A CT45 gene knock-in lung cancer mouse model and its construction method and application

Abstract

The present invention relates to a mouse model of CT45 gene knock-in and its construction method and application, belonging to the field of bioengineering technology. The present invention constructs a CT45 gene knock-in vector and injects it into a mouse to obtain an F0 mouse; the F0 mouse is hybridized with a Cre-LoxP mouse to obtain a CT45 gene expression mouse; finally, the CT45 gene expression mouse is hybridized with a Sftpc-Cre mouse to obtain a gene knock-in mouse that specifically expresses the CT45 gene in the lung, and a CT45 gene knock-in mouse model is successfully constructed. According to genotype identification and experimental verification, the knock-in and specific expression of the CT45 gene cause spontaneous lesions in the mouse lungs, and lung cells become cancerous, which can cause the occurrence of mouse lung cancer in 3-6 months, so the CT45 gene knock-in mouse is a brand-new lung cancer model animal, which can be applied to the preparation of lung cancer diagnosis products and therapeutic drugs, and the screening and development of new anticancer drugs.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a CT45 gene knock-in lung cancer mouse model and a construction method and application thereof. Background Art

[0002] Lung cancer can be divided into three types clinically and histopathologically: small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC) and large cell lung cancer. NSCLC accounts for about 85% of lung cancer cases. The incidence of lung cancer ranks first among all cancers in the world, and the five-year survival rate is only 18%. The main reason is that the pathogenesis of lung cancer has not been elucidated so far, and there is a lack of early diagnosis science and technology, as well as the lack of effective drugs that can cure the vast majority of lung cancer patients. Therefore, studying and clarifying the mechanism of lung cancer, developing new lung cancer diagnostic markers and effective anti-lung cancer drugs have become scientific and technological issues that need to be urgently addressed in the fields of medicine, oncology and pharmacy.

[0003] Animal models are important tools for lung cancer research and drug development. Although there are three types of lung cancer animal models, including chemical lung cancer mouse models, tumor-implanted mouse lung cancer models, and genetically engineered lung cancer mouse models, these models still have many shortcomings and cannot meet the needs of lung cancer research and cancer control drug development.

[0004] The chemical lung cancer mouse model uses carcinogens such as benzopyrene to induce malignant transformation and carcinogenesis of lung epithelial cells, leading to lung cancer. However, the variability of cancer occurrence in mice when chemical carcinogens induce lung cancer is very large, and it is difficult to control the onset time of mice, and the repeatability is poor. In addition, a variety of carcinogenic compounds such as benzopyrene are strictly controlled toxic and hazardous substances, and their purchase and use are strictly controlled and restricted. Furthermore, the smoking and cigarette smoke-induced lung cancer model also has limitations. Technically, special cigarette smoke mouse cages are required. The time required for smoking and cigarette smoke-induced lung cancer to occur is about one year, with poor repeatability and large variability. In view of the many technical and management reasons mentioned above, the chemical lung cancer mouse model is rarely used in practice.

[0005] The commonly used mouse models of lung cancer are made by using human lung cancer cell lines for ectopic or orthotopic transplantation (CDX), or by using tumor tissues of lung cancer patients for ectopic or orthotopic transplantation (PDX). Since mice with human lung cancer cell transplantation are easy to operate, they have been widely used; while the human lung cancer PDX model has few practical applications due to the high difficulty of modeling technology. However, the transplanted tumor mouse model must use immunodeficient mice, such as NOD mice, SCID mice, or NOD / SCID mice; it is difficult to generate transplanted tumor mice using mice with normal immune system function. It is well known that the immune system in the body plays a pivotal role in anti-tumor and tumor immunotherapy, so immunodeficient mice have great limitations in the study of lung cancer mechanism and drug treatment. In addition, although the human lung cancer cell transplanted tumor mouse model has a simple method and a high tumor formation rate, it has a low consistency with clinical practice. The orthotopic transplantation (PDX) model of lung cancer has a high consistency with clinical practice, but it is difficult to operate and can easily lead to pneumothorax and death in mice.

