Construction method and application of gastric adenocarcinoma transgenic mouse model

Abstract

The invention discloses a construction method and application of a gastric adenocarcinoma transgenic mouse model, and the construction method comprises the following steps: overexpressing or increasing the MYC gene expression quantity or MYC protein activity in gastric mucosa epithelial cells of a mouse by using transgenosis and hybridization technologies, and activating KRAS gene mutation in the gastric mucosa epithelial cells at the same time, thereby obtaining the gastric adenocarcinoma transgenic mouse model. The Kras protein is abnormal in function and is continuously in an activated state, and the finally obtained mouse is the gastric adenocarcinoma transgenic mouse model. According to the invention, the Myc gene overexpression and KRAS activation mutation double-gene mutation mouse spontaneous gastric cancer animal model is constructed, and the method is simpler. The gastric cancer tumor formation time of the mouse model is shortened, the tumor formation rate is up to 100%, and the tumor formation effect is stable. Only tumor formation is performed in the stomach through induction, and tumor formation has high specificity. The animal model is determined to be adenocarcinoma in gastric cancer through pathological section and immunohistochemical staining, has good clinical representativeness, and can be applied to later development or screening of gastric cancer drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of gastric cancer models, and particularly relates to a method for constructing a gastric adenocarcinoma transgenic mouse model and application thereof. Background Art

[0002] Gastric cancer is an important global disease and the fifth most diagnosed malignant tumor in the world. Since gastric cancer is often diagnosed in the late stage, the mortality rate is very high, making gastric cancer the third most common cause of cancer-related death. In Asia, the mortality rate of gastric cancer is second only to lung cancer. Early gastric cancer and advanced gastric cancer have diverse morphologies, and the histological classification is still increasing. The most commonly used classification is the 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. Gastric adenocarcinoma is more common in clinical practice. Experimental animal models can truly simulate a series of biological characteristics such as the occurrence, development, and metastasis of human gastric cancer tissue cells in the body, and are an important tool for studying gastric cancer. In recent years, basic research on digestive system tumors has made continuous breakthroughs, but gastric cancer is constrained by the lack of a well-representative animal model, and basic research progress has been relatively slow.

[0003] Compared with other digestive system tumor models, the construction of gastric cancer animal models has its own particularities. Although there are a few gastric cancer animal models reported abroad, they all use the construction method of three-gene or four-gene mutations, and generally have problems such as complex construction process, long tumor formation time, and low tumor formation rate. Summary of the invention

[0004] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a method for constructing a transgenic mouse model of gastric adenocarcinoma and its application. The present invention adopts the Cre / loxP system to specifically overexpress or increase the expression of the MYC gene in the epithelial cells of the gastric mucosal layer of mice, and activates the mutant Kras gene at the same time, so that the mutant Kras protein functions abnormally and is continuously in an activated state. The CreERT2 recombinase is expressed at the mouse Anxa10 locus. After induction by Tamoxifen, the Anxa10 gene is expressed in the epithelial cells of the gastric mucosal layer of adults, and the gastric mucosal epithelial cells specifically express the CreERT2 recombinase. The mouse model forms tumors in the stomach. The time for gastric cancer to form tumors in this mouse model is greatly shortened, the tumor formation rate is as high as 100%, and gastric adenocarcinoma is formed only in the stomach, and the tumor formation is highly specific.

[0005] Technical solution: To achieve the above purpose, the present invention adopts the following technical solution:

[0006] A method for constructing a gastric adenocarcinoma transgenic mouse model comprises the following steps:

[0007] By using transgenic and hybridization techniques, the MYC gene expression or MYC protein activity in the gastric mucosal epithelial cells of mice is overexpressed or increased, and the KRAS gene mutant in the gastric mucosal epithelial cells is activated, causing the Kras protein to function abnormally and remain in an activated state. The resulting mice are transgenic mouse models of gastric adenocarcinoma.

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

[0009] The Cre recombinase expression gene is implanted at the mouse gene site to establish mice that can inducibly express Cre recombinase; the MYC overexpression gene mouse is constructed by inserting the Myc overexpression gene; the Kras gene G12D conditional point mutation mouse is constructed; and then the above three transgenic mice are used to construct a mouse model with stable genotypes of CreERT2 heterozygous, MYC heterozygous, and KRAS heterozygous through hybridization technology, which is the gastric adenocarcinoma transgenic mouse model.

