A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Summary

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.

Abstract

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.

Introduction

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,<....

Protocol

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

1. Preparation of animals for surgery
   NOTE: Virtually any conditional ("floxed") mutation can be induced via the here described method. The use of mutations....

Results

If performed adequately, > 85% of the animals develop tumors. The mortality of the here presented surgical procedure is < 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.......

Discussion

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 Apc^Min mouse, which harbors a heterozygous null mutation in the Apc gene, therefore mimicking the human hereditary disease familial adenomatous polyposis (FAP).²¹ However, Apc^Min mice invariably develop multiple intestinal .......

Materials

List of materials used in this article

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