Construction method of gastric adenocarcinoma transgenic mouse model and application thereof

Abstract

The application discloses a kind of gastric adenocarcinoma transgenic mouse model construction method and its application, the construction method includes the following steps: using transgenic and hybridization technology, MYC gene expression quantity or MYC protein activity in mouse gastric mucosa layer epithelial cell is overexpressed or increased, KRAS gene mutant type in gastric mucosa layer epithelial cell is activated simultaneously, make Kras protein function abnormal, continuously in activated state, finally obtained mouse is gastric adenocarcinoma transgenic mouse model.The application constructs Myc gene overexpression and KRAS activation mutation double gene mutation mouse spontaneous gastric cancer animal model, method is simpler.This mouse model of gastric cancer tumorigenesis time is shortened, tumorigenic rate is as high as 100%, and tumorigenic effect is stable.And after induction, only in stomach tumorigenesis, tumorigenesis has high specificity.Pathological section and immunohistochemical staining determine that this animal model is adenocarcinoma in gastric cancer, has good clinical representativeness, can be applied to the application in later development or screening gastric cancer drug.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gastric cancer models, and particularly relates to a construction method of a gastric adenocarcinoma transgenic mouse model and application thereof. BACKGROUND

[0002] Gastric cancer is a global important disease and the fifth most diagnosed malignancy worldwide. Due to the fact that gastric cancer is often diagnosed at an advanced stage, the mortality rate is high, and gastric cancer is the third most common cause of cancer-related death. In Asia, the mortality rate of gastric cancer is only second to lung cancer. The morphology of early gastric cancer and advanced gastric cancer is diverse, and the histological classification is still increasing. The most commonly used classification is Laurén classification, which can be divided into intestinal type, diffuse type and mixed type. According to the histopathological classification, gastric cancer is generally divided into squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma and carcinoid, etc. Gastric adenocarcinoma is relatively common in clinical practice. Experimental animal models can truly simulate the occurrence, development and metastasis of human gastric cancer tissues and cells in vivo, and are an important tool for studying gastric cancer. In recent years, the basic research of the digestive system tumors has made breakthroughs, but the basic research of gastric cancer has been relatively slow due to the lack of a representative and good animal model.

[0003] Compared with other digestive system tumor models, the construction of a gastric cancer animal model has its particularity. Although a few gastric cancer animal models have been reported abroad, they all use a three-gene or four-gene mutation construction method, and generally have problems such as complex construction process, long tumorigenesis time and low tumorigenesis rate. SUMMARY

[0004] The application provides a construction method of a gastric adenocarcinoma transgenic mouse model and application thereof, and aims at the problems in the prior art. The application uses a Cre / loxP system to specifically overexpress or increase the expression amount of a MYC gene in the epithelial cells of the gastric mucosa layer of a mouse, and simultaneously activates a mutant Kras gene, so that the mutant Kras protein is abnormally functional and continuously in an activated state. After a CreERT2 recombinase is expressed at the Anxa10 gene locus of the mouse and is induced by Tamoxifen, the Anxa10 gene is expressed in the epithelial cells of the gastric mucosa layer of an adult, and the epithelial cells of the gastric mucosa layer specifically express the CreERT2 recombinase. The mouse model forms a tumor in the stomach. The gastric cancer tumorigenesis time of the mouse model is greatly shortened, the tumorigenesis rate is as high as 100%, and only gastric adenocarcinoma is formed in the stomach, so the tumorigenesis has high specificity.

[0005] TECHNICAL SCHEME

[0006] The application provides a construction method of a gastric adenocarcinoma transgenic mouse model.

[0007] The MYC gene expression or MYC protein activity in the gastric mucosa epithelial cells of the mouse is overexpressed or increased by using transgenic and hybridization techniques, and the KRAS gene mutation in the gastric mucosa epithelial cells is activated, so that the Kras protein is abnormally functional and continuously activated, and finally the mouse is a gastric adenocarcinoma transgenic mouse model.