[0010] Furthermore, the implantation of the Cre recombinase expression gene into the mouse gene locus is to implant the Cre recombinase expression gene into the mouse Anxa10 gene locus.

[0011] Furthermore, the construction of a MYC overexpression gene mouse by inserting a Myc overexpression gene is to construct a MYC overexpression gene mouse by inserting the Myc overexpression gene through a LoxP site.

[0012] Furthermore, the construction of the Kras gene G12D conditional point mutation mouse is carried out by replacing the exon2 of Kras with loxp-stop-loxp and exon2 containing G12D to construct the Kras gene G12D conditional point mutation mouse.

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

[0014] (1) Insert 2A-CreERT2-Wpre-pA into the stop codon of mouse Anxa10 gene to obtain Anxa10-CreERT2 recombinase transgenic mouse Anxa10-CreERT2 + / + mice; the CAG promoter-loxp-STOP-loxp-Myc-polyA conditional overexpression structure was inserted into the H11 site to establish the H11-LSL-Myc mouse model, which is a MYC overexpression gene mouse MYC LSL / LSL Mouse; loxp-stop-loxp and exon2 containing G12D replaced exon2 of Kras to establish the 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 among the F1 generation mice were selected and named 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 among the F1 generation mice were selected and named Anxa10-CreERT2 + / - ;Kras loxP / + Mice;

[0017] (4) Transfect the Anxa10-CreERT2 in the above F1 generation + / - ;MYC LSL / + Mice with Anxa10-CreERT2 + / - ;Kras loxP / + The mice were mated to obtain F2 mice, and the genotype of the F2 mice was selected to be Anxa-CreERT2 heterozygous, MYC heterozygous, and KRAS heterozygous mice, named Anxa10-CreERT2 + / - ;MYC LSL / + ;Kras loxP / + Mice;

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

[0019] As an alternative, the above method can also be used to construct Anxa10-CreERT2 + / + ;MYC LSL / LSL ;Kras loxP / + Mouse trigene homozygous mice or Anxa10-CreERT2 + / + ;MYC LSL / + ;Kras loxP / + Mouse or Anxa10-CreERT2 + / - ;MYC LSL / + ;KrasloxP / + Tri-gene heterozygous mice such as mice can be used as the gastric adenocarcinoma transgenic mouse model described in the present invention, as long as the MYC gene expression or MYC protein activity is overexpressed or increased based on Anxa10-CreERT2 expression, and the KRAS gene mutant in the gastric mucosal epithelial cells is activated, causing the Kras protein to function abnormally and remain in an activated state.

[0020] Anxa10-CreERT2 + / + The mice were homozygous for the Anxa10-CreERT2 recombinase transgenic mice. + / - The mice were Anxa10-CreERT2 recombinase transgenic heterozygous mice; MYC LSL / LSL Mice are homozygous for MYC overexpression. LSL / + MYC overexpression heterozygous mice; KRAS loxP / + The mice were heterozygous for the Kras gene G12D conditional point mutation.

[0021] The present invention also provides the above-mentioned MYC LSL / LSL Mice, Anxa10-CreERT2 recombinase transgenic mice, KRAS loxP / + Application of mice in constructing transgenic mouse model of gastric adenocarcinoma.

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

[0023] Finally, the present invention also provides the application of the transgenic mouse model constructed by the above method in screening gastric cancer cell lines.

[0024] The c-Myc gene (also called Myc) is abnormally expressed in many tumors, affecting cell proliferation, growth metabolism, gene instability, stimulating angiogenesis, cell malignant transformation, differentiation and apoptosis, and plays an extremely important regulatory role. The MYC proto-oncogene encodes a family of transcription factors and is one of the most common activated oncoproteins in human tumors. In fact, in the vast majority of cancers, MYC aberrations or MYC-related pathways are upregulated through alternative mechanisms. The MYC protein is the main regulator of cellular programs. Therefore, MYC-activated cancers trigger many cancer characteristics required for the autonomous growth of tumors. KRAS mutations can cause uncontrolled activation of RAS proteins. RAS proteins are located in the cell membrane and regulate cell growth, proliferation, movement, migration and angiogenesis. Based on this, the present invention constructs a spontaneous gastric cancer animal model of double-gene mutant mice based on Myc gene overexpression and KRAS activation mutation. This animal model is a transgenic animal model constructed using the Cre-LoxP system. It is constructed by inserting the Myc overexpression gene through the LoxP site and using a double gene mutation method based on Myc gene overexpression and KRAS activation mutation. Compared with other construction methods reported in the literature, this model is simpler. In addition, the mouse model only takes 2 months to form a tumor, with a tumor formation rate of up to 100%, and a stable tumor formation effect. And the tumor only forms in the stomach, and the tumor formation is highly specific. Pathological sections and immunohistochemical staining determined that this animal model is a common adenocarcinoma of gastric cancer, which has good clinical representativeness.

