tohum çizgisi mutasyonlarının aktivasyonu ile başarılabilir. Diğer pek çok tümör varlıklarının GEMM&#39;lerinde dokuya özgü hızlandırıcılar tarafından yönlendirilen germline (indüklenebilir) cre ifadesi kullanılırken, germline cre, kolondaki çok sayıda adenomaya yol açtığı için benign tümör yükü tarafından ölüm neden olur Çok genç yaş. Bu nedenle, burada tarif edilen modelde, cre&#39;yi ifade eden bir adenoviral vektör kısa bir kolon segmentini enfekte etmek için kullanılır. Bu, araştırmacı tarafından tanımlanan bir zaman noktasında, mukozanın bu segmenti içindeki tümörojenezin uyarılmasına yol açarak sonuçta invaziv ve metastatik karsinomaya ilerleyen adenomlarla sonuçlanır. Tümörler orijinal fare tümörleri olup bozulmamış bir mikro ortamda büyür ve bu nedenle tümör-konakçı etkileşimi ve metastatik kaskad da dahil olmak üzere kolorektal onkogenezin tamamını taklit edebilmektedir. Bu modelBu nedenle kanser biyolojisi ve klinik öncesi terapötik denemeler için çekici bir platform. CRC&#39;nin genetik olarak tasarlanmış fare modellerinin büyük bir dezavantajı teknik karmaşıklığıdır. Floxed Apc alelleri taşıyan farelerde rektal adeno-cre enema kullanan lokal cre doğum daha önce tanımlanmıştır; Bununla birlikte, bağırsak tümörlerinin insidansı, çokluğu ve lokalitesi, bu teknikle oldukça değişken olabilir. 15 Bu nedenle, adeno-cre enfeksiyonunun indüklenecek segmentin ameliyat klemplemesi ile sınırlandırılması tekniği geliştirilmiştir. 13 Bu prosedürü, hayvan refahını arttırmak için, ayrıca mortaliteyi ve ortaya çıkan tümör sayısını azaltmak için değiştirdik. Bu protokol ile, küçük kemirgen cerrahide deneyime sahip tüm laboratuarlar, modeli çoğaltmalı ve kolonoskopi için kolaylıkla erişilebilen ve oldukça tekrarlanabilir olan tümörler üretmelidir. Koşullu m&#39;ye bağlı olarakTümörigenez için kullanılan utasyonlar, adenomun tam spektrumu, invaziv karsinom ve metastazlar gözlenebilir. Tümörler distal kolondaki konumundan dolayı bu modelde seri endoskopik değerlendirme kolayca mümkündür.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents / consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Life Technologies GmbH</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%, Phenol-Red)</td>
			<td>Life Technologies GmbH</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline 0.9% (E154)</td>
			<td>Serumwerk Bernburg AG</td>
			<td>10013</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua ad injectabilia</td>
			<td>B. Braun Melsungen AG</td>
			<td>235144</td>
			<td></td>
		</tr>
		<tr>
			<td>Ad5CMV-Cre (adenovirus, c = 2E+11 PFU/mL)</td>
			<td>Gene Transfer Vector Core 
			University of Iowa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL, 50 mL centrifuge tubes</td>
			<td>Greiner Bio-One GmbH</td>
			<td>188271/227270</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes 1.5 mL/ 2 mL</td>
			<td>Sarstedt AG &#38; Co.</td>
			<td>72,695,400</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish PS 100/15 mm (sterile, Nuclon)</td>
			<td>Fisher Scientific GmbH</td>
			<td>10508921/ NUNC150350</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Syringe (without dead volume) - Injekt-F SOLO</td>
			<td>Braun/neoLab</td>
			<td>194291661</td>
			<td></td>
		</tr>
		<tr>
			<td>30G injection needle</td>
			<td>BECTON DICKINSON</td>
			<td>304000</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Analgesia / anesthesia</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Sevoflurane (Sevoflurane AbbVie)</td>
			<td>AbbVie Germany GmbH &#38; Co. KG</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical oxygen</td>
			<td>Air Liquide Medical GmbH</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine (Temgesic)</td>
			<td>Indivior Eu Ltd.</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Bepanthen - ophthalmic ointment</td>
			<td>Bayer Vital GmbH</td>
			<td>10047757</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Research Anesthesia Machine x/O2 Flush w/ Sevoflurane Vaporizer</td>
			<td>Parkland Scientific</td>
			<td>V3000PS/PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Surgical Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulose swabs</td>
			<td>Lohmann &#38; Rauscher Deutschland</td>
			<td>13356</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe EMG 1 mL (with 30G cannula)</td>
			<td>B. Braun Melsungen AG</td>
			<td>9161627S</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Bore Tubing (bore: 0.28 mm/ diameter: 0.61mm)</td>
			<td>Smiths Medical Deutschland</td>
			<td>800/100/100</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps</td>
			<td>Fine Science Tools</td>
			<td>11018-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissor - ToughCut</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holder</td>
			<td>Fine Science Tools</td>
			<td>12002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoClip Kit</td>
			<td>Fine Science Tools</td>
			<td>12020-00</td>
			<td></td>
		</tr>
		<tr>
			<td>PDS Z1012H 6/0 C1 (surgical suture)</td>
			<td>Johnson &#38; Johnson Medical GmbH</td>
			<td>Z1012H</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Micro Serrefine Vascular Clamp</td>
			<td>Fine Science Tools</td>
			<td>18055-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Fogarty Spring Clips</td>
			<td>Edwards</td>
			<td>CDSAFE 6</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot Plate 062</td>
			<td>Labotect</td>
			<td>13854</td>
			<td></td>
		</tr>
		<tr>
			<td>Isis - Hair shaver</td>
			<td>Aesculap - Braun</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Colonoscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cold Light Fountain XENON 175 SCB</td>
			<td>Karl Storz</td>
			<td>20132101-1</td>
			<td>Karl Storz Coloview System Mainz</td>
		</tr>
		<tr>
			<td>Fiber Optic Light Cable</td>
			<td>Karl Storz</td>
			<td>69495NL</td>
			<td>Karl Storz Coloview System Mainz</td>
		</tr>
		<tr>
			<td>TRICAM Three-Chip Camera Head</td>
			<td>Karl Storz</td>
			<td>20221030</td>
			<td>Karl Storz Coloview System Mainz</td>
		</tr>
		<tr>
			<td>TRICAM SLII Camera Control Unit</td>
			<td>Karl Storz</td>
			<td>20223011-1</td>
			<td>Karl Storz Coloview System Mainz</td>
		</tr>
		<tr>
			<td>15&#34; Flat Screen Monitor EndoVue</td>
			<td>Karl Storz</td>
			<td>9415NN</td>
			<td>Karl Storz Coloview System Mainz</td>
		</tr>
		<tr>
			<td>HOPKINS Straight Forward Telescope 
			diameter 1.9 mm; length 10 cm 
			autoclavable 
			fiber optic light transmission incorporated</td>
			<td>Karl Storz</td>
			<td>64301AA</td>
			<td></td>
		</tr>
		<tr>
			<td>Protection and Examination Sheath</td>
			<td>Karl Storz</td>
			<td>61029C</td>
			<td></td>
		</tr>
	</tbody>
</table>