[0008] As a specific embodiment, the construction method comprises:

[0009] A Cre recombinase expression gene is implanted on a mouse gene site to establish a mouse capable of inducing Cre recombinase expression; a MYC overexpression gene mouse is constructed by inserting a Myc overexpression gene; a Kras gene G12D conditional point mutation mouse is constructed; and then the three transgenic mice are hybridized to construct a mouse model with stable genotypes of CreERT2 heterozygosity, MYC heterozygosity and KRAS heterozygosity, which is the gastric adenocarcinoma transgenic mouse model.

[0010] Further, the Cre recombinase expression gene is implanted on the Anxa10 gene site of the mouse.

[0011] Further, the MYC overexpression gene mouse is constructed by inserting a Myc overexpression gene at a LoxP site.

[0012] Further, the Kras gene G12D conditional point mutation mouse is constructed by replacing the exon2 of Kras with loxp-stop-loxp and exon2 containing G12D.

[0013] As a more specific embodiment, the construction method of the gastric cancer transgenic mouse model comprises the following steps:

[0014] (1) inserting 2A-CreERT2-Wpre-pA into the stop codon of the Anxa10 gene of the mouse to obtain Anxa10-CreERT2 recombinase transgenic mouse Anxa10-CreERT2 + / + mouse; inserting CAG promoter-loxp-STOP-loxp-Myc-polyA conditional overexpression structure into H11 site to establish H11-LSL-Myc mouse model, which is MYC overexpression gene mouse MYC LSL / LSL mouse; replacing the exon2 of Kras with loxp-stop-loxp and exon2 containing G12D to establish Kras gene G12D conditional point mutation mouse strain KRAS loxP / + mouse.

[0015] (2) MYC LSL / LSL mice were mated with Anxa10-CreERT2 recombinase transgenic mice to obtain F1 generation mice, and heterozygous mice in the F1 generation mice were selected and named as Anxa10-CreERT2 + / - ; MYC LSL / + mice.

[0016] (3) KRAS loxP / + mice were mated with Anxa10-CreERT2 recombinase transgenic mice to obtain F1 generation mice, and heterozygous mice in the F1 generation mice were selected and named as Anxa10-CreERT2 + / - ; Kras loxP / + mice.

[0017] (4) Anxa10-CreERT2 + / - ; MYC LSL / + mice in the above F1 generation were mated with Anxa10-CreERT2 + / - ; Kras loxP / + mice to obtain F2 generation mice, and Anxa-CreERT2 heterozygous, MYC heterozygous, KRAS heterozygous mice in the F2 generation mice were selected and named as Anxa10-CreERT2 + / - ; MYC LSL / + ; Kras loxP / + mice.

[0018] (5) Anxa10-CreERT2 + / - ; MYC LSL / + ; Kras loxP / + mice with the same genotype in the F2 generation were mated to obtain F3 generation mice, and Anxa10-CreERT2 + / - ; MYC LSL / LSL ; Kras loxP / + mice in the F3 generation mice were selected, that is, the gastric adenocarcinoma transgenic mouse model.

[0019] As an alternative, Anxa10-CreERT2 + / + ; MYC LSL / LSL ; Kras loxP / + triple homozygous mice or Anxa10-CreERT2 + / + ; MYC LSL / + ; Kras loxP / + mice or Anxa10-CreERT2 + / - ; MYC LSL / + ; KrasloxP / + The mouse is a three-gene hybrid mouse, and as long as the MYC gene expression or MYC protein activity is overexpressed or increased based on Anxa10-CreERT2 expression, and the KRAS gene mutation in the gastric mucosa epithelial cells is activated, the Kras protein function is abnormal, and the mouse is continuously in an activated state, the mouse can be used as the gastric adenocarcinoma transgenic mouse model.

[0020] Anxa10-CreERT2 + / + The mouse is an Anxa10-CreERT2 recombinase transgenic homozygous mouse, Anxa10-CreERT2 + / - The mouse is an Anxa10-CreERT2 recombinase transgenic hybrid mouse; MYC LSL / LSL The mouse is a MYC overexpression homozygous mouse, MYC LSL / + The mouse is a MYC overexpression hybrid mouse; KRAS loxP / + The mouse is a Kras gene G12D conditional point mutation hybrid mouse.

