Construction method and application of animal model

Abstract

The invention discloses a construction method and application of a mouse colorectal cancer model. The construction method specifically comprises the following steps of: constructing GT11ML; the method comprises the following steps: enabling an animal of Hprt-Cre to mate with a TG11ML animal and a P53flox animal, and finally stably obtaining the TG11ML animal and the P53flox animal; a GT11ML; and Hprt-Cre animals. The animal model constructed by the method is stable in character, and the success rate reaches 100%; compared with a traditional animal model, the method has the advantages that the whole process of occurrence and development of diseases can be simulated more truly, the transfer or abnormal behaviors of mutant cells can be monitored and the like, and the method is of great significance to development of nutrients and medicines in related fields.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a method for constructing an animal model and its application. Background Art

[0002] Colorectal cancer is one of the most common cancers in humans and one of the main causes of death. The high incidence of colorectal cancer will bring a heavy treatment burden to society and patients' families. If discovered in the early stages, the prognosis of early treatment of colorectal cancer is good, and the 5-year survival rate of stage I is over 75%. Early intervention is crucial. Therefore, the prevention and control of colorectal cancer through foodborne polysaccharide immune regulation is of great research significance. The most widely used and studied model in the scientific community is the genetically engineered mouse model with APC gene mutation. However, after the loss of the APC gene as a driver mutation, colorectal cancer progresses rapidly across the early pathological stage. There is a lack of an early prevention and control window period, and it is impossible to effectively evaluate the early colorectal cancer prevention and control efficacy of drugs and nutrients.

[0003] Transgenic mouse models of colorectal cancer (CRC) are important tools for studying tumor biology and testing new treatments. The following are some of the commonly used transgenic mouse models of colorectal cancer:

[0004] APC Min / + (Multiple Intestinal Neoplasia) model: APC gene mutations are closely related to the occurrence of colorectal cancer. In this model, mice carry mutations in the APC gene, which leads to the development of spontaneous small intestinal and colorectal adenomas. Mice in the APC Min / + model exhibit tumor characteristics similar to those of human familial adenomatous polyposis (FAP). However, tumors in this model mainly occur in the small intestine, while human colorectal cancer occurs more frequently in the colorectal area. This difference may affect the applicability of the model in certain studies. The APC Min / + model provides an important platform for studying the genetics, molecular mechanisms, and treatment strategies of colorectal cancer. However, due to the differences in the site of tumor occurrence from human colorectal cancer, data need to be interpreted with caution when using this model.

[0005] P53 mutant colorectal cancer model: In this model, mice exhibit accelerated tumorigenesis, which is highly similar to tumors associated with P53 mutations in humans. Tumors in the P53 mutant model show high metastasis, especially to the liver and lungs, which provides an important tool for studying the metastatic mechanism of tumors. The problem is that p53 is homozygous knockout in all epithelial cells in the intestine.

[0006] In addition to transgenic models, there is also a chemically induced AOM / DSS colorectal cancer model. The AOM / DSS model demonstrates a multi-stage carcinogenesis process similar to that of human CRC in replicating inflammation-driven colorectal cancer (CRC). From inflammation-induced epithelial damage to the development of adenomas and cancers, this model reveals a direct link between tumor progression and chronic inflammation. However, this model has disadvantages: since tumor development depends on chemical induction, this may affect the applicability of the model and the interpretation of the results; and when AOM-DSS is used for induction, it will form tumors, and the value of tumor formation is very limited. Specifically, the microenvironment epithelial cells surrounding the tumor cells are also cells with p53 gene mutations, which is seriously inconsistent with the process of human colorectal cancer.

[0007] In summary, traditional models cannot simulate how tumor cells evolve with the tumor microenvironment during the development of human colorectal tumors. A mouse model that can more realistically simulate the development of tumors (from gene mutation to tumor development) is urgently needed. Understanding the early development of tumors will greatly accelerate the analysis of the pathogenesis of tumors and provide a theoretical basis for targeted immune intervention with drugs and nutrients. Summary of the invention

[0008] The purpose of the present invention is to use MADM technology to achieve precise control of p53 gene activity in intestinal stem cells, and further observe the changes in cell behavior after the p53 gene state changes through the inflammatory environment induced by AOM, and construct a spontaneous MADM-p53 mutation early intestinal cancer animal model. The model of the present invention can achieve the distinction between microenvironment cells and gene mutation cells. More importantly, MADM technology realizes the linkage between genotype and color lineage tracing, and the genotype can be judged by color, so the green color can be used to determine that it is a p53 gene mutation cell; at the same time, because it can be directly distinguished by color, it is better to analyze the tumor.