[0025] Beneficial effects: Compared with other existing experimental animal models, the present invention constructs a spontaneous gastric cancer animal model of double-gene mutation mice with Myc gene overexpression and KRAS activation mutation based on the Cre-LoxP system. It is simpler to construct the model. The tumor formation time of gastric cancer in this mouse model is shortened, the tumor formation rate is as high as 100%, and the tumor formation effect is stable. After induction, tumors are only formed in the stomach, and the tumor formation is highly specific. Through pathological sectioning and immunohistochemical staining, it was determined that this animal model is the most common gastric adenocarcinoma in gastric cancer, with good clinical representativeness, and can be used in the later development or screening of gastric cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the electrophoresis diagram of the product amplified using the Anxa10-CreERT2 primer, where lanes 1, 2 and 3 represent three different samples.

[0027] Figure 2 This is the electrophoresis diagram of the product amplified using MYC primers, where lanes 1, 2 and 3 represent three different samples.

[0028] Figure 3 The electrophoresis diagram of the product amplified using KRAS primers, where lanes 1, 2 and 3 represent three different samples.

[0029] Figure 4 These are the validation results of the gastric adenocarcinoma transgenic mouse model, including: A: gastric tumor in the gastric adenocarcinoma mouse model; B: survival curve of gastric adenocarcinoma mice; C: KI67, P-ERK, and MYC histochemistry of gastric adenocarcinoma mice; D: CDX2 and MUC2 histochemistry of gastric adenocarcinoma mice. DETAILED DESCRIPTION

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

[0031] Example 1 Method for constructing a gastric adenocarcinoma transgenic mouse model

[0032] 1. Gene sources and construction principles of each transgenic mouse

[0033] 1. Obtaining Anxa10-CreERT2 mice

[0034] 2A-CreERT2-Wpre-pA was inserted into the stop codon of mouse Anxa10 gene to obtain Anxa10-CreERT2 recombinase transgenic mouse Anxa10-CreERT2 + / + Mice. The mice were purchased from Shanghai Model Organisms Technology Co., Ltd.

[0035] Anxa10-2A-CreERT2 gene:

[0036] NCBI ID: 26359

[0037] MGI ID: 1347090

[0038] Ensembl ID: ENSMUSG00000031635

[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-marked cell types: gastric epithelial cells, skin hair follicle cells

[0044] This mouse expresses CreERT2 recombinase at the Anxa10 locus. When Anxa10-2A-CreERT2 is bred with reporter mice, the reporter gene is expressed in the gastric epithelial cells of the adult after Tamoxifen induction; when bred with mice containing loxP flanking sequences, Tamoxifen-induced recombination will lead to the deletion of the floxed sequence in the gastric epithelial cells. It can be used for gastric cancer research.

[0045] 2. Obtaining MYC overexpressing mice

[0046] The CAG promoter-loxp-STOP-loxp-Myc-polyA conditional overexpression structure was inserted into the H11 locus to establish the H11-LSL-Myc mouse model, which is a MYC overexpression gene mouse MYC LSL / LSL Homozygous mice were purchased from Shanghai Model Organisms 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, cell malignant transformation, differentiation and apoptosis. The MYC proto-oncogene encodes a family of transcription factors and is one of the most common activated oncoproteins in human tumors. In fact, in most cancers, MYC aberrations or MYC-related pathways are upregulated through alternative mechanisms. MYC protein is a major regulator of cellular programs. The c-Myc gene (also called Myc) is abnormally expressed in many tumors, affecting cell proliferation, growth metabolism, gene instability, stimulating angiogenesis, cell malignant transformation, differentiation and apoptosis. The CAGpromoter-loxp-STOP-loxp-Myc-polyA conditional overexpression structure was inserted into the H11 site to establish the H11-LSL-Myc mouse model. The H11 site is located on mouse chromosome 11. This site has been shown to be similar to Rosa26 and can be used to express exogenous genes more widely. H11-LSL-Myc can highly express the Myc gene in Cre-expressing tissues after mating with Cre-expressing mice. It can be used to establish tumor models and conduct tumor research.