[0006] Genetically engineered lung cancer mouse models use gene editing technology to change genomic DNA, generate gene knock-in or gene knock-out mouse genes for targeted design, produce oncogenes or tumor suppressor gene deletions in mice, and thus generate spontaneous lung cancer mouse models. Genetically engineered lung cancer mouse model animals have received increasing attention and are increasingly used. Common gene mutation lung cancer mouse models include KRAS mutations, EGFR mutations, ALK gene rearrangements, BRAF mutations, PIK3CA mutations, etc. These spontaneous lung cancer animal models are all modeled with immune-normal mice, which can well simulate the entire pathogenesis of clinical lung cancer patients. At the same time, they also help to simulate the formation of tumor microenvironment during tumorigenesis, and can track the various stages of lung cancer initiation and evolution in vivo and determine the tumor progression stage. Their application is increasing. However, the existing genetically engineered lung cancer mouse models also have certain limitations. Due to the low incidence of tumorigenic gene mutations in human lung cancer, for example, KRAS mutations are 15-25%, EGFR mutations are 10-50%, ALK gene rearrangements are 3-7%, BRAF mutations are only 1-3%, and PIK3CA mutations are only 1-3%. This results in that the animal model of lung cancer with a single mutant gene cannot well represent the pathogenesis and evolution of most lung cancer patients. In addition, practice has shown that it takes a long time, often more than a year, for the above-mentioned single gene mutant mice to develop lung cancer. In fact, the above-mentioned mutant gene is prone to lung cancer in mice when the p53 gene is missing. Therefore, it is necessary to use tumorigenic genes with high tumor specificity and sensitivity to establish a highly versatile lung cancer mouse model that can simulate the tumor occurrence and evolution process of most lung cancer patients. How to establish such a mouse model has become a scientific and technological problem that needs to be solved urgently. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a CT45 gene knock-in mouse model and its construction method and application. The present invention constructs a CT45 gene knock-in vector and injects it into mice to obtain F0 mice; the F0 mice are hybridized with Cre-LoxP mice to obtain CT45 gene expression mice; finally, the CT45 gene expression mice are hybridized with Sftpc-Cre mice to obtain gene knock-in mice that specifically express the CT45 gene in the lungs, and a new lung cancer mouse model is successfully constructed.

[0008] The first object of the present invention is to provide a CT45 gene knock-in mouse model, wherein the CT45 gene is knocked into the ROSA26 site of the mouse genome.

[0009] Furthermore, the CT45 gene is specifically expressed in mouse alveolar type II epithelial cells.

[0010] The inventors previously studied 284 lung cancer patient samples and found that 187 of them were positive for CT45, with a positive rate of up to 65.8%. Moreover, it was highly expressed in various lung cancer subtypes, with a positive rate of 61.6% in adenocarcinoma, 75.7% in squamous cell carcinoma, and 48.1% in small cell lung cancer. However, it was not expressed in normal lung and pneumonia tissues, with high lung cancer specificity and sensitivity, and abnormally high expression of CT45 was closely related to poor prognosis of lung cancer patients. Therefore, the CT45 gene was selected to construct a lung cancer mouse model.

[0011] Furthermore, the specific expression of CT45 gene in alveolar type II epithelial cells was achieved through the alveolar surfactant protein C (Sftpc) promoter.

[0012] The second object of the present invention is to provide a gene knock-in vector for constructing a CT45 gene knock-in mouse, wherein the gene knock-in vector comprises a human CT45 encoding cDNA fragment hCT4CDS and a regulatory element LOXP-STOP-LOXP.

[0013] Furthermore, the gene knock-in vector also includes a WPRE element.

[0014] Furthermore, the nucleotide sequence of the human CT45 encoding cDNA fragment hCT4CDS is shown in SEQ ID NO.1.

[0015] Furthermore, the LOXP-STOP-LOXP fragment is used to achieve conditional knock-in of the CT45 gene. After the expression element is knocked into the mouse genome, the STOP fragment needs to be removed by Cre recombinase in the mouse body to achieve stable expression of the target gene.