a genetically engineered mouse model of sporadic colorectal cancer

Kolay uygulanabilirlik ve maliyet etkinliğinin avantajlarına rağmen, tümör hücresi enjeksiyonuna dayanan kolorektal kanserli fare modelleri ciddi sınırlamalara sahiptir ve tümör biyolojisi ve tümör hücresi yayılımını doğru şekilde simüle etmez. Genetik olarak tasarlanmış fare modelleri bu sınırlamaların üstesinden gelmek için sunulmuştur; Bununla birlikte, bu tür modeller, özellikle tek bir tümörün arzulanan kolon gibi büyük organlarda teknik olarak talep edilmektedir. Sonuç olarak, oldukça uyumlu tümörler geliştiren ve tümör biyolojisi çalışmaları ve terapötik denemeler için kullanılabilen, bağışıklık kazandırıcı, genetik olarak tasarlanmış kolorektal kanserli fare modeli geliştirildi. Tümör gelişimi, bileşik koşullu mutant farelerde distal kolonun adeno-cre virüsü ile cerrahi, segmental enfeksiyonu ile başlatılır. Kolonoskopi ile tümörler kolayca tespit edilebilir ve izlenebilir. Burada segmental adeno-cre enfeksiyonunun cerrahi tekniğini tanımlıyoruzKolon, yüksek çözünürlüklü kolonoskopi ile tümörün gözetimi ve oluşan kolorektal tümörler.

Yeterli şekilde uygulandığında, hayvanlar&gt; n% 85&#39;inde tümörler gelişir. Burada sunulan cerrahi prosedürün mortalitesi &lt;% 5, kolonoskopinin mortalitesi neredeyse mevcut değildir. Farelerin çoğunda tek bir lezyon tespit edilmiştir; Yaklaşık% 30 2-3 küçük adenom tespit edilebilir, bunlar genellikle tümör indüksiyonundan 2-3 hafta sonra tek bir tümöre bağlanır. Ortaya çıkan tümörlerin fenotip...

Watch this Scientific Journal Video about A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer at JoVE.com

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Sporadik Kolorektal Kanserin Genetik Olarak Geliştirilmiş Fare Modeli

Segmental adeno-cre enfeksiyonu ile genetik olarak tasarlanmış kolorektal kanserli fare modeli kurulması ve yüksek çözünürlüklü kolonoskopi ile gözlemlenmesi için bir protokol sunulmuştur.