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

[0052] The loxp-stop-loxp and exon2 containing G12D replaced the exon2 of Kras 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 use K19-CreERT mice and Loxp-STOP-Loxp-K-Ras (G12D) mice to obtain mice that conditionally express K-Ras (G12D). These mice also have 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 continuously in an activated state, leading to the continuous proliferation of tumor cells. The loxp-stop-loxp and exon2 containing G12D replace the Kras exon2 to establish a Kras gene G12D conditional point mutation mouse strain. After mating with Cre mice, G12D point mutation mice can be obtained.

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

[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 / +Mouse.

[0059] (2) KRAS conditional point mutation activated mice - KRAS loxP / + The mice were mated with Anxa10-CreERT2 recombinase transgenic mice that specifically expressed CreERT2 recombinase in gastric epithelial cells to obtain F1 mice; heterozygous mice among the F1 mice were selected and named Anxa10-CreERT2 + / - ;Kras loxP / + Mouse.

[0060] (3) Transfecting the Anxa10-CreERT2 in the F1 generation + / - ;MYC LSL / + Anxa10-CreERT2 in mice and the F1 generation + / - ;KRAS loxP / + The mice were mated to obtain F2 mice; the F2 mice were selected to have the genotype of Anxa-CreERT2 heterozygous, MYC heterozygous, and KRAS heterozygous, and their genotype was named Anxa10-CreERT2 + / - ;MYC LSL / + ;Kras loxP / + Mice;

[0061] (4) Select the genotype of F2 mice to be Anxa10-CreERT2 + / - ;MYC LSL / + ;Kras loxP / + The mice were mated to obtain F3 mice, and the genotype of the F3 mice was selected to be Anxa10-CreERT2

[0062] + / - ;MYC LSL / LSL ;Kras loxP / + The mouse is a gastric epithelial cell-specific MYC overexpression and Kras activation mutation mouse, which is the gastric adenocarcinoma transgenic mouse model;

[0063] Anxa10-CreERT2 + / + The mice were homozygous for the Anxa10-CreERT2 recombinase transgenic mice. + / - The mice were Anxa10-CreERT2 recombinase transgenic heterozygous mice; MYC LSL / LSL Mice are homozygous for MYC overexpression. LSL / + MYC overexpression heterozygous mice; KRAS loxP / + The mice were heterozygous for the Kras gene G12D conditional point mutation.

[0064] The constructed Anxa10-CreERT2 + / - ;MYC LSL / LSL ;Kras loxP / + Mice, for adult mice, tamoxifen is injected intraperitoneally at 6 weeks of age. Dissolve tamoxifen in corn oil at a concentration of 20 mg / ml and shake overnight at 37 degrees Celsius. Store it at 4°C during injection. The injection dose is determined by weight, approximately 200 mg / kg body weight. Administer tamoxifen by intraperitoneal injection (using an ACUC-approved injection procedure) once every 24 hours for 3-5 consecutive days. For safety, disinfect the injection site with 70% ethanol before injection. After the last injection, mice should be isolated for 24 hours before returning to their normal animal room.

[0065] 3. Mouse genotype identification method

[0066] The genotype identification of gastric mucosal epithelial cell-specific Anxa10-CreERT2; MYC overexpression and Kras activation point mutation mice and the PCR identification method for obtaining heterozygous mice are as follows:

[0067] Take 2-3 mm of the tail of the offspring mouse and extract the genomic DNA of the tail using standard procedures. Use the genomic DNA of the tail as a template and use gene-specific primers to perform polymerase chain reaction (PCR) to identify the mouse genotype. Use the genomic DNA of the tail of wild-type mice C57 mice as a control.

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

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

[0070] The mutant type primer sequences are:

[0071] 5'-CAGGGCACTATGAGAAAGCACTAC-3' and

[0072] 5'-AGCCCGGACCGACGATGAAGC-3'.

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

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

[0075] The mutant type primer sequences are:

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

[0077] 3) The sequence of the wide type primer for identifying the KRAS point mutation gene is:

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

[0079] The 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, 2xTaq (buffer): 7.5ul, H2O: 5.5ul, mouse tail DNA: 1.5ul, Total: 15ul.

[0083] PCR instrument loading procedure:

[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 5Goto2 x35, Step6 72℃00:05:00(5min), Step7 12℃Forever.

[0086] 2) Kras identification:

[0087] Volume: 15ul, Step1 94℃00:03:00(3min), Step2 94℃00:00:15(15s), Step358'C00:00:15(15s), Step4 72℃00:00:40(40s), Step 5Goto2 x30, Step6 72℃00:05:00(5min), Step7 10'℃Forever.

