Name: A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer (Video) | JoVE Journal
Uploaded: 2017-07-06T12:30:00.000+00:00
Duration: 6 min 1 s
Description: Learn to establish a genetically engineered mouse model of colorectal cancer using segmental adeno-cre infection. This protocol enables reproducible colon-localized tumors for studying CRC biology and treatment.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work is dedicated to the memory of Professor Moritz Koch.

The animal experiments presented here were independently reviewed and approved by an institutional and a governmental Animal Care and Use Committee and were conducted according to Federation of Laboratory Animal Science Associations (FELASA) guidelines. All possible measures were taken to minimize suffering including anesthesia and analgesia or, if necessary, premature euthanasia.
1. Local Tumor Induction via Surgical Adeno-cre Infection
<ol>
	<li>Preparation of animals for surgery 
	NOTE: Virtually any conditional (&#34;floxed&#34;) mutation can be induced via the here described method. The use of mutations in genes relevant in colorectal cancer such as Apc, Kras or Tp53 is recommended. The efficiency of cre recombination is dependent on the size of the construct to be excised. Large floxed sequences are excised less efficiently. The recombination of all alleles should be confirmed in the tumors by PCR.
	<ol>
		<li>For the development of colorectal tumors, use a cross of the following conditional alleles for a basic model of CRC (MGI database number in brackets): 
		Apctm2Rak (MGI: 3688435)16 
		Krastm4Tyj (MGI: 2429948)17 
		Tp53tm2Tyj (MGI: 3039263)18</li>
		<li>If a fluorescence reporter allele is required (e.g., to detect micrometastases), use the following allele: 
		Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze (MGI: 3809522)19 
		NOTE: All above strains are available via the NCI Mouse Repository or the Jackson Laboratory. No fasting is required as all remaining fecal matter can be flushed out prior to the adenoviral infection. Preoperative fasting leads to higher perioperative mortality and constitutes tremendous stress for small rodents.</li>
		<li>Use sevoflurane at 3 - 3.5 vol% for general anesthesia. A loss of the toe pinch reflex indicates sufficient anesthesia.</li>
		<li>Prior to the first incision, inject 0.05 mg/kg of buprenorphine subcutaneously.</li>
		<li>Cover the eyes of the anesthetized mouse with ophthalmic ointment to avoid desiccation of the cornea.</li>
		<li>Place the mouse in a supine position on a small table. Use non-traumatic adhesive tapes to restrain the mouse.</li>
		<li>Shave the abdomen with an electric shaver (depilatory cream can be used alternatively) and disinfect with alcohol swabs or iodine. Use the contact time recommended by the manufacturer.</li>
		<li>Cover the surgical field with sterile drapes. 
		NOTE: The use of perioperative antibiotics is optional and subject to institutional guidelines.</li>
		<li>Use sterile single use or sterilized instruments for all surgical procedures.</li>
	</ol>
	</li>
	<li>Midline laparotomy and exposure of the colon
	<ol>
		<li>Use scissors (scalpels can be used alternatively) to make a midline incision (~ 15 mm) of the skin on the lower abdomen.</li>
		<li>Pick up the abdominal wall musculature with forceps and carefully incise it with scissors, thus opening the abdominal cavity.</li>
		<li>Identify the distal colon, only touch it with atraumatic forceps. Clamp the colon with a delicate clamp (e.g., a Micro Serrefine vascular clamp) approximately 15 mm proximally of the anus. 
		NOTE: Give special attention to the vulnerability of the colon at all times. Perforation inevitably leads to peritonitis and sepsis and requires euthanasia of the animal.</li>
	</ol>
	</li>
	<li>Segmental colon infection with Adeno-cre virus
	<ol>
		<li>Insert a flexible Teflon tube transanally and carefully advance it until it reaches the lumen occlusion achieved by the clamp previously placed at 15 mm from the anal verge. Do not use excess force as this may lead to perforation.</li>
		<li>Cannulate the tube with a 30G cannula, connect a standard 1 mL syringe and flush the colon with normal saline in order to evacuate remaining fecal matter. This may require several mL of saline.</li>