[0021] The application also provides the above-mentioned MYC LSL / LSL The mouse, the Anxa10-CreERT2 recombinase transgenic mouse, and the KRAS loxP / + The application also provides application of the mouse in construction of a gastric adenocarcinoma transgenic mouse model.

[0022] The application also provides application of the transgenic mouse model constructed by the above-mentioned method in screening of drugs for preventing and / or treating gastric adenocarcinoma.

[0023] The application also provides application of the transgenic mouse model constructed by the above-mentioned method in screening of gastric cancer cell lines.

[0024] The c-Myc gene (also called Myc) is abnormally expressed in many tumors, and plays an extremely important regulatory role in cell proliferation, growth metabolism, gene instability, stimulation of malignant transformation, differentiation and apoptosis. The MYC proto-oncogene encodes a family of transcription factors, and is one of the most common activated cancer proteins in human tumors. In fact, in most cancers, MYC is aberrant or MYC-related pathways are up-regulated through alternative mechanisms. MYC protein is a major regulator of cell programming. Therefore, MYC-activated cancer triggers many cancer characteristics required for autonomous tumor growth. KRAS mutation can cause uncontrolled activation of RAS protein. RAS protein is located in the cell membrane and regulates cell growth, proliferation, movement, migration and angiogenesis. Based on this, the present application constructs a double gene mutation mouse spontaneous gastric cancer animal model based on Myc gene overexpression and KRAS activating mutation. The animal model is a transgenic animal model constructed by the Cre-LoxP system, and the Myc overexpression gene is inserted through the LoxP site. The double gene mutation method based on Myc gene overexpression and KRAS activating mutation is used for construction. Compared with other construction methods reported in the literature, the constructed model is simpler. In addition, the gastric cancer tumor formation time of the mouse model is only 2 months, the tumor formation rate is as high as 100%, and the tumor formation effect is stable. And only in the stomach, the tumor formation has high specificity. After pathological sectioning and immunohistochemical staining, it is determined that the animal model is the gastric cancer of the most common adenocarcinoma in gastric cancer, and has good clinical representativeness.

[0025] Beneficial effects: Compared with other existing experimental animal models, the present application constructs a double gene mutation mouse spontaneous gastric cancer animal model based on Myc gene overexpression and KRAS activating mutation based on the Cre-LoxP system. The constructed model is simpler. The gastric cancer tumor formation time of the mouse model is shortened, the tumor formation rate is as high as 100%, and the tumor formation effect is stable. And only in the stomach, the tumor formation has high specificity. After pathological sectioning and immunohistochemical staining, it is determined that the animal model is the most common gastric adenocarcinoma in gastric cancer, and has good clinical representativeness, and can be applied to the development or screening of gastric cancer drugs. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is an electrophoresis diagram of a product amplified by using Anxa10-CreERT2 primers, wherein: lanes 1, 2 and 3 represent three different samples.

[0027] Figure 2 It is an electrophoresis diagram of a product amplified by using MYC primers, wherein: lanes 1, 2 and 3 represent three different samples.

[0028] Figure 3 It is an electrophoresis diagram of a product amplified by using KRAS primers, wherein: lanes 1, 2 and 3 represent three different samples.

[0029] Figure 4 Verification results of gastric adenocarcinoma transgenic mouse model, wherein: A: gastric tumor of gastric adenocarcinoma mouse model; B: survival curve of gastric adenocarcinoma mouse; C: gastric adenocarcinoma mouse KI67, P-ERK, MYC group; D: gastric adenocarcinoma mouse CDX2, MUC2 group. DETAILED DESCRIPTION

[0030] The application will be further described below in conjunction with specific examples, but the application is not limited to the following examples. Unless otherwise specified, the experimental methods used in the examples are conventional methods, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0031] Example 1 Construction method of gastric adenocarcinoma transgenic mouse model

[0032] I. Gene source and construction principle of each transgenic mouse

[0033] 1. Obtaining of Anxa10-CreERT2 mouse

[0034] Insert 2A-CreERT2-Wpre-pA into the stop codon of mouse Anxa10 gene to obtain Anxa10-CreERT2 recombinase transgenic mouse Anxa10-CreERT2 + / + Mouse. The mouse was purchased from Nanmodel (Shanghai Nanmodel Biotechnology Co., Ltd.).