[0016] The third object of the present invention is to provide a method for constructing a CT45 gene knock-in mouse model, comprising the following steps:

[0017] Step S1, constructing a gene knock-in vector, and injecting it into mice to obtain F0 mice;

[0018] Step S2, hybridizing the F0 mice with the Cre-LOXP mice to obtain CT45+ / +Cre+ / + mice expressing the CT45 gene;

[0019] Step S3, hybridizing CT45+ / +Cre+ / + mice with Sftpc-Cre mice, and self-pollinating the obtained heterozygous mice to obtain lung-specifically expressed CT45 gene knock-in mice.

[0020] Furthermore, it includes genotyping of gene knock-in mice.

[0021] Furthermore, the nucleotide sequences of the primers used for genotype identification are shown in SEQ ID NO.2-3.

[0022] Specifically, the forward primer was 5′-AGTCGCTCTGAGTTGTTATCAG-3′;

[0023] Specifically, the reverse primer was 5'-AGTCCCTATTGGCGTTACTATGG-3'.

[0024] Furthermore, genotype identification was performed using genomic DNA from the tail of CT45 gene knock-in mice.

[0025] The fourth object of the present invention is to provide the application of the above mouse model, the above gene knock-in vector or the above construction method in the study of CT45 gene expression.

[0026] The fifth object of the present invention is to provide the use of the above mouse model, the above gene knock-in vector or the above construction method in screening drugs that inhibit the expression of CT45 gene.

[0027] The sixth object of the present invention is to provide the use of the above-mentioned mouse model, the above-mentioned gene knock-in vector or the above-mentioned construction method in the preparation of lung cancer auxiliary diagnosis products.

[0028] Beneficial effects of the present invention:

[0029] The present invention has established a CT45 gene lung-specific knock-in mouse, and a series of experimental studies have shown that a single CT45 gene knock-in can cause lung cancer and death in mice within 3-6 months. The lungs of the CT45 gene lung-specific knock-in mice can produce spontaneous lesions, and lung cells undergo canceration, resulting in 100% of mice developing lung cancer, while none of the mice in the control group developed lesions or died. The present invention has established a new lung cancer model animal of the CT45 gene lung-specific knock-in mouse, which is used to screen CT45 inhibitors and prepare lung cancer diagnostic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0031] Figure 1 The design strategy for constructing the CT45 gene knock-in mouse in Example 1 of the present invention, wherein the WT allele is the DNA structure of the wild-type mouse chromosome ROSA26, and the CT45 KI allele is the DNA structure of the CT45 exon DNA knock-in mouse ROSA26;

[0032] Figure 2 Genotyping of CT45 lung-specific knock-in mice in Example 2 of the present invention;

[0033] Figure 3 The expression of CT45 in various tissues of the CT45 gene knock-in mice in Example 3 of the present invention, wherein A is the expression of CT45 in the heart, liver, spleen, lung, kidney and other organs of the CT45 gene knock-in mice detected by immunoblotting, and B is the expression of CT45 in wild-type mice and knock-in mice verified by QT-PCR;

[0034] Figure 4 The photographic records and weighing results of the mouse lung tissue in Example 4 of the present invention, wherein A is a photograph of the lungs of wild-type mice and a photograph of the lungs of CT45 knock-in mice after the onset of the disease, B is a statistical result of the lung weights of 11 CT45 knock-in mice and 11 wild-type mice that have developed the disease, C is a photograph of the lungs of p53 knockout p53- / - mice and CT45+ / +p53- / - mice, and D is a statistical result of the lung weights of p53 knockout p53- / - mice and CT45+ / +p53- / - mice;

[0035] Figure 5The results of the study on lung tissue of CT45 gene knock-in mice in Example 5 of the present invention, wherein A is the result of observation under a 200X and 1000X microscope by hematoxylin-eosin (H&E) staining, the arrows point to tumor tissues and cells at 200X and 1000X magnification, and B is the expression levels of tumor-specific markers Ki67 and CEA shown by real-time quantitative PCR;

[0036] Figure 6 The lung sections and staining results of the mice in Example 6 of the present invention, wherein A is the CT45 immunohistochemical staining results of the wild-type and gene knock-in mice, B is the Ki67 immunohistochemical staining results of the wild-type and gene knock-in mice, and C and D are the CEA1 immunohistochemical staining results of the wild-type and gene knock-in mice;