[0009] The technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides a method for constructing a mouse colorectal cancer model, comprising the following steps: constructing a P53 mutation-MADM animal model, and then inducing it using AOM.

[0011] In some embodiments of the present invention, the method for constructing a P53 mutation-MADM mouse model includes: constructing a genetically edited mouse with p53 flox that can induce recombination at the p53 genomic site on chromosome 11 of the mouse, and then mating MADM-Chr11 mice with p53flox mutation genetic mice to obtain P53 mutation-MADM mice.

[0012] In some embodiments of the present invention, the method for constructing a P53 mutation-MADM mouse model specifically comprises the following steps:

[0013] (1) Construction of Stock I mice: GT11ML mice were mated with HPRT-Cre mice to obtain GTML11;Hprt-Cre double heterozygous mice, and GTML11;Hprt-Cre double heterozygous mice were self-fertilized to obtain Stock I mice with the genotype of GT11ML;HPRT-Cre. Construction of Stock II mice: TG11ML mice were mated with P53 flox mice to produce TG11ML,P53 flox double heterozygous positive mice, and the double heterozygous positive mice were mated with TG11ML mice to obtain Stock II mice with the genotype of TG11ML,P53 flox;

[0014] (2) Stock I mice were mated with Stock II mice to obtain animals with the genotype of TG11ML, P53 flox; GT11ML; Hprt-Cre, i.e., the P53 mutant-MADM mouse model;

[0015] Among them, GT11ML and TG11ML are MADM genes; G stands for green fluorescent protein, T stands for red fluorescent protein, 11 represents the MADM allele knocked into the animal chromosome 11, and ML stands for multiple flox site; GT11ML and TG11ML are located on different chromosomes.

[0016] In some embodiments of the invention, the green fluorescent protein comprises GFP.

[0017] In some embodiments of the present invention, the green fluorescent protein is GFP.

[0018] In some embodiments of the present invention, the red fluorescent protein includes tdTomato.

[0019] In some embodiments of the present invention, the red fluorescent protein is tdTomato.

[0020] In some embodiments of the present invention, the single injection dose of AOM is 5-20 mg / kg

[0021] In some embodiments of the present invention, the AOM is administered once or more times.

[0022] In some embodiments of the present invention, the AOM is administered once a week for 2 consecutive weeks.

[0023] In some embodiments of the present invention, the AOM administration methods include but are not limited to intravenous injection, oral administration, intramuscular injection, subcutaneous injection, oral gavage, intraperitoneal injection and the like.

[0024] In some embodiments of the present invention, the mice are 4-50 weeks old, with no particular limitation on gender.

[0025] In some embodiments of the present invention, in the construction method, the mice are raised at a rate of 4-6 mice per cage in an SPF-level barrier facility with free access to food.

[0026] The second aspect of the present invention provides the use of an animal model constructed by the construction method described in the first aspect of the present invention in studying the molecular mechanism of disease occurrence and development and / or screening drugs that can prevent, alleviate or treat diseases.

[0027] In some embodiments of the invention, the disease comprises a tumor.

[0028] In some embodiments of the invention, the tumor is colorectal cancer.

[0029] The present invention also provides a method for screening drugs that can prevent, alleviate or treat colorectal cancer, comprising administering the drug to be tested to mice, detecting the tumor status of the mice before and after administration; and selecting the drug that can prevent, alleviate or treat colorectal cancer.

[0030] MADM cancer models have three unique advantages that make them ideal tools for studying the origin of cancer cells and tumor development:

[0031] (1) MADM is based on the loss-of-heterozygosity gene mutation and produces a small number of cancerous cells. This process highly simulates the process of spontaneous tumor cell generation in the body;

[0032] (2) When the initial mutation is introduced into a cell, the cell and its progeny cells are permanently labeled with GFP, thereby enabling high-resolution observation of mutant cell behavior and cell lineage tracing analysis at the early stages of cancer that cannot be detected by traditional pathological analysis.

[0033] (3) For every green mutant cell that is generated, a red normal cell is also generated. By comparing the two colored labeled cells, we can obtain accurate information about the abnormal behavior of mutant cells. Using the unique advantages of MADM, we can study the evolution of the tumor microenvironment.

[0034] Based on MADM technology, the present invention constructs a new MADM-P53 colorectal cancer model (gene knock-in on chromosome 11). In this model, cells labeled with green fluorescent protein are p53 gene knockout cells, and cells labeled with red fluorescent protein are normal cells (refer to Figure 1 ). Since intestinal inflammation is closely related to the occurrence and development of colorectal cancer, the risk of patients with ulcerative colitis and Crohn's disease developing colorectal cancer is significantly higher than that of the normal population. Therefore, the present invention aims to use azomethane (AOM) to induce intestinal inflammation based on the new MADM-P53 model, promote the clonal expansion of p53 gene knockout green cells, and thus obtain a new model of colorectal cancer of different pathological stages (early-mid-stage).

[0035] In tumor biology research, it is crucial to understand the functions of specific genes such as p53 in intestinal stem cells and their role in tumor development. The present invention develops an innovative colorectal cancer model by combining HPRT-Cre-induced MADM (Mosaic Analysis with DoubleMarkers) recombination technology and the use of chemical inducer azomethane (AOM), which can simulate the development process of cancer from early to mid-stage.

[0036] MADM technology is an advanced genetic tool that allows accurate control of gene expression at the single cell level, particularly in complex tissues with strict cell type specificity requirements. In the present application, by combining the HPRT promoter with the expression of the Cre recombinase, it is possible to specifically induce the knockout or overexpression of the p53 gene in intestinal stem cells. This is achieved by the Cre dependent recombination in the MADM system, which allows the target gene to be knocked out or activated at a specific stage during cell mitosis, thereby producing a mosaic cell clone containing a mark. Simultaneously, because MADM of the present invention is coupled to mouse chromosome 11, its mosaic allele needs to be located at chromosome 11, linked to MADM11, and p53 is located at mouse chromosome 11 (chr11:69,471,185-69,482,698) site, which satisfies the condition of genetic linkage with MADM11.

[0037] Generally speaking, MADM technology is widely used in the field of neuroscience (such as the brain), and its application in other tissues such as the intestine is relatively rare, and may face some specific challenges. For example, the expression level and activity of Cre enzyme directly affect the efficiency of MADM recombination. The expression level of Cre enzyme controlled by HPRT promoter in intestinal cells just meets the requirement of inducing MADM recombination while producing very diluted stem cell markers, which is the core basis for azoxymethane (AOM) to induce rapid expansion of p53 homozygous knockout cells.

[0038] By using azomethane (AOM), a chemical known to induce intestinal inflammation and promote the development of colorectal cancer, this model further simulates the pathological environment of colorectal cancer. The application of AOM not only accelerated the clonal expansion of intestinal stem cells with p53 mutations, but also replicated the environmental stress and cellular dynamics of early to mid-stage cancer development. This combined genetic and chemical induction approach allows us to observe how p53 dysfunction drives the progression of colorectal cancer in a real intestinal microenvironment.

[0039] The beneficial effects of the present invention are:

[0040] The present invention provides a method for constructing a spontaneous colorectal cancer mouse model; specifically, mice with genotypes of GT11ML; Hprt-Cre are mated with mice with TG11ML, P53 flox, and finally mice with genotypes of TG11ML, P53 flox, GT11ML, and Hprt-Cre can be stably obtained. The genotype mice constructed by the method of the present invention specifically produce colorectal tumors in the colorectal region after 8 weeks of modeling with azoxymethane (AOM). The genotype mice constructed by the construction method of the present invention can quickly produce colorectal cancer shapes, with an incidence rate of 100% and stable traits.

[0041] The innovation of the present invention lies in the use of MADM technology to achieve precise control of the activity of the p53 gene in intestinal stem cells, and the changes in cell behavior after the change of the p53 gene state were further observed through the AOM-induced inflammatory environment, and a spontaneous MADM-p53 mutation early intestinal cancer animal model was constructed; compared with the traditional chemical drug induced mouse model, it is closer to and more suitable for the development of early intestinal cancer related treatment methods, drugs and nutrients; compared with other models constructed by gene editing modeling or chemical drug modeling, the mouse colorectal cancer model provided by the present invention has the process from gene mutation, tumor occurrence to tumor development, and can more realistically simulate the entire process of tumor occurrence and development, can specifically produce tumors in the colorectum, and can monitor the metastasis or abnormal behavior of mutant cells, etc., which is of great significance for the development of nutrients and drugs in related fields.