[0035] Anxa10-2A-CreERT2 gene:

[0036] NCBI ID: 26359

[0037] MGI ID: 1347090

[0038] Ensembl ID: ENSMSUSG00000031635

[0039] Pubmed: Anxa10

[0040] Anxa10 gene:

[0041] Anxa10 annexin A10 [Mus musculus (house mouse)]

[0042] NCBI Gene ID: 26359, updated on 11-Sep-2023

[0043] Examples of gene marker cell types: gastric mucosal epithelial cells, hair follicle cells of the skin

[0044] This mouse expresses CreERT2 recombinase at the Anxa10 locus. When bred to reporter mice, upon Tamoxifen induction, the reporter is expressed in the epithelial cells of the gastric mucosal layer in adults; when bred to mice containing loxP flanked sequences, Tamoxifen-induced recombination will result in the deletion of the floxed sequences in the epithelial cells of the gastric mucosal layer. Can be used for gastric cancer research.

[0045] 2. Generation of MYC overexpression mice

[0046] A CAG promoter-loxp-STOP-loxp-Myc-polyA conditional overexpression construct was inserted into the H11 site to generate the H11-LSL-Myc mouse model, which is a MYC overexpression mouse. LSL / LSL Homozygous mice. The mice were purchased from Nanjing Biomedical (Shanghai Nanjing Biomedical Technology Co., Ltd.).

[0047] MYC gene:

[0048] MGI ID: 5461148

[0049] https: / / www.modelorg.com / index.php / portal / article / index / id / 858 / post_type / 3.html

[0050] The c-Myc gene (also called Myc) is abnormally expressed in many tumors, affecting cell proliferation, growth metabolism, gene instability, stimulating angiogenesis, malignant transformation of cells, differentiation and apoptosis. MYC proto-oncogene encodes a family of transcription factors, which is one of the most common activated cancer proteins in human tumors. In fact, in most cancers, MYC is abnormal or MYC-related pathways are up-regulated through alternative mechanisms. MYC protein is a major regulator of cell programming. The c-Myc gene (also called Myc) is abnormally expressed in many tumors, affecting cell proliferation, growth metabolism, gene instability, stimulating angiogenesis, malignant transformation of cells, differentiation and apoptosis. A CAG promoter-loxp-STOP-loxp-Myc-polyA conditional overexpression construct was inserted into the H11 site to generate the H11-LSL-Myc mouse model. The H11 site is located on mouse chromosome 11, which has been shown to be similar to Rosa26 and can be used to express foreign genes more widely. H11-LSL-Myc can highly express Myc gene in tissues expressing Cre after mating with Cre tool mice. It can be used for the establishment of tumor models and tumor research.

[0051] 3.Kras gene G12D conditional point mutation mouse strain KRAS loxP / + Acquisition of mice

[0052] The loxp-stop-loxp and G12D-containing exon2 were used to replace Kras exon2 to establish the Kras gene G12D conditional point mutation mouse strain KRAS loxP / + Mice. The mice were purchased from Jackson lab.

[0053] Kras gene:

[0054] Kras Kirsten rat sarcoma viral oncogene homolog[Mus musculus(housemouse)];

[0055] NCBI Gene ID:16653, updated on 11-Oct-2023

[0056] KRAS mutations can lead to uncontrolled activation of RAS protein. RAS protein is located in the cell membrane and regulates cell growth, proliferation, movement, migration and angiogenesis. KRAS mutations will significantly increase the invasiveness of gastric cancer. KRAS expression is high in the Lauren classification of intestinal type. The most common model is to mate K19-CreERT mice with Loxp-STOP-Loxp-K-Ras (G12D) mice to obtain mice that conditionally express K-Ras (G12D). These mice also develop multiple dysplasia, metaplasia and adenocarcinoma in the stomach. The activation / inactivation effect of wild-type Kras is controlled, while the mutant Kras protein is dysfunctional and remains in an activated state, leading to the continuous proliferation of tumor cells. By replacing Kras exon2 with loxp-stop-loxp and exon2 containing G12D, a mouse strain with G12D conditional point mutation of the Kras gene was established. After mating this mouse with Cre mice, G12D point mutation mice can be obtained.