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

The overall goal of this procedure is to achieve segmental adeno-cre infection of the colon in a genetically engineered mouse model. This method can help answer key questions in the field of colorectal cancer, or CRC, as it provides an excellent model for studying the biology and treatment of CRC. The main advantage of this technique is that any lab with small rodent surgery experience can use this model to produce highly reproducible and easily accessible colon-localized tumors.Begin by placing an anesthetized mouse in the supine position and apply ointment to the animal's eyes. Tape the mouse in a secure position and then confirm the appropriate level of sedation by toe pinch. Make an approximately 15-millimeter midline incision in the skin of the lower abdomen.Then, use forceps to grasp the abdominal wall musculature and carefully incise the muscle with scissors to open the abdominal cavity. After identifying the distal colon, carefully clinch the tissue with a delicate clamp approximately 15 millimeters proximal to the anus. Next, insert a flexible Teflon tube transanally carefully advancing the tube until it reaches the luminal occlusion.Encannulate the tube with a 30-gauge cannula. Connect a standard one-milliliter syringe to the cannula and flush the colon with several milliliters of normal saline until all of the fecal matter has been evacuated. Once the distal colon is empty, replace the saline tube with a new Teflon tube and use a second clamp to occlude the colon approximately three millimeters distal to the first clamp.Using a second one-milliliter syringe equipped with a 30-gauge cannula, carefully inject 50 to 80 microliters of 0.25%trypsin-EDTA into the clamped colon segment. The colon should inflate enough to break up the mucosal barrier without perforating the clamped tissue segment. After 10 minutes, remove the distal clamp.Then, remove the trypsin tube and flush the distal colon with 500 microliters of normal saline. After inserting a new Teflon tube, clamp the colon again and inflate the tissue with 50 to 80 microliters of the adenoviral solution. After 30 minutes, remove the clamps and the tube and close the abdominal wall with 6-0 rapidly absorbable running sutures.Close the skin with surgical wound clips and monitor the animal on a heating pad until it is fully recovered. To monitor tumor growth via colonoscopy, place the anesthetized tumor-bearing animal in the supine position on a heating pad and transanally insert the endoscope into the intestinal tract. Gently insufflate air under visual control to distend the colon.Then, carefully push the scope forward until a mucosal lesion can be identified in the distal colon saving each endoscopic image as it is acquired for later evaluation. When all of the images have been obtained, slowly remove the endoscope and place the mouse on a 38-degree-Celsius heating pad until fully recovered. The tumors can be easily and repeatedly monitored via colonoscopy without major stress for the animals.The disease manifestations are very similar to the human colorectal cancer with the animals first developing adenomas followed ultimately by adenocarcinoma development in the distal colon. Depending on the conditional genotype of the animals, the tumors also metastasize to the peritoneum and the liver. If a fluorescent cre-reporter allele is used, the tumor manifestations can be easily identified by fluorescent protein expression which depending on the reporter allele, may be bright enough to be visualized in daylight.Histologically, 95%of the developing tumors are adenocarcinomas closely resembling human colorectal cancer and feature the entire spectrum of tumor development from non-invasive adenoma to adenocarcinoma invading the surrounding structures. Once mastered, this technique can be completed in 50 minutes if it's performed properly. While attempting this procedure, it is important to remember to give special attention to the vulnerability of the colon at all times.Perforation inevitably leads to peritonitis and sepsis and requires a termination of the enema. Don't forget that working with adenovirus requires biosafety level two safety measures as the virus can be hazardous.

Research

JoVE Journal

Cancer Research

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pankreas Kanseri Floresan Ortotopik Fare Modeli

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Kanser Araştırmaları

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Periferik Radyasyonla Bağlı Yorgunluk A Fare Modeli

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Kolorektal kanserli bir Ortotopik Fare Modelinde Dolaşan Tümör Hücrelerinin İzolasyonu

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

Yüksek çözünürlüklü ultrason monitör tümör başlangıçlı ve büyüme genetiği pankreas kanseri modellerinde kullanmak

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

Metastatik kolorektal kanser hastalarında dolaşımdaki mikroRNA özel Panel tespiti

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

Adeno-Associated Virus-Cas9 Sistemi Kullanılarak Genetiği Değiştirilmiş Bir Murine Baş ve Boyun Kanseri Hücre Hattı In Vitro Kurulması

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Establishing a Competing Risk Regression Nomogram Model for Survival Data

Survival Data için Rakip Risk Regresyon Nomogram Modeli Oluşturma

The Kaplan&#8211;Meier method and Cox proportional hazards regression model are the most common analyses in the survival framework. These are relatively ...

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Braf/Pten Genetiği Mühendisliğiyle Tasarlanmış Melanom Fare Modelinde Ultra Yüksek Frekanslı Ultrason Görüntüleme ile Lenf Düğümü Hacminin Analizi

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Karaciğer Metastazı Stromasını İncelemek için Kolorektal Kanser Organoidlerinin Portal Ven Enjeksiyonu

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor

Gastrointestinal Stromal Tümörün Genetiği Değiştirilmiş Bir Fare Modelinde Moleküler ve İmmünolojik Teknikler

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor ...