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

Dette arbeidet er dedikert til minnet av professor Moritz Koch.

Dyreforsøkene som presenteres her, ble uavhengig gjennomgått og godkjent av en institusjonell og en statlig dyrepleie- og brukskomité og ble utført i henhold til retningslinjer fra FELASA (Federation of Laboratory Animal Science Associations). Alle mulige tiltak ble tatt for å minimere lidelse, inkludert anestesi og analgesi eller om nødvendig for tidlig dødshjelp. 1. Lokal tumorinduksjon via kirurgisk adeno-cre-infeksjon <ol><li> Forberedelse av dyr for kirurgi</st...

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2016.">Cancer statistics, 2016.</a> CA Cancer J Clin. 66 (1), 7-30 (2016).</li><li>Weitz, J., 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=Colorectal+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+relevance+of+minimal+residual+disease+in+colorectal+cancer.">Prognostic relevance of minimal residual disease in colorectal cancer.</a> World J Gastroenterol. 20 (30), 10296-10304 (2014).</li><li>Steinert, G., Sch&#246;lch, S., Koch, M., Weitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+significance+of+circulating+and+disseminated+tumour+cells+in+colorectal+cancer.">Biology and significance of circulating and disseminated tumour cells in colorectal cancer.</a> Langenbecks Arch Surg. 397 (4), 535-542 (2012).</li><li>Garc&#237;a, S. 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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dominant+mutation+that+predisposes+to+multiple+intestinal+neoplasia+in+the+mouse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+mouse+Msh2+gene+results+in+mismatch+repair+deficiency,+methylation+tolerance,+hyperrecombination,+and+predisposition+to+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+intestinal+carcinomas+and+skin+neoplasms+in+Msh2-deficient+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+operating+procedures+for+describing+and+performing+metabolic+tests+of+glucose+homeostasis+in+mice.">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., S&#248;rensen, D. B., Mikkelsen, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fasting+of+mice:+a+review.">Fasting of mice: a review.</a> Lab Anim. 47 (4), 225-240 (2013).</li></ol>

Mens de generelt er enkle å generere og vedlikeholde, er klassiske CRC-musemodeller basert på injeksjon av cellelinjer kunstige og kan ikke fullt ut rekapulere den menneskelige sykdommen. Som en konsekvens er GEMMer blitt utviklet. Den første CRC GEMM var Apc Min- musen, som har en heterozygot null mutasjon i Apc-genet, og etterligner derfor den menneskelige arvelige sykdommen familial adenomatous polyposis (FAP). 21 Apc Min- mus utvikler imidlertid alltid flere intestina...