[0057] 2. Specific process of constructing a transgenic mouse model of gastric adenocarcinoma

[0058] (1) MYC overexpression gene mice LSL / LSL Anxa10-CreERT2 transgenic mice expressing CreERT2 recombinase specifically in gastric epithelial cells + / + Mice were mated to obtain F1 generation mice; heterozygous mice among the F1 generation mice were selected and named Anxa10-CreERT2 + / - ;MYC LSL / +Mice.

[0059] (2) KRAS conditional point mutation-activated mice - KRAS loxP / + The mice were mated with AnxalO-CreERT2 recombinase transgenic mice which specifically express CreERT2 recombinase in gastric epithelial cells to obtain F1 generation mice; heterozygous mice in the F1 generation mice were selected and named AnxalO-CreERT2 + / - ; Kras loxP / + Mice.

[0060] (3) The F1 generation AnxalO-CreERT2 + / - ; MYC LSL / + Mice and the F1 generation AnxalO-CreERT2 + / - ; KRAS loxP / + Mice were mated to obtain F2 generation mice; AnxalO-CreERT2 + / - ; MYC LSL / + ; Kras loxP / + Mice with genotype of AnxalO-CreERT2

[0061] (4) The F2 generation mice with genotype of AnxalO-CreERT2 + / - ; MYC LSL / + ; Kras loxP / + Mice were mated to obtain F3 generation mice, and the F3 generation mice with genotype of AnxalO-CreERT2

[0062] + / - ; MYC LSL / LSL ; Kras loxP / + Mice were selected as gastric epithelial cell-specific MYC overexpression and Kras activating mutation mice, i.e. the gastric adenocarcinoma transgenic mouse model;

[0063] AnxalO-CreERT2 + / + Mice are AnxalO-CreERT2 recombinase transgenic homozygous mice, AnxalO-CreERT2 + / - Mice are AnxalO-CreERT2 recombinase transgenic heterozygous mice; MYC LSL / LSL Mice are MYC overexpression homozygous mice, MYC LSL / + Mice are MYC overexpression heterozygous mice; KRAS loxP / + Mice are Kras gene G12D conditional point mutation heterozygous mice.

[0064] The constructed Anxa10-CreERT2 + / - ; MYC LSL / LSL ; Kras loxP / + Mice, for adult mice, tamoxifen intraperitoneal injection was performed at 6 weeks of age. Tamoxifen was dissolved in corn oil at a concentration of 20 mg / ml and shaken overnight at 37 degrees Celsius. It was stored at 4 degrees Celsius during injection. The injection dose was determined by weight, approximately 200 mg / kg body weight. Tamoxifen was administered by intraperitoneal injection (using ACUC approved injection procedures), once every 24 hours, for 3-5 consecutive days. For safety, the injection site was disinfected with 70% ethanol before injection. After the last injection, the mice should be isolated for 24 hours before returning to their normal animal room.

[0065] Three, mouse genotype identification method

[0066] Genotype identification of gastric mucosa epithelial cell specific Anxa10-CreERT2; MYC overexpression and Kras activating point mutation mice, PCR identification of heterozygous mice is as follows:

[0067] Take the offspring mouse tail 2-3 mm, extract the tail genomic DNA in the standard procedure. Using tail genomic DNA as a template, use gene-specific primers to perform polymerase chain reaction (PCR) to identify mouse genotypes. Wild-type mouse C57 mouse tail genomic DNA was used as a control.

