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

Este trabajo está dedicado a la memoria del profesor Moritz Koch.

Los experimentos con animales presentados aquí fueron revisados ​​y aprobados de forma independiente por un Comité institucional y gubernamental de Cuidado y Uso de Animales y se llevaron a cabo de acuerdo con las directrices de la Federación de Asociaciones de Ciencia Animal de Laboratorio (FELASA). Todas las medidas posibles fueron tomadas para minimizar el sufrimiento incluyendo anestesia y analgesia o, si es necesario, eutanasia prematura. 1. Inducción local del tumor a través...

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LDB1+overexpression+is+a+negative+prognostic+factor+in+colorectal+cancer.">LDB1 overexpression is a negative prognostic factor in colorectal cancer.</a> Oncotarget. 7 (51), 84258-84270 (2016).</li><li>van Noort, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Drug+Candidates+for+the+Treatment+of+Metastatic+Colorectal+Cancer+through+Global+Inverse+Gene-Expression+Profiling.">Novel Drug Candidates for the Treatment of Metastatic Colorectal Cancer through Global Inverse Gene-Expression Profiling.</a> Cancer Res. 74 (20), 5690-5699 (2014).</li><li>Nanduri, L. K., Garc&#237;a, S., Weitz, J., Sch&#246;lch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Models+of+Colorectal+Cancer-Derived+Circulating+Tumor+Cells.">Mouse Models of Colorectal Cancer-Derived Circulating Tumor Cells.</a> Med Chem (Los Angeles). 6 (7), 497-499 (2016).</li><li>Taketo, M. M., Edelmann, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+colon+cancer.">Mouse models of colon cancer.</a> Gastroenterology. 136 (3), 780-798 (2009).</li><li>Sch&#246;lch, S., 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=Circulating+tumor+cells+exhibit+stem+cell+characteristics+in+an+orthotopic+mouse+model+of+colorectal+cancer.">Circulating tumor cells exhibit stem cell characteristics in an orthotopic mouse model of colorectal cancer.</a> Oncotarget. 7 (19), 27232-27242 (2016).</li><li>Sch&#246;lch, S., 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=Radiotherapy+combined+with+TLR7/8+activation+induces+strong+immune+responses+against+gastrointestinal+tumors.">Radiotherapy combined with TLR7/8 activation induces strong immune responses against gastrointestinal tumors.</a> Oncotarget. 6 (7), 4663-4676 (2015).</li><li>Corbett, T. H., Griswold, D. P., Roberts, B. J., Peckham, J. C., Schabel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+induction+relationships+in+development+of+transplantable+cancers+of+the+colon+in+mice+for+chemotherapy+assays,+with+a+note+on+carcinogen+structure.">Tumor induction relationships in development of transplantable cancers of the colon in mice for chemotherapy assays, with a note on carcinogen structure.</a> Cancer Res. 35 (9), 2434-2439 (1975).</li><li>Roper, J., Hung, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priceless+GEMMs:+genetically+engineered+mouse+models+for+colorectal+cancer+drug+development.">Priceless GEMMs: genetically engineered mouse models for colorectal cancer drug development.</a> Trends Pharmacol Sci. 33 (8), 449-455 (2012).</li><li>Hung, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+mouse+model+for+sporadic+and+metastatic+colon+tumors+and+its+use+in+assessing+drug+treatment.">Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment.</a> Proc Natl Acad Sci USA. 107 (4), 1565-1570 (2010).</li><li>Sharpless, N. E., Depinho, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mighty+mouse:+genetically+engineered+mouse+models+in+cancer+drug+development.">The mighty mouse: genetically engineered mouse models in cancer drug development.</a> Nat Rev Drug Discov. 5 (9), 741-754 (2006).</li><li>Shibata, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+colorectal+adenoma+formation+initiated+by+conditional+targeting+of+the+Apc+gene.">Rapid colorectal adenoma formation initiated by conditional targeting of the Apc gene.</a> Science. 278 (5335), 120-123 (1997).</li><li>Kuraguchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenomatous+polyposis+coli+(APC)+is+required+for+normal+development+of+skin+and+thymus.">Adenomatous polyposis coli (APC) is required for normal development of skin and thymus.</a> PLoS Genet. 2 (9), e146 (2006).</li><li>Jackson, E. 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. P., 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=Mutant+p53+gain+of+function+in+two+mouse+models+of+Li-Fraumeni+syndrome.">Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome.</a> Cell. 119 (6), 847-860 (2004).</li><li>Madisen, 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=A+robust+and+high-throughput+Cre+reporting+and+characterization+system+for+the+whole+mouse+brain.">A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.</a> Nature Neurosci. 13 (1), 133-140 (2010).</li><li>Becker, C., Fantini, M. C., Neurath, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+colonoscopy+in+live+mice.">High resolution colonoscopy in live mice.</a> Nat Protoc. 1 (6), 2900-2904 (2006).</li><li>Moser, A. R., Pitot, H. C., Dove, W. 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Mientras que son generalmente fáciles de generar y de mantener, los modelos clásicos del ratón del CRC basados ​​en la inyección de la línea celular son artificiales y no pueden recapitulate completamente la enfermedad humana. Como consecuencia, se han desarrollado GEMMs. El primer CRC GEMM fue el ratón Apc Min , que alberga una mutación nula heterocigótica en el gen Apc, por lo tanto imitando la enfermedad hereditaria humana poliposis adenomatosa familiar (FAP). Sin embargo, los ratones Apc ...