[0037] Figure 7 The expression of tumor-related genes in CT45 gene knock-in mice in Example 7 of the present invention, wherein each point in C is the average value of the experimental results of three repeated experiments on one mouse. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0039] Example 1: Construction of CT45 gene knock-in mice

[0040] The cDNA encoding human CT45 (hCT45CDS) was cloned, and the CT45 gene was inserted into the gene knock-in vector using CRSPR / Cas9 technology to obtain the CAG-LOXP-STOP-LOXP-hCT45CDS-WPRE-polyA vector plasmid ( Figure 1). Subsequent microinjection and other operations were performed to insert the CAG-LOXP-STOP-LOXP-hCT45CDS-WPRE-polyA fragment into the ROSA26 gene site of the mouse genome to generate F0 mice. After breeding, 4 CT45 gene knock-in mice were obtained. The ROSA26 gene is a non-coding gene composed of three exons. It is often used for safe gene knock-in and can ensure the normal and stable expression of the transferred gene. The LOXP-STOP-LOXP fragment is used to achieve conditional gene knock-in. Since the transcription stop signal box (Transcription STOP cassette) of LOXP-STOP-LOXP exists in the above CT45 knock-in fragment, CT45 cannot be expressed after the fragment is knocked into the mouse genome. The STOP fragment needs to be removed by Cre recombinase in the mouse body to achieve stable expression of the target gene. The CAG promoter is a gene promoter designed to achieve a higher level of expression of the inserted gene, and the IRSE-EGFP box is designed to monitor the expression of the inserted gene in mice. The nucleotide sequence of CT45 exon DNA (hCT45CDS) is shown in SEQ ID NO.1.

[0041] After obtaining four CT45 knock-in mice, these mice were hybridized with Cre-LOXP mice to remove the DNA sequence of the transcription termination signal in the mice, resulting in mice expressing CT45 (CT45+ / +; Cre+ / +). Afterwards, in order to achieve the specific expression of CT45 in mouse lung tissue, the above CT45 knock-in mice were hybridized with mice with the alveolar surfactant protein C (Sftpc, SPC) promoter-Cre gene linkage. Since the SPC promoter is only present in alveolar type Ⅱ (AT Ⅱ) epithelial cells in vivo, the Cre recombinase can only be expressed in the lung tissue of mice with the SPC promoter after hybridization, thereby achieving the specific expression of the CT45 gene in alveolar type Ⅱ epithelial cells. First, CT45+ / -Cre+ / - heterozygous mice were obtained, and then the heterozygous mice were self-crossed to obtain CT45+ / +Cre+ / + mice (all subsequent CT45+ / + mice were SPC-Cre+ / + mice), that is, mice with lung-specific expression of CT45.

[0042] Example 2: Genotyping of CT45 knock-in mice

[0043] The tail of the CT45 gene knock-in newborn mouse was cut within 7-14 days after birth, and the genomic DNA was extracted using a lysis buffer, and the genotype of the mouse was identified by PCR. The primer sequences are shown in SEQ ID NO.2-3. The PCR primers of CT45 were synthesized again to identify the knock-in status of the mouse CT45 and SPC-Cre genes. The primer sequences are shown in SEQ ID NO.4-5.

[0044] The results showed that homozygous mice (CT45+ / +), heterozygous mice (CT45+ / -), and wild-type mice (CT45- / -, the mouse genome does not have the CT45 gene) with the CT45 gene knocked in were constructed. Figure 2 A), homozygous mice (CT45+ / +Cre+ / +) and heterozygous mice (CT45+ / -+Cre+ / -) ( Figure 2 B) Cre positivity indicates that a stop codon inserted into the CT45 gene has been removed and the CT45 gene can be expressed. The CT45 knock-in band size is 456 bp, while the wild-type band size is 296 bp ( Figure 2 A); The size of the Cre knock-in band is 739 bp, and the wild-type band is 386 bp ( Figure 2 B). The molecular weight and size of the bands amplified by PCR can be used to determine whether the mouse is a CT45 lung-specific knock-in mouse. Only when there are Cre-positive bands and CT45-positive bands can the mouse lung specifically express CT45. The results of the genotype identification of newborn mice showed that both CT45 and Cre had been knocked into these mice, indicating that the CT45 gene lung-specific knock-in mouse was successfully constructed, and the CT45+ / + mice were able to reproduce normally and produce offspring.