[0068] 1) The Wide type primer sequence for identifying Anxa10-CreERT2 recombinase gene is:

[0069] 5'-ATCTCCCATCCTCCCTAACTT-3' and 5'-ATCGCATCACCTTCAAACTCT-3'.

[0070] The Mutant type primer sequence is:

[0071] 5'-CAGGGCACTATGAGAAAGCACTAC-3' and

[0072] 5'-AGCCCGGACCGACGATGAAGC-3'.

[0073] 2) The Wide type primer sequence for identifying MYC gene is:

[0074] 5'-ATAAGCCATTCTCCATTTCATAA-3' and 5'-CCCCTTGTTCCCTTTCTGC-3'.

[0075] Mutant type primer sequence is:

[0076] 5'-TTTGCCTTTGTTACCTGTTCCATC-3' and 5'-CTCCCCCGTGCCTTCCTTGAC-3'.

[0077] 3) Wide type primer sequence for identifying KRAS point mutation gene is:

[0078] 5'-CTGCATAGTACGCTATACCCTGT-3' and 5'-TGTCTTTCCCCAGCACAGT-3'.

[0079] Mutant type primer sequence is

[0080] 5'-CTGCATAGTACGCTATACCCTGT-3' and 5'-GCAGGTCGAGGGACCTAATA-3'.

[0081] Genotype identification system:

[0082] F (upstream primer): 0.25ul, R (downstream primer): 0.25ul, 2x Taq (buffer): 7.5ul, H2O: 5.5ul, mouse tail DNA: 1.5ul, Total: 15ul.

[0083] PCR instrument loading program:

[0084] 1) ANXA identification:

[0085] Volume: 15ul, Step1 94℃ 00:05:00 (5min), Step2 94℃ 00:00:30 (30s), Step3 60℃ 00:00:30 (30s), Step4 72℃ 00:01:00 (1min), Step 5 Goto 2 x 35, Step6 72℃ 00:05:00 (5min), Step7 12℃ Forever.

[0086] 2) Kras identification:

[0087] Volume: 15ul, Step1 94°C 00:03:00 (3 min), Step2 94°C 00:00:15 (15 s), Step3 58°C 00:00:15 (15 s), Step4 72°C 00:00:40 (40 s), Step5 Goto2 x30, Step6 72°C 00:05:00 (5 min), Step7 10°C Forever.

[0088] 3) MYC identification:

[0089] Volume: 15ul, Step1 94°C 00:05:00 (5 min), Step2 94°C 00:00:30 (30 s), Step3 65°C 00:00:30 (30 s), Step4 72°C 00:01:00 (1 min), Step5 Goto2 x34, Step6 72°C 00:05:00 (5 min), Step7 12°C Forever

[0090] The genotype identification results of mice are shown in Figures 1-3 , three lanes in the figure represent three samples, Figure 1 Anxa10-CreERT2 primer amplification product electrophoresis, if a specific 436bp fragment can be amplified, it is Anxa10-CreERT2 positive; a specific 823bp fragment can be amplified, it is Anxa10-CreERT2 negative.

[0091] Figure 2 MYC primer amplification product electrophoresis, if a specific 434bp fragment can be amplified, it is MYCLSL / LSL homozygote, if a specific 428bp fragment can be amplified, it is MYC negative, if both positive and negative bands are amplified, it is heterozygous MYCLSL / +.

[0092] Figure 3 KRAS primer amplification product electrophoresis, if a specific 100bp fragment can be amplified, and a specific 200bp fragment can be amplified, it is KRAS loxP / + heterozygote, if a specific 200bp fragment can be amplified, it is KRAS negative, wild type.

[0093] Figures 1-3The genotype of sample 1 is Anxa10-CreERT2+ / -; MYC LSL / +, the genotype of sample 2 is Anxa10-CreERT2+ / -; MYC LSL / LSL; Kras loxP / +, and the genotype of sample 3 is Anxa10-CreERT2+ / +; MYC LSL / +. Therefore, sample 2 is the desired mouse model of gastric epithelial cell-specific MYC overexpression and Kras activating point mutation.