		<li>Once the distal colon is empty remove the tube and replace it with a fresh Teflon tube and again position it directly distal to the clamp as described above.</li>
		<li>Occlude the colon with a second clamp ~ 3 mm distal to the proximal clamp (i.e., over the inserted tube, ~ 12 mm from the anal verge), resulting in a 3 mm isolated segment to be infected. 
		NOTE: For distal occlusion of the segment Fogarty coronary artery clips have proven most suitable as they are rubberized, leading to tight occlusion of the colon despite the intraluminal tube between the clamp&#39;s branches.</li>
		<li>Use a second syringe (standard 1 mL with a 30G cannula) to carefully inject 50 - 80 &#181;L of 0.25% Trypsin-EDTA into the clamped colon segment and incubate for 10 min. Leave the cannula and the syringe attached to the Teflon tube to prevent the fluid from leaking back. 
		NOTE: The colon must be inflated to break up the mucosal barrier and to reach the crypt stem cells, yet not too much inflated to avoid perforation of the clamped segment.</li>
		<li>First remove the distal clamp, and then the trypsin tube.</li>
		<li>Flush the distal colon with ~ 500 &#181;L of normal saline to remove remaining trypsin.</li>
		<li>Insert a new Teflon tube, put the distal clamp back in place and inflate the colon segment with 50 - 80 &#181;L of adenoviral solution (1011 plaque-forming units (PFU)/mL in phosphate-buffered saline) and incubate for 30 min (Figure 2A). 
		NOTE: Do not spill viral solution as contact with adeno-cre may lead to tumor development in any tissue of conditionally mutant mice.</li>
		<li>Remove the clamps and the tube.</li>
	</ol>
	</li>
	<li>Closure of the abdomen and postoperative recovery
	<ol>
		<li>Close the abdominal wall with 6-0 rapidly absorbable running sutures (e.g., polydioxanone (PDS)).</li>
		<li>Close the skin with surgical wound clips.</li>
		<li>Place the mouse on heating pad set to 38 &#176;C until it has fully recovered from the anesthesia.</li>
		<li>Administer another bolus of 0.05 mg/kg buprenorphine i.p. 12 h after surgery, followed by additional buprenorphine boluses every 12 h&#160;if needed.</li>
		<li>Monitor the mice at least once daily for signs of distress due to tumor growth.</li>
	</ol>
	</li>
</ol>
2. Colonoscopy
NOTE: Depending on the conditional mutations used, adenoviral infection leads to endoscopically visible tumors within 2 - 4 weeks. Therefore, perform the first postoperative colonoscopy 2 weeks after the adenoviral induction and repeat every 2 weeks. A commercially available system is recommended for murine colonoscopy.20
<ol>
	<li>Preparation of animals for colonoscopy 
	NOTE: No fasting is required. Remaining fecal matter is usually well-formed in the distal colon and can be pushed beyond the tumor during colonoscopy, thus making the stressful process of repeated fasting unnecessary.
	<ol>
		<li>Use sevoflurane at 3 - 3.5 vol% for general anesthesia. A loss of the toe pinch reflex indicates sufficient anesthesia.</li>
		<li>Cover the eyes of the anesthetized mice with ophthalmic ointment to avoid desiccation of the cornea.</li>
		<li>Restrain the mice in a supine position on a small table.</li>
	</ol>
	</li>
	<li>Colonoscopy
	<ol>
		<li>Insert the scope (diameter 1.9 mm; length 10 cm) into the intestinal tract through the anus and carefully insufflate air under visual control to distend the colon. Do not insufflate more air than required for the examination. 
		NOTE: For air insufflation, the anti-fog air pump of the colonoscopy system may be used. If no anti-fog air pump is available, any other air pump with very low pressure settings can be used, or pressurized air with a delicate pressure-reducing valve. Carbon dioxide (CO2) easily leads to acidosis in small rodents and must therefore be avoided.</li>
		<li>Carefully push the scope forward until a mucosal lesion in the distal colon can be identified (Figure 1A - 1D).</li>
		<li>Save endoscopic images for later evaluation. An endoscopic scoring system for intraluminal tumors has been described before.20</li>
		<li>Carefully remove the scope and place the mouse on heating pad set to 38 &#176;C until it has fully recovered from the anesthesia.</li>
	</ol>
	</li>
</ol>