[0045] Example 3: CT45 protein in CT45 gene knock-in mice is highly expressed only in lung tissue

[0046] After the mice were anesthetized and killed, their organs (heart, liver, spleen, lung, and kidney) were removed and their proteins and RNA were collected. The expression of CT45 in each tissue was detected by Western Blot and QT-PCR. The results showed that CT45 protein was only expressed in the lung tissue of mice ( Figure 3 A), and CT45 mRNA levels were also very high in mouse lung tissue ( Figure 3 B) shows that lung-specific knock-in mice of the CT45 gene have been established.

[0047] Example 4: CT45 gene knock-in causes lung lesions in mice

[0048] In the observation of mouse phenotype, it was found that CT45+ / + mice showed weight loss, lower body temperature, rapid breathing at 3-6 months of age. The mice were also observed to have symptoms of severe coughing, suggesting that the mouse lungs may be abnormal. The average time of death after the onset of the disease was 17.4 weeks ± 5.6 weeks. The CT45+ / + diseased mice were paired with normal control group mice of similar age and taken out. The mice were killed by overdose of anesthesia, the blood in the blood vessels was cleared by cardiac perfusion, the lungs were dissected out, and the lungs were perfused and cleaned. The lungs were photographed and recorded and weighed.

[0049] The results showed that there were obvious differences in lung weight and overall morphology between the CT45+ / + disease group mice and the normal control group CT45- / - mice after cardiac perfusion. The lungs of the CT45 gene knock-in disease group mice were dark yellow or even dark red, and the lungs also lost their original normal morphology and became dense and swollen. The lungs of the normal control group mice were milky white after cardiac perfusion, and the lungs were loose and soft ( Figure 4 A). Weighing the lung tissues of CT45+ / + mice and CT45- / - control mice revealed that the average weight of the lung tissues of the control mice was 0.23 grams, while the average weight of the lungs of CT45 mice was 0.53 grams, indicating that the mice had abnormal lung enlargement ( Figure 4 B). These phenotypes suggest that CT45+ / + mice have lesions in their lung tissues. In addition, the disease status of the constructed CT45+ / +p53- / - mice was observed. The results showed that CT45+ / +p53- / - mice developed the disease at 3-5 months of age, with lesions and tumors inside and outside the lungs ( Figure 4 C). The average weight of the lungs of mice in the CT45+ / +p53- / - group was 1.25 g, and the average weight of the lung tissues of mice in the CT45- / -p53- / - control group was 0.25 g ( Figure 4 D), indicating that CT45 accelerated and aggravated lung lesions and tumorigenesis in mice.

[0050] Example 5: CT45 gene knock-in mice become a new lung cancer model animal

[0051] After the wild-type and simple CT45 knock-in mice were killed by overdose anesthesia, the mouse lung tissue was removed by dissecting scissors, weighed, and fixed in 4% paraformaldehyde for 48 hours. After removal, it was placed in a tissue embedding box and rinsed with running water. After rinsing, the embedding box containing the tissue was placed in the 75% ethanol prepared in advance and soaked for 2 hours or overnight. Then the tissue was placed in 85-100% ethanol for dehydration for 2 hours each, and finally placed in two xylenes for 30 minutes each to replace the anhydrous ethanol in the tissue. Finally, the tissue was placed in the pre-melted paraffin and soaked overnight at 60°C. After the paraffin was completely melted, the tissue was embedded. The paraffin tissue block was sliced ​​at a thickness of 4 μm on the microtome, and the slice was placed in a spreader for spreading at a temperature of 53°C, and finally placed on a 58°C drying machine for drying. Finally, the paraffin slices were stored in the slice box at room temperature. During HE staining, the paraffin sections are soaked in xylene, 75-100% ethanol, and double distilled water for 5 minutes in turn to achieve dewaxing and hydration. Then add hematoxylin to cover the tissue, stain for about 10 minutes, observe the coloring of the cell nucleus under a microscope, and rinse with tap water to reverse blue. Finally, add eosin dye solution to stain for 10-30 seconds and rinse with tap water. Finally, the sections are placed in 75-100% ethanol and xylene in turn to dehydrate and make them transparent, and finally add neutral gum to seal the sections. Take pictures with an upright microscope.