[0094] Example 2 Verification of the gastric adenocarcinoma transgenic mouse model

[0095] (1) The constructed Anxa10-CreERT2 + / - ; MYC LSL / LSL ; Kras loxP / + model mice, and wild-type mice were fed. After the mice grew up, tamoxifen was intraperitoneally injected into the two groups of mice at the age of 6 weeks. The tamoxifen was dissolved in corn oil at a concentration of 20 mg / ml and shaken at 37 degrees Celsius overnight. It was stored at 4 degrees Celsius during the injection period. The injection dose was determined by weight, and the calculation was about 200 mg / kg body weight. Tamoxifen was administered by intraperitoneal injection (using ACUC-approved injection procedures) once every 24 hours for 3-5 consecutive days. For safety, the injection site was disinfected with 70% ethanol before injection. After the last injection, the two groups of mice should be isolated for 24 hours before they can return to their normal animal room and be fed with ordinary mouse growth feed.

[0096] (2) The constructed model mice, after 2-5 days of tamoxifen intraperitoneal injection at the age of 6 weeks, the mice developed tumors in the stomach 5-7 weeks later, while the wild-type mice did not develop tumors. The experimental animal dissection was performed on the MYC overexpression and Kras activating mutation mice. The experimental results showed that the mice developed tumors in the stomach, as shown in Figure 4 . Among them, Figure A is the gastric tumor of the gastric adenocarcinoma mouse model, and the results show that the genotype mice were dissected after 5-7 weeks, and the stomach showed obvious tumor formation. Figure B is the survival curve of the gastric adenocarcinoma mouse, and the results show that the survival rate of the gastric adenocarcinoma mouse decreases significantly with time. Figure C is the KI67, P-ERK, MYC group of gastric adenocarcinoma mouse, and the results prove that the MYC overexpression and Kras activating mutation of the genotype mice. KI67 staining can see the gastric tumor, which proves that the mouse model is caused by MYC overexpression and Kras activating mutation. Figure D is the CDX2, MUC2 group of gastric adenocarcinoma mouse, and from the figure it can be seen that the stomach has both intestinal and diffuse type tumors. The above results prove that the model mouse belongs to the gastric adenocarcinoma mouse.

[0097] The present application specifically includes the use of the Cre / loxP system to specifically overexpress or increase the expression of the MYC gene in mouse gastric epithelial cells, while activating the Kras gene, the mutant Kras protein is dysfunctional and remains in an activated state, leading to continuous proliferation of tumor cells. After induction by Tamoxifen, the Anxa10 gene is expressed in the epithelial cells of the adult gastric mucosa layer, and the gastric mucosa layer epithelial cells specifically express the CreERT2 recombinase. Cre-ERT2 mice are a type of mouse that expresses a fusion protein containing a mutant ligand binding region of the estrogen receptor (ERT) and Cre recombinase. Cre-ERT2 is in an inactive state in the cytoplasm without Tamoxifen induction; when Tamoxifen is induced, the metabolite 4-OHT (estrogen analog) of Tamoxifen binds to ERT, allowing Cre-ERT2 to enter the nucleus and exert Cre recombinase activity. Using different promoters, Cre expression can be specifically regulated in different tissues or cells. After induction by Tamoxifen, the recombinase induced by Tamoxifen will cause the deletion of the floxed sequence in the gastric mucosa layer epithelial cells.

[0098] The animal model of the present application is a transgenic animal model constructed using the Cre-LoxP system. First, the present application uses the uniquely expressed gene Anxa10 in the gastric epithelium, and implants the tamoxifen-induced Cre recombinase (CreERT2) at the Anxa10 gene site of the mouse to establish an inducible Anxa10-creert2 mouse. By inserting the Myc overexpression gene at the LoxP site, a double-gene mutant mouse model is constructed using a method based on Myc gene overexpression and KRAS activating mutation. Finally, an inducible gastric adenocarcinoma transgenic mouse is established. The transgenic mouse develops adenocarcinoma within two months and eventually dies after 6 weeks of age. The inducible gastric adenocarcinoma mouse model constructed using the method described in the present application can be used for the development or screening of gastric cancer drugs.