[0088] 3) MYC identification:

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

[0090] The genotype identification results of mice are shown in Figure 1-3 , the three lanes in the figure represent three samples, Figure 1 The electrophoresis of the product amplified by Anxa10-CreERT2 primers was performed. If a 436bp fragment was specifically amplified, it was Anxa10-CreERT2 positive; if an 823bp fragment was specifically amplified, it was Anxa10-CreERT2 negative.

[0091] Figure 2 For the electrophoresis of the product amplified with MYC primers, if a 434bp fragment can be specifically amplified, it is MYC LSL / LSL homozygous, if a 428bp fragment can be specifically amplified, it is MYC negative, and if a negative-positive band is amplified, it is heterozygous MYC LSL / +.

[0092] Figure 3 The product amplified by KRAS primers is electrophoresed. If a 100bp fragment can be specifically amplified and a 200bp fragment can be amplified, it is a KRAS loxP / + heterozygote. If a 200bp fragment can be specifically amplified, it is KRAS negative and wild type.

[0093] Figure 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 required gastric epithelial cell-specific MYC overexpression and Kras activation point mutation mouse.

[0094] Example 2 Validation of Gastric Adenocarcinoma Transgenic Mouse Model

[0095] (1) The constructed Anxa10-CreERT2 + / - ;MYC LSL / LSL ;Kras loxP / + Model mice, as well as wild-type mice, were fed separately. After the mice became adults, two groups of mice were given an intraperitoneal injection of tamoxifen 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°C during the injection. The injection dose was determined by weight, approximately 200 mg / kg body weight. Tamoxifen was administered by intraperitoneal injection (using an ACUC-approved injection procedure) once every 24 hours for 3-5 consecutive days. For safety reasons, the injection site was disinfected with 70% ethanol before injection. After the last injection, both groups of mice should be isolated for 24 hours before returning to their normal animal room and fed with ordinary mouse growth feed.

[0096] (2) The model mice constructed above were injected intraperitoneally with tamoxifen for 2-5 days at 6 weeks of age. After 5-7 weeks, the mice developed stomach tumors, while wild-type mice did not develop tumors. Experimental animal dissection was performed on mice with MYC overexpression and Kras activation mutation. The experimental results showed that the mice developed stomach tumors, as shown in Figure 4 . Among them, Figure A is a gastric tumor in a gastric adenocarcinoma mouse model. The results show that the genotype mice were dissected after 5-7 weeks of experiment, and the stomach showed obvious tumor formation. Figure B is the survival curve of gastric adenocarcinoma mice. The results show that with the increase of time, the survival rate of gastric adenocarcinoma mice significantly decreases. Figure C is the KI67, P-ERK, and MYC histochemistry of gastric adenocarcinoma mice. The results prove that MYC is overexpressed and Kras activates mutations in this genotype of mice. KI67 staining can show gastric tumors, proving that the mouse model is caused by MYC overexpression and Kras activation mutations. Figure D is the CDX2 and MUC2 histochemistry of gastric adenocarcinoma mice. It can be clearly seen from the figure that intestinal and diffuse tumors appear in the stomach at the same time. The above results prove that this model mouse belongs to gastric adenocarcinoma mice.

[0097] The specific embodiment of the present invention specifically includes using the Cre / loxP system to specifically overexpress or increase the expression of the MYC gene in mouse gastric epithelial cells, and at the same time mutating and activating the Kras gene. The mutant Kras protein is dysfunctional and continuously in an activated state, resulting in the continuous proliferation of tumor cells. The CreERT2 recombinase is expressed at the mouse Anxa10 locus. After induction by Tamoxifen, the Anxa10 gene is expressed in the gastric mucosal epithelial cells of the adult, and the gastric mucosal epithelial cells specifically express the CreERT2 recombinase. Cre-ERT2 mice are a type of mouse that expresses a fusion protein containing a mutant of the ligand binding region (ERT) of the estrogen receptor (ER) and the Cre recombinase. Cre-ERT2 is inactive in the cytoplasm without Tamoxifen induction; when Tamoxifen is induced, the metabolite 4-OHT (estrogen analogue) of Tamoxifen binds to ERT, which can enable 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. CreERT2 recombinase is expressed at the mouse Anxa10 locus. After induction with Tamoxifen, when it is bred with mice containing loxP flanking sequences, Tamoxifen-induced recombination will lead to the deletion of the floxed sequence in the epithelial cells of the gastric mucosal layer.