<ol><li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+statistics%2C+2016%5BTitle%5D)+AND+%22CA+Cancer+J+Clin%22%5BJournal%5D)">Cancer statistics, 2016.</a> CA Cancer J Clin. 66 (1), 7-30 (2016).</li>
<li>Weitz, J., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/15639298">Colorectal cancer.</a> Lancet. 365 (9454), 153-165 (2005).</li>
<li>Bork, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prognostic+relevance+of+minimal+residual+disease+in+colorectal+cancer%5BTitle%5D)+AND+%22World+J+Gastroenterol%22%5BJournal%5D)">Prognostic relevance of minimal residual disease in colorectal cancer.</a> World J Gastroenterol. 20 (30), 10296-10304 (2014).</li>
<li>Steinert, G., Schölch, S., Koch, M., Weitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biology+and+significance+of+circulating+and+disseminated+tumour+cells+in+colorectal+cancer%5BTitle%5D)+AND+%22Langenbecks+Arch+Surg%22%5BJournal%5D)">Biology and significance of circulating and disseminated tumour cells in colorectal cancer.</a> Langenbecks Arch Surg. 397 (4), 535-542 (2012).</li>
<li>García, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LDB1+overexpression+is+a+negative+prognostic+factor+in+colorectal+cancer%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">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/pubmed?term=((Novel+Drug+Candidates+for+the+Treatment+of+Metastatic+Colorectal+Cancer+through+Global+Inverse+Gene-Expression+Profiling%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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., García, S., Weitz, J., Schölch, S. <a target="_blank" href="https://www.omicsonline.org/peer-reviewed/mouse-models-of-colorectal-cancerderived-circulating-tumor-cells-76740.html">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="https://www.ncbi.nlm.nih.gov/pubmed/19263594">Mouse models of colon cancer.</a> Gastroenterology. 136 (3), 780-798 (2009).</li>
<li>Schölch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+tumor+cells+exhibit+stem+cell+characteristics+in+an+orthotopic+mouse+model+of+colorectal+cancer%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">Circulating tumor cells exhibit stem cell characteristics in an orthotopic mouse model of colorectal cancer.</a> Oncotarget. 7 (19), 27232-27242 (2016).</li>
<li>Schölch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Radiotherapy+combined+with+TLR7%2F8+activation+induces+strong+immune+responses+against+gastrointestinal+tumors%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">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. Jr <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumor+induction+relationships+in+development+of+transplantable+cancers+of+the+colon+in+mice+for+chemotherapy+assays%2C+with+a+note+on+carcinogen+structure%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Priceless+GEMMs%3A+genetically+engineered+mouse+models+for+colorectal+cancer+drug+development%5BTitle%5D)+AND+%22Trends+Pharmacol+Sci%22%5BJournal%5D)">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/pubmed?term=((Development+of+a+mouse+model+for+sporadic+and+metastatic+colon+tumors+and+its+use+in+assessing+drug+treatment%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">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/pubmed?term=((The+mighty+mouse%3A+genetically+engineered+mouse+models+in+cancer+drug+development%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">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/pubmed?term=((Rapid+colorectal+adenoma+formation+initiated+by+conditional+targeting+of+the+Apc+gene%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Adenomatous+polyposis+coli+%28APC%29+is+required+for+normal+development+of+skin+and+thymus%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">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/pubmed?term=((Analysis+of+lung+tumor+initiation+and+progression+using+conditional+expression+of+oncogenic+K-ras%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">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/pubmed?term=((Mutant+p53+gain+of+function+in+two+mouse+models+of+Li-Fraumeni+syndrome%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((A+robust+and+high-throughput+Cre+reporting+and+characterization+system+for+the+whole+mouse+brain%5BTitle%5D)+AND+%22Nature+Neurosci%22%5BJournal%5D)">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/pubmed?term=((High+resolution+colonoscopy+in+live+mice%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">High resolution colonoscopy in live mice.</a> Nat Protoc. 1 (6), 2900-2904 (2006).</li>
<li>Moser, A. R., Pitot, H. C., Dove, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+dominant+mutation+that+predisposes+to+multiple+intestinal+neoplasia+in+the+mouse%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse.</a> Science. 247 (4940), 322-324 (1990).</li>
<li>de Wind, N., Dekker, M., Berns, A., Radman, M., te Riele, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inactivation+of+the+mouse+Msh2+gene+results+in+mismatch+repair+deficiency%2C+methylation+tolerance%2C+hyperrecombination%2C+and+predisposition+to+cancer%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Inactivation of the mouse Msh2 gene results in mismatch repair deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer.</a> Cell. 82 (2), 321-330 (1995).</li>
<li>Reitmair, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spontaneous+intestinal+carcinomas+and+skin+neoplasms+in+Msh2-deficient+mice%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Spontaneous intestinal carcinomas and skin neoplasms in Msh2-deficient mice.</a> Cancer Res. 56 (16), 3842-3849 (1996).</li>
<li>Ayala, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standard+operating+procedures+for+describing+and+performing+metabolic+tests+of+glucose+homeostasis+in+mice%5BTitle%5D)+AND+%22Dis+Model+Mech%22%5BJournal%5D)">Standard operating procedures for describing and performing metabolic tests of glucose homeostasis in mice.</a> Dis Model Mech. 3 (9-10), 525-534 (2010).</li>
<li>Jensen, T. L., Kiersgaard, M. K., Sørensen, D. B., Mikkelsen, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fasting+of+mice%3A+a+review%5BTitle%5D)+AND+%22Lab+Anim%22%5BJournal%5D)">Fasting of mice: a review.</a> Lab Anim. 47 (4), 225-240 (2013).</li>
</ol>