[0042] Moreover, the modeling method of the present invention is innovative and the results are stable, filling the model gap in the field of early colorectal cancer in mice; the introduction of this method also provides a new perspective for studying the molecular mechanism of colorectal cancer, especially for exploring the role of tumor suppressor genes such as p53 in the development of early colorectal cancer. In addition, the development of this model provides an experimental platform for possible therapeutic targets in the future, especially in the development of intervention strategies for early to mid-stage colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the tumor occurrence and development pattern in the MADM-P53 model. Green represents gene mutation cells, red represents wild-type cells, and white represents cells that have not undergone chromosome exchange.

[0044] Figure 2 The figure is a comparison of tumor formation rates in mice with different genotypes.

[0045] Figure 3 The colorectal tumor morphology of mice bearing the MADM-P53 colorectal cancer model under a fluorescent stereomicroscope.

[0046] Figure 4 Pathological HE staining analysis of tumor tissues and colon tissues of MADM-P53 genotype mice induced by single injection of AOM at doses of 10 mg / kg and 15 mg / kg, respectively, and the saline control group.

[0047] Figure 5 Immunostaining analysis of tumor proliferation activity in MADM-P53 genotype mice induced by single injection of AOM at 10 mg / kg and 15 mg / kg, respectively. DETAILED DESCRIPTION

[0048] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0049] To simplify the nomenclature, when referring to genotypes in this article, bold fonts represent homozygotes, non-bold fonts represent heterozygotes, commas represent adjacent alleles are located on the same chromosome (genetic linkage), and semicolons represent adjacent alleles are located on different chromosomes. This unified nomenclature is used below.

[0050] Example 1

[0051] A method for constructing a mouse colorectal cancer model based on chimera double-labeling technology;

[0052] 1. Construction of Stock I mice (GT11ML; HPRT-Cre):

[0053] In this example, heterozygous GT11ML mice and heterozygous Hprt-Cre mice were mated to obtain Stock I mice (homozygous GT11ML; heterozygous Hprt-Cre). In which, G in GT11ML is the abbreviation of GFP, T is the abbreviation of tdTomato, 11 represents the knock-in of MADM allele on mouse chromosome 11 (p53 gene locus is located on mouse chromosome 11), and ML is multiple floxsite.

[0054] To produce mice of the desired genotype, two rounds of mating are required. The specific steps are as follows:

[0055] First round: heterozygous GT11ML mice (purchased from Jicui Yaokang Biotechnology Co., Ltd.) were mated with heterozygous HPRT-Cre mice (purchased from Jicui Yaokang Biotechnology Co., Ltd.) to generate double heterozygous mice GTML11;Hprt-Cre;

[0056] Second round: The offspring of the double heterozygous mice GT11ML; Hprt-Cre produced in the first round were self-crossed to obtain Stock I mice (GT11ML (homozygous); HPRT-Cre (heterozygous)).

[0057] 2. Construction of Stock II mice (TG11ML, P53 flox):

[0058] In this example, heterozygous TG11ML mice were mated with heterozygous P53 flox mice to obtain Stock II mice (TG11ML (homozygous), P53 flox (heterozygous)).

[0059] To produce mice of the desired genotype, two rounds of mating are required. The specific steps are as follows:

[0060] First round: Heterozygous TG11ML mice were mated with P53 flox mice (purchased from Jicui Yaokang Biotechnology Co., Ltd.) to produce double heterozygous mice TG11ML, P53 flox.

[0061] Second round: Double heterozygous mice obtained in the first round were mated with TG11ML mice (purchased from Jicui Yaokang Biotechnology Co., Ltd.) to obtain Stock II mice (TG11ML (homozygous), P53 flox (heterozygous)).

[0062] 3. The offspring produced by mating Stock I mice with Stock II mice were genotyped and the mice that could produce tumors (homozygous TG11ML, heterozygous P53 flox; homozygous GT11ML; heterozygous Hprt-Cre) were retained. For the sake of brevity, they will be referred to as MADM-P53 mice.

[0063] The offspring produced by them have the genotype of homozygous TG11ML and heterozygous P53 flox; the mice with homozygous GT11ML are called MADM mice and used as the control of MADM-P53 mice.