[0052] Microscopic observation showed that the lung tissue of wild-type mice showed a normal reticular structure, with regular alveolar morphology and a cavity in the middle. The alveolar wall was composed of a thin layer of transparent epithelial cells arranged in a regular and orderly manner. However, the lung tissue of mice with simple CT45 gene knock-in (CT45+ / +) showed obvious abnormal cell proliferation, and these cells had larger nuclei, darker staining, less cytoplasm, and an increased nuclear-cytoplasmic ratio, which were all consistent with the characteristics of tumor cells. At the same time, the lung tissue lost its original alveolar reticular structure, forming a dense structure, and caused abnormal morphological changes and functional impairment of the lung ( Figure 5 A). In addition, real-time quantitative PCR (QT-PCR) also showed that the expression levels of tumor-specific markers Ki67 and CEA were greatly increased ( Figure 5 B). These results are consistent with the above mouse phenotypes, indicating that CT45 gene knock-in can induce lung cancer in mice. A new mouse model of lung cancer was successfully established by the above method.

[0053] Example 6: CT45 gene knock-in mice for lung cancer diagnosis

[0054] The paraffin sections were soaked in xylene, 75-100% ethanol, and double distilled water for 5 minutes in turn to achieve dewaxing and hydration. The section antigens were repaired using the microwave antigen repair method, and the microwave temperature was heated to 90°C for 20 minutes. Finally, the antigen repair solution containing the sections was taken out of the microwave and cooled at room temperature. Then the sections were taken out and blocked with 5% BSA for one hour. CT45, proliferating cell nuclear antigen (Ki67) and carcinoembryonic antigen (CEA) primary antibodies were added at a ratio of 1:1000, respectively, and kept at 4°C overnight. The next day, PBS was washed 3 times for 3 minutes each time, and then enzyme-labeled anti-mouse / rabbit IgG polymer was added for 30 minutes, PBS was washed 3 times for 3 minutes each time, and freshly prepared DAB color development solution was added for color development. Stain with hematoxylin for 10-30 seconds and rinsed with tap water. Finally, the sections were placed in 75-100% ethanol and xylene in turn for dehydration and transparency, and neutral gum was added to seal the sections.

[0055] The results of CT45 immunohistochemical staining showed that the nuclei of many CT45-positive cells were significantly larger than those of the surrounding cells; at the same time, CT45-positive cells mostly grew in clusters, which was consistent with the growth characteristics of tumor cells ( Figure 6 A). In addition, many CT45-positive cells were found in the mouse lung tissue in the mitotic phase, suggesting that these cells proliferated abnormally actively, which was consistent with the characteristics of tumor cells.

[0056] Proliferating cell nuclear antigen Ki67 is a cell cycle protein that is often used to assess tumor growth rate and prognosis. The higher the Ki67 expression level, the faster the cell proliferation and tumor growth, but at the same time the poorer the cell differentiation. Tumors with high Ki67 expression are insensitive to chemotherapy. In human non-small cell lung cancer, the Ki67 positivity rate is usually greater than 60%, so it can be used as an important molecular marker for identifying the occurrence of lung cancer. Immunohistochemical staining results showed that Ki67 was strongly positive in lung cells of CT45 knock-in mice, with a positivity rate greater than 60% ( Figure 6 B), indicating that 60% of the lung tissue cells of mice are tumor cells. In addition, most of the Ki67-positive cells are distributed in clusters, which is consistent with the phenomenon observed by HE staining before. By performing grayscale scanning analysis on the captured images, and then plotting the obtained data using Graphpad 9.0 and performing T-test, it was shown that the Ki67 expression level in CT45 knock-in mice was 32 times higher than that in wild-type mice. It is worth mentioning that in clinical practice, if the Ki67 level of patients is more than 5 times higher than that of normal people, it indicates that there is a malignant tumor in the body, and if it is more than 10 times higher, it can be confirmed that there is a malignant tumor in the body.