The authors have nothing to disclose.

While they are generally easy to generate and maintain, classical CRC mouse models based on cell line injection are artificial and are not able to fully recapitulate the human disease. As a consequence, GEMMs have been developed. The first CRC GEMM was the ApcMin mouse, which harbors a heterozygous null mutation in the Apc gene, therefore mimicking the human hereditary disease familial adenomatous polyposis (FAP).21 However, ApcMin mice invariably develop multiple intestinal ...

Colorectal cancer (CRC) continues to be one of the leading causes of cancer-related death in western countries.1 While the prognosis of patients with early stage disease is good, many tumors are diagnosed at later stages in which, despite numerous treatment options, the prognosis is limited.2,3,4,5
The majority of current mouse models of CRC are based on the implantation of tumor cells derived from cell lines or patient tumors into immunodeficient mice.6,7,8 This leads to local and, depending on the injection site and the tumor cells used for injection, sometimes metastatic tumors.9,10 However, the resulting xenograft models have major limitations. They must be established in immunodeficient mice, thus eliminating the complex interaction between the tumor and the host immune system. In addition, as the tumor stroma is derived from host cells, the interaction between human tumor parenchyma and murine stroma is defective and therefore not representative of the disease. These deficiencies can be avoided by the use of murine cell lines for injection. However, only few murine CRC cell lines are available and, similar to most available human CRC cell lines, are monoclonal and highly anaplastic.11 In summary, most currently available CRC mouse models are highly artificial and not fully representative of the human disease.
Genetically engineered mouse models (GEMMs) of CRC can avoid these drawbacks as they feature genuine mouse tumors which are created via induction of key mutations of CRC in the colon.12,13,14 This can be achieved by the activation of conditional (floxed) germline mutations by cre recombinase within the colorectal mucosa. While in GEMMs of many other tumor entities germline (inducible) cre expression driven by tissue-specific promoters is used, germline cre cannot be used in the colon as this leads to a great number of adenomas throughout the colon causing death by benign tumor load at a very young age. Therefore, in the here described model an adenoviral vector expressing cre is used to infect a short colon segment. This leads to the induction of tumorigenesis within this segment of the mucosa at a time point defined by the investigator, resulting in adenomas ultimately progressing to invasive and metastatic carcinoma. The tumors are genuine mouse tumors, grow in an intact microenvironment and are therefore able to simulate the entirety of colorectal oncogenesis including tumor&#8211;host interaction and the metastatic cascade. This model is therefore an attractive platform for studies of cancer biology and preclinical therapeutic trials.
A major disadvantage of genetically engineered mouse models of CRC is their technical complexity. Local cre delivery using rectal adeno-cre enemas in mice carrying floxed Apc alleles has been described before; however, the incidence, multiplicity and location of the intestinal tumors can be highly variable with this technique.15 Therefore, the technique of confining the adeno-cre infection by surgical clamping of the segment to be induced has been developed.13 We have modified this procedure in order to improve animal welfare, as well as reduce mortality and the number of resulting tumors. With this protocol, all labs with experience in small rodent surgery should be able to reproduce the model and to produce tumors which are highly reproducible and easily accessible to colonoscopy. Depending on the conditional mutations used for tumorigenesis, the full spectrum of adenoma, invasive carcinoma and metastases can be observed. As the tumors are located in the distal colon, serial endoscopic assessment is easily possible in this model.