[0064] After one week of adaptive feeding, the obtained mice were randomly given AOM to induce colon cancer.

[0065] Example 2

[0066] A method for constructing a mouse colorectal cancer model based on chimera double labeling technology comprises the following steps:

[0067] AOM was used to induce MADM-P53 mice to obtain a colon cancer mouse model.

[0068] The specific steps are:

[0069] MADM-P53 mice (8-10 weeks old) were randomly divided into 2 groups, 10 mice in each group, and AOM was subcutaneously injected in the first and second weeks (10 mg / kg body weight in the first group and 15 mg / kg body weight in the second group). At the same time, 0.1 ml / 10 g body weight of normal saline was subcutaneously injected as a blank control group. MADM mice were used as the experimental control group to compare the tumor formation rate.

[0070] After the last drug injection, mice in each group continued to be raised normally for 8 weeks.

[0071] The number and location of the tumors were observed and recorded, and then the tumor incidence and average number of tumors were calculated according to the following formula:

[0072] Tumor incidence (%) = number of tumor-bearing mice / number of experimental mice × 100%;

[0073] The average number of tumors = number of tumors / number of experimental mice.

[0074] The colorectum of mice was observed using a fluorescent stereoscope, and then each tumor and its adjacent cancerous tissue and normal tissue were excised, fixed with 4% paraformaldehyde, embedded in paraffin, and tissue sections were prepared. After hematoxylin-eosin staining, the histological characteristics of the tumor tissue were observed under an optical microscope, and Ki-67 antibody was used for incubation for tumor proliferation detection.

[0075] The experimental results are:

[0076] Figure 2 Comparison of tumor formation rates between MADM-P53 genotype mice and control mice.

[0077] It can be found that in MADM mice without P53 mutation, the tumor induction rate was 50% after 10 mg / kg AOM induction twice (once a week), and the tumor induction rate was 60% after 15 mg / kg AOM induction twice (once a week). In MADM mice with homozygous P53 knockout (i.e., MADM-P53 mice), the tumor induction rate was 100% after 10 mg / kg and 15 mg / kg AOM induction twice (once a week), reflecting the advantages of the model.

[0078] Figure 3 The figure shows the morphology of colorectal tumors in MADM-P53 genotype mice observed under a stereomicroscope.

[0079] It can be found that in the model constructed based on MADM-P53 mice, because the P53 mutation is coupled with green fluorescent protein, whether a tumor has formed can be clearly observed under a fluorescent microscope.

[0080] The results showed that both 10mg / kg and 15mg / kg of AOM could induce the proliferation of GFP-positive P53 cells in MADM-P53 mice and form cell clones, thus generating strong green fluorescence (such as Figure 3 In addition, the number of tumors formed at 15 mg / kg was greater than that at 10 mg / kg. In MADM non-P53 mutant mice, the same dose of AMO was induced for 8 weeks. Because there was no GFP fluorescent protein to guide, it was impossible to determine whether tumors were formed under a fluorescent microscope, which reflects the model advantage of MADM-P53.

[0081] Figure 4 After 8 weeks of AOM exposure, pathological HE staining analysis of colon tissues of MADM-p53 genotype mice in tumor tissues induced by 10 mg / kg and 15 mg / kg AOM and in the saline control group was performed.

[0082] The results showed that in the colorectal mucosa of the saline control group, the cells were arranged neatly to form a regular glandular structure. The glandular ducts were straight and evenly distributed in the mucosal layer. The epithelial cells were columnar, with round or oval nuclei located at the base, pale nuclei, and inconspicuous nucleoli. The cytoplasm was abundant and evenly stained. The epithelial cells were arranged tightly and orderly without abnormal proliferation or cell atypia. The cells were closely connected, showing good tissue structural integrity. The submucosal structure was intact, containing blood vessels, smooth muscle cells and connective tissue, with no obvious signs of lesions.

[0083] The tumor tissue induced by 10mg / kg and 15mg / kg mainly showed abnormal morphological cells under the microscope, including significant variations in cell size and shape. Nuclear atypia: cell nuclei vary in size and shape, and obvious nucleoli may appear. Abnormal nuclear division images can be seen, and the ratio of the cell nucleus to the cytoplasm is increased. Tumor cells destroy the normal colorectal mucosal structure and are arranged in a disordered manner. Tumor cells can be seen penetrating the basement membrane and invading the surrounding tissues. In addition, in the tissue close to the core area of ​​the tumor tissue, the crypt volume is enlarged, the glandular cell membrane is thickened, the staining is deepened, the crypt volume is increased, and the colorectal abnormal hyperplasia is enlarged. The cell morphology is relatively normal, the basement membrane is clear, and no invasive growth occurs.