[0098] The animal model of the present invention is a transgenic animal model constructed using the Cre-LoxP system. First, the present invention uses the gene Anxa10 uniquely expressed in the gastric epithelium, implants the Cre recombinase (CreERT2) induced by tamoxifen at the Anxa10 gene site of the mouse, and establishes an inducible Anxa10-creert2 mouse. And through the LoxP site, the Myc overexpression gene is inserted, and a double gene mutation mouse model is constructed based on the Myc gene overexpression and KRAS activation mutation. Finally, an inducible gastric adenocarcinoma transgenic mouse is established. When the mouse is 6 weeks old, Tamoxifen 200mg / kg is injected intraperitoneally for 2-5 consecutive days, and the transgenic mice develop adenocarcinoma within two months, and eventually all die. The inducible gastric adenocarcinoma mouse model constructed using the method described in the present invention can be applied to the application in the later development or screening of gastric cancer drugs.

[0099] The above embodiments are the best implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for constructing a gastric adenocarcinoma transgenic mouse model, characterized in that: The following steps are involved: By using transgenic and hybridization techniques, the MYC gene expression or MYC protein activity in the gastric mucosal epithelial cells of mice is overexpressed or increased, and the KRAS gene mutant in the gastric mucosal epithelial cells is activated, causing the Kras protein to function abnormally and remain in an activated state. The resulting mice are transgenic mouse models of gastric adenocarcinoma.

2. The method for constructing a gastric adenocarcinoma transgenic mouse model according to claim 1, characterized in that: The construction method comprises: The Cre recombinase expression gene is implanted at the mouse gene site to establish mice that can inducibly express Cre recombinase; the MYC overexpression gene mouse is constructed by inserting the Myc overexpression gene; the Kras gene G12D conditional point mutation mouse is constructed; and then the above three transgenic mice are used to construct a mouse model with stable genotypes of CreERT2 heterozygous, MYC heterozygous, and KRAS heterozygous through hybridization technology, which is the gastric adenocarcinoma transgenic mouse model.

3. The method for constructing a gastric adenocarcinoma transgenic mouse model according to claim 2, characterized in that: The implantation of the Cre recombinase expression gene into the mouse gene locus is to implant the Cre recombinase expression gene into the mouse Anxa10 gene locus.

4. The method for constructing a gastric adenocarcinoma transgenic mouse model according to claim 2, 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.

5. The method for constructing a gastric cancer transgenic mouse model according to claim 2, characterized in that: The Kras gene G12D conditional point mutation mouse is constructed by replacing the Kras exon2 with loxp-stop-loxp and exon2 containing G12D to construct the Kras gene G12D conditional point mutation mouse.

6. 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 locus to establish the H11-LSL-Myc mouse model, which is a MYC overexpression gene mouse MYC LSL / LSL mice; 2A-CreERT2-Wpre-pA was inserted into the stop codon of the mouse Anxa10 gene to obtain Anxa10-CreERT2 recombinase transgenic mice; loxp-stop-loxp and exon2 containing G12D replaced exon2 of Kras 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 above F1 generation + / - ;MYC LSL / + Mice with Anxa10-CreERT2 + / - ;Kras loxP / + The mice were mated to obtain F2 mice, and the genotype of the F2 mice was selected to be Anxa-CreERT2 heterozygous, MYC heterozygous, and KRAS heterozygous mice, named Anxa10-CreERT2 + / - ;MYC LSL / + ;Kras loxP / + Mice; (5) The Anxa10-CreERT2 with the same genotype in the F2 generation + / - ;MYC LSL / + ;Kras loxP / + The mice were mated to obtain F3 mice, and the genotype of the F3 mice was selected to be Anxa10-CreERT2 + / - ;MYC LSL / LSL ;Kras loxP / + The mouse is the gastric adenocarcinoma transgenic mouse model.

7. The MYC according to claim 6 LSL / LSL Mice, Anxa10-CreERT2 recombinase transgenic mice, KRAS loxP / + Application of mice in constructing transgenic mouse model of gastric adenocarcinoma.

8. Use of the transgenic mouse model constructed by the method according to any one of claims 1 to 6 in screening drugs for preventing and / or treating gastric adenocarcinoma.

9. Use of the transgenic mouse model constructed by the method according to any one of claims 1 to 6 in screening gastric cancer cell lines.

Images