<table><tbody><tr><td>&lt;strong&gt;Reagents / consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;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&lt;br/&gt; 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 &amp;amp; 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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Analgesia / anesthesia&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sevoflurane (Sevoflurane AbbVie)</td><td>AbbVie Germany GmbH &amp;amp; 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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Surgical Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cellulose swabs</td><td>Lohmann &amp;amp; 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 &amp;amp; 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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Colonoscopy&lt;/strong&gt;</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&quot; 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&lt;br/&gt; diameter 1.9 mm; length 10 cm&lt;br/&gt; autoclavable&lt;br/&gt; 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 Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

If performed adequately, &gt; 85% of the animals develop tumors. The mortality of the here presented surgical procedure is &lt; 5%, mortality of the colonoscopy is virtually non-existent. In the majority of mice, a single lesion is detected; in about 30% 2 - 3 small adenomas can be detected which usually fuse to a single tumor within 2 - 3 weeks after tumor induction.
The phenotype and biological behavior of the resulting tumors...

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

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.

a-genetically-engineered-mouse-model-of-sporadic-colorectal-cancer

Research

JoVE Journal

Cancer Research

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Orthotopic Mouse Model of Colorectal Cancer

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

This video describes the protocol for generating a patient-derived orthotopic xenograft colorectal cancer mouse model to study the progression of colorectal cancer cells in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. This murine model helps in understanding primary tumor growth and metastases pattern.

Generating PDOX Mouse Model of Colorectal Cancer: Studying Progression of Colorectal Cancer in a Murine Model

This video describes the protocol to establish a spontaneous metastasis mouse model of colorectal cancer patient-derived xenografts or PDXs. These xenograft models partially mimic the features of human carcinomas and understand the critical aspects of human malignancies, including invasion and metastasis.

Spontaneous Metastasis Mouse Models: A Platform to Study the Metastatic Potential of Orthotopically Injected Colorectal Cancer Cells

This video describes the generation of a colorectal mouse model by segmental adeno-cre infection. The infected colon segment leads to tumorigenesis within this segment of the mucosa, resulting in adenomas ultimately progressing to invasive and metastatic carcinoma. This model is an attractive platform for studies of cancer biology and preclinical therapeutic trials.

Segmental Adeno-Cre Infection: A Technique to Generate Isolated Colorectal Cancer using Genetically Engineered Mouse Models

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

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

The goal of this manuscript is to describe the KitV558Δ/+ mouse model and techniques for successful dissection and processing of mouse specimens.

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

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

This study focuses on the in vivo subcutaneous induction of luminescent colorectal cancer cells in a murine model and the subsequent luminescence measurement using an imaging system. The procedure aims to establish a reproducible tumor growth model for real-time monitoring and therapeutic evaluation.

Subcutaneous Injection of Human Colorectal Cancer Cells in Athymic Nude Mice to Evaluate Antitumor Efficacy

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.



Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Learn to establish a genetically engineered mouse model of colorectal cancer using segmental adeno-cre infection. This protocol enables reproducible colon-localized tumors for studying CRC biology and treatment.

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Explore More Videos