[0084] Figure 5 This is an immunostaining analysis of the proliferation activity of tumor tissue induced by two doses of AOM, 10 mg / kg and 15 mg / kg. Ki67 is a nuclear protein, and its high expression is usually associated with rapid proliferation and strong invasiveness of tumor cells. In the immunostaining results, Ki67-positive cells will show strong staining in the cell nucleus. Iba1 (Ionized calcium-binding adapter molecule 1) is a marker specific for macrophages and microglia. In colorectal cancer tissues, the presence and distribution of tumor-associated macrophages (TAMs) can be observed by Iba1 staining. These cells are usually concentrated in the tumor microenvironment, especially at the tumor invasion front and necrotic areas. Through the immunostaining of Ki67 and Iba1, important information about the proliferation activity of colorectal cancer cells and the immune status of the tumor microenvironment can be obtained, which is of great significance for understanding the tumor biological characteristics of this model.

[0085] In the tumor tissues induced by 10 mg / kg and 15 mg / kg, there were a large number of Ki67-positive tumor cells and extensive infiltration of Iba1-positive tumor-associated macrophages, indicating that the model constructed in this example can simulate the tumor biology and microenvironment characteristics of colorectal cancer.

[0086] The above specific implementations have been described in detail for the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for constructing a mouse colorectal cancer model, comprising constructing a P53 mutation-MADM mouse model and then inducing it using AOM; The method for constructing the P53 mutation-MADM mouse model comprises: At the p53 genomic locus on chromosome 11 of mice, genetic gene-edited mice with inducible recombination of p53 flox were constructed, and then MADM-Chr11 mice were mated with p53 flox mutant genetic mice to obtain P53 mutant-MADM mice.

2. The construction method according to claim 1, characterized in that: The method for constructing a P53 mutation-MADM mouse model specifically comprises the following steps: (1) Construction of Stock I mice: GTML11 was obtained by mating GT11ML mice with HPRT-Cre mice; Hprt-Cre double heterozygous mice: GTML11; Hprt-Cre double heterozygous mice were selfed to obtain Stock I mice with the genotype of GT11ML; HPRT-Cre; Stock II mice were constructed: TG11ML mice were mated with P53 flox mice to produce TG11ML, P53 flox double heterozygous mice, and TG11ML, P53 flox double heterozygous mice were mated with TG11ML mice to obtain Stock II mice with the genotype of TG11ML, P53 flox; (2) Stock I mice were mated with Stock II mice to obtain the genotype TG11ML, P53 flox; GT11ML; Hprt-Cre mice, a P53 mutant-MADM mouse model.

3. The construction method according to claim 2, characterized in that: The GT11ML and TG11ML are MADM genes; wherein G is green fluorescent protein, T is red fluorescent protein, 11 represents the MADM allele knocked into the animal chromosome 11, and ML is multiple flox site.

4. The construction method according to claim 3, characterized in that: The green fluorescent protein includes GFP; the red fluorescent protein includes tdTomato.

5. The construction method according to claim 1, characterized in that: The single injection dose of AOM is 5-20 mg / kg.

6. The construction method according to claim 1, characterized in that: The AOM is administered once or more times.

7. The construction method according to any one of claims 1 to 6, characterized in that: The AOM administration method includes intravenous injection, oral administration, intramuscular injection, subcutaneous injection, oral gavage or intraperitoneal injection.

8. Use of the construction method according to any one of claims 1 to 7 in studying the molecular mechanism of disease occurrence and development and / or screening drugs capable of preventing, alleviating or treating rectal cancer.

9. Use of the animal model constructed by the construction method according to any one of claims 1 to 7 in studying the molecular mechanism of disease occurrence and development and / or screening drugs capable of preventing, alleviating or treating rectal cancer.

10. A method for screening drugs that can prevent, alleviate or treat colorectal cancer, comprising administering the drug to be tested to a colorectal cancer model mouse constructed by the construction method described in any one of claims 1-7, detecting the tumor status of the mouse before and after administration; and selecting drugs that can prevent, alleviate or treat colorectal cancer.

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