Colorectal cancer (CRC) er fortsatt en av de viktigste årsakene til kreftrelatert død i vestlige land. 1 Mens prognosen til pasienter med tidlig stadiumssykdom er god, diagnostiseres mange svulster ved senere stadier der, til tross for mange behandlingsalternativer, er prognosen begrenset. 2 , 3 , 4 , 5 Flertallet av nåværende musemodeller av CRC er basert på implantering av tumorceller som er avledet fra cellelinjer eller pasienttumorer i immunodeficiente mus. 6 , 7 , 8 Dette fører til lokal og, avhengig av injeksjonsstedet og tumorcellene som brukes til injeksjon, noen ganger metastaserende tumorer. 9 , 10 Imidlertid har de resulterende xenograftmodellene majoR begrensninger. De må etableres i immunodeficiente mus, og eliminerer dermed det komplekse samspillet mellom svulsten og vertsimmunsystemet. I tillegg, da tumorstroma er avledet fra vertsceller, er samspillet mellom humant tumorparenchyma og murint stroma defekt og derfor ikke representativt for sykdommen. Disse manglene kan unngås ved bruk av murine cellelinjer for injeksjon. Imidlertid er bare få muse-CRC-cellelinjer tilgjengelige, og er lik monoklonale og svært anaplastiske, som de fleste tilgjengelige humane CRC-cellelinjer. 11 Sammendrag er de fleste tilgjengelige CRC-musemodellene svært kunstige og ikke fullt representative for den menneskelige sykdommen. Genetisk utviklede musemodeller (GEMMs) av CRC kan unngå disse ulempene da de har ekte musetumorer som er opprettet via induksjon av nøkkelmutasjoner av CRC i kolon. 12 , 13 ,14 Dette kan oppnås ved aktivering av betingede (floxed) germline mutasjoner av cre recombinase i kolorektal mucosa. Mens det i GEMMer av mange andre tumor-enheter er kimlinje (inducerbart) cre-uttrykk drevet av vevsspesifikke promotorer, kan germline cre ikke brukes i tykktarmen da dette fører til et stort antall adenomer gjennom tykktarmen som forårsaker død ved godartet tumorbelastning ved En veldig ung alder. Derfor, i den her beskrevne modellen, brukes en adenoviral vektor-uttrykkende cre til å infisere et kort tarmsegment. Dette fører til induksjon av tumorigenese innenfor dette segmentet av slimhinnen på et tidspunkt definert av etterforskeren, noe som resulterer i adenomer i ferd med å utvikle seg til invasiv og metastatisk karsinom. Tumorene er ekte musetumorer, vokser i intakt mikromiljø og kan derfor simulere hele kolorektal onkogenesen, inkludert tumor-vert-interaksjon og metastatisk kaskade. Denne modellen erDerfor en attraktiv plattform for studier av kreftbiologi og prekliniske terapeutiske studier. En stor ulempe med genetisk utviklede musemodeller av CRC er deres tekniske kompleksitet. Lokal kreverlevering ved hjelp av rektale adeno-cre-enemaser i mus som bærer flomede Apc-alleler, er tidligere beskrevet; Imidlertid kan forekomsten, multiplikasjonen og plasseringen av tarmtumorene være svært variabel med denne teknikken. Derfor er teknikken for å begrense adeno-cre-infeksjonen ved kirurgisk klemming av segmentet som skal induseres, blitt utviklet. 13 Vi har endret denne prosedyren for å forbedre dyrevelferd, samt redusere dødelighet og antall resulterende svulster. Med denne protokollen skal alle laboratorier med erfaring i liten gnagekirurgi kunne reproducere modellen og produsere svulster som er svært reproduserbare og lett tilgjengelige for koloskopi. Avhengig av betinget mUtgaver som brukes til tumorigenese, det fullstendige spektrum av adenom, invasiv karsinom og metastaser kan observeres. Da svulstene befinner seg i distal kolon, er seriell endoskopisk vurdering lett mulig i denne modellen.

<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>
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a genetically engineered mouse model of sporadic colorectal cancer

Til tross for fordelene ved enkel brukbarhet og kostnadseffektivitet, har muskelmodeller med tykktarmskreft basert på tumorcelleinjeksjon alvorlige begrensninger og simulerer ikke nøyaktig simulering av tumorbiologi og tumorcelle. Genetisk utviklede musemodeller er innført for å overvinne disse begrensningene; Imidlertid er slike modeller teknisk krevende, spesielt i store organer som tykktarmen der bare en enkelt tumor er ønsket. Som et resultat ble en immunokompetent, genetisk utviklet musemodell av kolorektal kreft utviklet som utvikler høyt ensartede tumorer og kan brukes til tumorbiologi studier samt terapeutiske studier. Tumorutvikling initieres ved kirurgisk, segmental infeksjon i distal kolon med adeno-cre-virus i sammensatte betingede mutante mus. Tumorene kan lett oppdages og overvåkes via koloskopi. Vi beskriver her kirurgisk teknikk for segmental adeno-cre infeksjon avKolon, overvåkning av svulsten via kolonoskopi med høy oppløsning og presentere de resulterende kolorektale svulstene.

Hvis det utføres tilstrekkelig, utvikler&gt; 85% av dyrene svulster. Dødeligheten av den her presenterte kirurgiske prosedyren er &lt;5%, dødeligheten av koloskopien er praktisk talt ikke-eksisterende. I de fleste mus, oppdages en enkelt lesjon; I ca 30% kan 2 - 3 små adenomer detekteres som vanligvis smelter til en enkelt tumor innen 2 - 3 uker etter tumorinduksjon. Fenotypen og biologisk oppførsel av de resulterende svu...

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

En genetisk utviklet musemodell av sporadisk kolorektal kreft

En protokoll for etablering av en genetisk utviklet musemodell av kolorektal kreft ved segmentert adeno-cre infeksjon og overvåkning via kolonoskopi med høy oppløsning presenteres.

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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