[0099] The above examples are the best embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included within the scope of the present application.

Claims

1. A method for constructing a gastric adenocarcinoma transgenic mouse model, characterized in that: The following steps are involved: A Cre recombinase expression gene is implanted at the mouse gene locus to generate mice with inducible Cre recombinase expression; a MYC overexpression gene mouse is constructed by inserting a Myc overexpression gene; a Kras gene G12D conditional point mutation mouse is constructed; and then, using the above three transgenic mice, a mouse model with a stable genotype of CreERT2 heterozygous, MYC heterozygous, and KRAS heterozygous is constructed through hybridization technology, which is the gastric adenocarcinoma transgenic mouse model.

2. The method for constructing a gastric adenocarcinoma transgenic mouse model according to claim 1, characterized in that: The said implanting of the Cre recombinase expression gene into the mouse gene site is implanting the Cre recombinase expression gene into the mouse Anxa10 gene site.

3. The method for constructing a gastric adenocarcinoma transgenic mouse model according to claim 1, characterized in that: The method of constructing a MYC overexpression gene mouse by inserting the Myc overexpression gene is to construct a MYC overexpression gene mouse by inserting the Myc overexpression gene at the LoxP site.

4. The method for constructing a gastric adenocarcinoma transgenic mouse model according to claim 1, characterized in that: The Kras gene G12D conditional point mutation mouse is constructed by replacing Kras exon2 with loxp-stop-loxp and exon2 containing G12D to construct the Kras gene G12D conditional point mutation mouse.

5. The method for constructing a gastric adenocarcinoma transgenic mouse model according to claim 1, characterized in that: The following steps are involved: (1) Insert the CAG promoter-loxp-STOP-loxp-Myc-polyA conditional overexpression structure into the H11 site to establish the H11-LSL-Myc mouse model, which is a MYC overexpression gene mouse model. LSL / LSL mice; 2A-CreERT2-Wpre-pA was inserted into the stop codon of the mouse Anxa10 gene to generate Anxa10-CreERT2 recombinase transgenic mice; loxp-stop-loxp and exon2 containing G12D replaced Kras exon2 to establish the Kras gene G12D conditional point mutation mouse strain KRAS loxP / + mice; (2) MYC LSL / LSL Mice were mated with Anxa10-CreERT2 recombinase transgenic mice to obtain F1 generation mice, and heterozygous mice among the F1 generation mice were selected and named Anxa10-CreERT2 + / - ;MYC LSL / + mice; (3) KRAS loxP / + Mice were mated with Anxa10-CreERT2 recombinase transgenic mice to obtain F1 generation mice, and heterozygous mice among the F1 generation mice were selected and named Anxa10-CreERT2 + / - Kras loxP / + mice; (4) Transfect the Anxa10-CreERT2 in the F1 generation + / - ;MYC LSL / + Mice with Anxa10-CreERT2 + / - Kras loxP / + Mice were mated to obtain F2 mice, and the F2 mice with the genotype of Anxa-CreERT2 heterozygous, MYC heterozygous, and KRAS heterozygous were selected and named Anxa10-CreERT2 + / - ;MYC LSL / + ;Kras loxP / + mice; (5) The Anxa10-CreERT2 with the same genotype in the F2 generation + / - ;MYC LSL / + ;Kras loxP / + Mice were mated to obtain F3 generation mice, and the genotype of the F3 generation mice was selected to be Anxa10-CreERT2 + / - ;MYC LSL / LSL ;Kras loxP / + The mouse is the gastric adenocarcinoma transgenic mouse model.

6. MYC according to claim 5 LSL / LSL Mice, Anxa10-CreERT2 recombinase transgenic mice, KRAS loxP / + Application of mice in the construction of transgenic mouse models of gastric adenocarcinoma.

7. Use of the transgenic mouse model constructed by the method according to any one of claims 1 to 5 in screening drugs for preventing and / or treating gastric adenocarcinoma.

8. Use of the transgenic mouse model constructed by the method according to any one of claims 1 to 5 in screening gastric cancer cell lines.

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