[0057] Carcinoembryonic antigen (CEA) is an acidic glycoprotein complex specific for human embryonic antigens. It is usually expressed in the fetal embryonic endoderm epithelium and is not expressed or very low in the normal human body after birth. However, CEA is highly expressed in a variety of malignant tumors, such as melanoma, non-small cell lung cancer, gastric cancer, etc., and is often used as a broad-spectrum tumor marker. In this example, CEA was used as a marker for identifying tumors. The results showed that the immunohistochemical staining of CEA in wild-type control mice was negative or weakly positive, but it was strongly positive in the lung tissue cells of CT45 knocked-in diseased mice ( Figure 6 C) The grayscale scanning analysis of the images was performed, and the obtained data were plotted using Graphpad 9.0 and subjected to T test, showing that the expression level of CEA in the lung tissue of CT45 gene knock-in mice was 21 times higher than that of the control group ( Figure 6 D) Clinically, if the patient's CEA level is more than 5 times higher than that of a normal person, it indicates the presence of a malignant tumor in the body, and if it is more than 10 times higher, it can be confirmed that a malignant tumor is present in the body.

[0058] In summary (Examples 3-6), CT45 can induce lung epithelial cells to become carcinomatous, and CT45 gene knock-in mice are a new lung cancer model animal that can be used for lung cancer diagnosis.

[0059] Example 7: Application of CT45 gene knock-in mice in the study of the pathogenesis of lung cancer

[0060] Lung tissues of CT45 knock-in mice and wild-type mice were collected in Trizol solution for mRNA sequencing. RNA was extracted from a portion of the same batch by the chloroform-isopropanol method and reverse transcribed into cDNA using a kit, and then related genes were detected by RT-PCR and QT-PCR.

[0061] The results showed that 117 tumorigenic genes were abnormally overexpressed in the lung tissues of CT45 knock-in mice, including c-MYC, Twist1, Rab20, etc. ( Figure 7 A. Figure 7 B), indicating that CT45 induces the high expression of numerous tumorigenic genes, which is an important reason for its induction of lung cancer. Therefore, CT45 gene knock-in mice can be used to study the pathogenesis of lung cancer. C-MYC is a recognized proto-oncogene, which is highly expressed in a variety of malignant tumors. As a transcription factor, c-MYC promotes the high expression of more than 100 tumorigenic genes. RT-PCR, QT-PCR and immunohistochemical staining were used to detect the expression level of the proto-oncogene cMYC. The results showed that CT45 gene knock-in led to high expression of the lung cancer marker cMYC in mouse lung tissue cells ( Figure 7C), indicating that CT45 promotes lung cell carcinogenesis and is related to the promotion of high expression of cMYC, suggesting that CT45 driving high expression of cMYC is one of the important mechanisms for causing lung cancer. These research results show that CT45 gene knock-in mice can be used to study the pathogenesis of lung cancer.

[0062] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for constructing a CT45 gene knock-in lung cancer mouse model, characterized in that: The CT45 gene is knocked into the ROSA26 site of the mouse genome, and the construction method comprises the following steps: Step S1, constructing a gene knock-in vector, and injecting it into mice to obtain F0 mice, wherein the gene knock-in vector includes a human CT45 encoding cDNA fragment hCT4CDS and a regulatory element LOXP-STOP-LOXP; Step S2, hybridizing the F0 mice with the Cre-LoxP mice to obtain CT45+ / +Cre+ / + mice expressing the CT45 gene; Step S3, hybridizing CT45+ / +Cre+ / + mice with Sftpc-Cre mice, and self-pollinating the obtained heterozygous mice to obtain lung-specifically expressed CT45 gene knock-in mice.

2. The construction method according to claim 1, characterized in that: The gene knock-in vector further comprises a WPRE element.

3. The construction method according to claim 1, characterized in that: Includes genotyping of knock-in mice.

4. The construction method according to claim 3, characterized in that: The nucleotide sequences of the primers used for genotype identification are shown in SEQ ID NO.2-3.

5. Application of the construction method according to any one of claims 1 to 4 in the study of CT45 gene expression.

6. Use of the construction method according to any one of claims 1 to 4 in screening drugs for inhibiting CT45 gene expression.

7. Use of the construction method according to any one of claims 1 to 4 in the preparation of lung cancer auxiliary diagnosis products.

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