El cáncer colorrectal (CRC) sigue siendo una de las principales causas de muerte relacionada con el cáncer en los países occidentales. 1 Aunque el pronóstico de los pacientes con enfermedad en estadio temprano es bueno, muchos tumores son diagnosticados en etapas posteriores en los que, a pesar de las numerosas opciones de tratamiento, el pronóstico es limitado. 2 , 3 , 4 , 5 La mayoría de los actuales modelos de CRC de ratones se basan en la implantación de células tumorales derivadas de líneas celulares o tumores de pacientes en ratones inmunodeficientes. 6 , 7 , 8 Esto conduce a local y, dependiendo del sitio de inyección y de las células tumorales utilizadas para inyección, a veces tumores metastásicos. 9 , 10 Sin embargo, los modelos de xenoinjerto resultantes tienen majoR limitaciones. Deben establecerse en ratones inmunodeficientes, eliminando así la interacción compleja entre el tumor y el sistema inmune del huésped. Además, como el estroma tumoral se deriva de las células huésped, la interacción entre el parénquima tumoral humano y el estroma murino es defectuosa y por lo tanto no es representativa de la enfermedad. Estas deficiencias pueden evitarse mediante el uso de líneas de células murinas para inyección. Sin embargo, sólo hay pocas líneas de células de CRC murinas disponibles y, similares a la mayoría de las líneas de células CRC humanas disponibles, son monoclonales y altamente anaplásticas. En resumen, la mayoría de los modelos de ratón CRC actualmente disponibles son altamente artificiales y no son totalmente representativos de la enfermedad humana. Modelos de ratón genéticamente modificados (GEMM) de CRC pueden evitar estos inconvenientes, ya que presentan verdaderos tumores de ratón que se crean a través de la inducción de las principales mutaciones de CRC en el colon. 12 , 13 ,Esto puede lograrse mediante la activación de las mutaciones de la línea germinal condicional (floxed) por cre recombinasa dentro de la mucosa colorrectal. Mientras que en GEMMs de muchas otras entidades tumorales germline (inducible) cre expresión impulsada por promotores específicos de tejidos se utiliza, línea germinal cre no se puede utilizar en el colon, ya que esto conduce a un gran número de adenomas en todo el colon causando la muerte por tumor benigno carga a Una edad muy joven. Por lo tanto, en el modelo descrito aquí se utiliza un vector adenoviral que expresa cre para infectar un segmento de colon corto. Esto conduce a la inducción de la tumorigénesis dentro de este segmento de la mucosa en un punto de tiempo definido por el investigador, dando lugar a adenomas en última instancia, progresando a carcinoma invasivo y metastásico. Los tumores son verdaderos tumores de ratón, crecen en un microambiente intacto y por lo tanto son capaces de simular la totalidad de la oncogénesis colorrectal incluyendo la interacción tumor-huésped y la cascada metastásica. Este modelo esPor lo tanto, una plataforma atractiva para estudios de biología del cáncer y ensayos terapéuticos preclínicos. Una de las principales desventajas de los modelos de CRC modificados genéticamente es su complejidad técnica. Se ha descrito anteriormente la administración local de crema usando enemas de adeno-crema rectales en ratones que llevan alelos de Apc floxed; Sin embargo, la incidencia, multiplicidad y localización de los tumores intestinales puede ser altamente variable con esta técnica. Por lo tanto, se ha desarrollado la técnica de confinar la infección adeno-cre por pinzamiento quirúrgico del segmento a inducir. 13 Hemos modificado este procedimiento con el fin de mejorar el bienestar animal, así como reducir la mortalidad y el número de tumores resultantes. Con este protocolo, todos los laboratorios con experiencia en cirugía de roedores pequeños deben ser capaces de reproducir el modelo y producir tumores que sean altamente reproducibles y de fácil acceso a la colonoscopia. Dependiendo del condicional mUvas utilizadas para la tumorigénesis, se puede observar el espectro completo de adenoma, carcinoma invasivo y metástasis. Como los tumores se localizan en el colon distal, la evaluación endoscópica seriada es fácilmente posible en este modelo.

<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>
		</tr>
		<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

A pesar de las ventajas de la fácil aplicabilidad y rentabilidad, los modelos de ratones de cáncer colorrectal basados ​​en la inyección de células tumorales tienen limitaciones severas y no simulan con precisión la biología tumoral y la diseminación de células tumorales. Se han introducido modelos de ratón genéticamente modificados para superar estas limitaciones; Sin embargo, tales modelos son técnicamente exigentes, especialmente en órganos grandes como el colon en el que sólo se desea un solo tumor. Como resultado, se desarrolló un modelo inmunocompetente, modificado genéticamente, de cáncer colorrectal que desarrolla tumores muy uniformes y puede usarse para estudios de biología tumoral así como para ensayos terapéuticos. El desarrollo tumoral se inicia mediante la infección quirúrgica segmentaria del colon distal con el virus adeno-cre en ratones mutantes condicionalmente compuestos. Los tumores pueden ser fácilmente detectados y monitorizados vía colonoscopia. Aquí describimos la técnica quirúrgica de la infección segmentaria adeno-cre deEl colon, la vigilancia del tumor vía colonoscopia de alta resolución y presentan los tumores colorrectales resultantes.

Si se realiza adecuadamente,&gt; 85% de los animales desarrollan tumores. La mortalidad del procedimiento quirúrgico aquí presentado es &lt;5%, la mortalidad de la colonoscopia es prácticamente inexistente. En la mayoría de los ratones, se detecta una sola lesión; En aproximadamente el 30% se pueden detectar 2 - 3 adenomas pequeños que usualmente se fusionan a un solo tumor dentro de 2 a 3 semanas después de la inducción del tumor. <p class="jove_content" fo:keep-together.wit...

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

Un modelo de ratón de ingeniería genética de cáncer colorrectal esporádico

Se presenta un protocolo para el establecimiento de un modelo genético de ratón de cáncer colorrectal por infección segmentaria de adenovirus y su vigilancia mediante colonoscopia de alta resolución.

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.

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