A method for constructing a transgenic mouse model of lung adenocarcinoma with overexpression of MR-1

Abstract

The present invention relates to a method for constructing a transgenic lung adenocarcinoma mouse model with overexpression of MR-1, and the method comprises the following steps: Step 1, constructing a PNKD gene knock-in model mouse; Step 2, obtaining cKP mice; Step 3, mating the cKP mice obtained in Step 2 with the PNKD gene knock-in mice obtained in Step 1; Step 4, inducing genotype change with tamoxifen; Step 5, detecting.

Description

Technical Field

[0001] The present invention relates to a transgenic animal, and particularly to a transgenic lung adenocarcinoma mouse model overexpressing MR-1 for medical test purposes and a method for constructing the same. Background Art

[0002] Myofibrillogenesis regulator 1 (MR1) is a gene cloned and discovered by the Metabolic Engineering Laboratory of the Institute of Medical Biotechnology, Chinese Academy of Medical Sciences. It is located at 2q35, with a full-length mRNA of 755 bp, encoding a protein composed of 142 amino acids, and is highly expressed in the heart, skeletal muscle, kidney and liver. This gene is almost completely identical in location and sequence to the shortest of the three transcriptional splicing isomers of the paroxysmal nonkinesigenic dyskinesia (PNKD) gene. Therefore, the transgenic lung adenocarcinoma mouse model overexpressing MR-1 described in the present invention can also be referred to as a transgenic lung adenocarcinoma mouse model overexpressing PNKD.

[0003] Lung adenocarcinoma is a type of lung cancer and belongs to non-small cell carcinoma. Different from squamous cell lung cancer, lung adenocarcinoma is more likely to occur in women and non-smokers, and its incidence is lower than that of squamous cell carcinoma and undifferentiated carcinoma.

[0004] In patients with lung adenocarcinoma, the MR-1 gene is overexpressed. Therefore, lung adenocarcinoma can be diagnosed by detecting the MR-1 gene, and can be treated by inhibiting the MR-1 gene. Replicating lung adenocarcinoma in animals and constructing a model overexpressing the MR-1 gene can provide a medical test model for the diagnosis and treatment of lung adenocarcinoma, and can be used for the screening of targeted therapeutic drugs.

[0005] Currently, the following several methods are commonly used for establishing experimental animal models of lung adenocarcinoma:

[0006] 1. Tumor cell transplantation model: Lung adenocarcinoma cells are transplanted subcutaneously into mice to form tumors. This model can simulate different subtypes and mutation situations by selecting different lung adenocarcinoma cell lines. The advantages are easy to establish and operate, and can quickly form tumors. The disadvantage is the lack of interaction between the tumor microenvironment and the immune system, and it may not be able to fully simulate the complexity of the tumor.

[0007] 2. Chemical induction model: Chemical substances (such as carcinogens) are used to induce lung adenocarcinoma in mice. This model can simulate the influence of certain environmental factors on the occurrence of lung adenocarcinoma. The advantages are relatively simple and easy to perform, and can simulate lung adenocarcinoma caused by environmental exposure. The disadvantage is that the selection and dosage of chemical substances need to be carefully controlled, and it may not be able to fully simulate the diversity of human lung adenocarcinoma.

[0008] 3. Genetically engineered mouse models: Mutations or overexpressions of genes related to lung adenocarcinoma are introduced through gene editing techniques (such as CRISPR / Cas9) or transgenic techniques. Currently, the most commonly used method is to introduce the Kras-G12D allele mutation and the p53 loss-of-function mutation gene to construct a mouse model of lung adenocarcinoma. The disadvantages are that it takes a long time to establish and maintain these models and the incidence of lung adenocarcinoma is still relatively low. Summary of the Invention

[0009] The present invention provides an overexpressing MR-1 transgenic mouse model of lung adenocarcinoma and a method for constructing the same.

[0010] The method of the present invention can improve the success rate of constructing a mouse model of lung adenocarcinoma, advance the onset time, shorten the experimental period, and accurately simulate the development process of human lung adenocarcinoma.

[0011] To this end, the present invention provides a method for constructing an overexpressing MR-1 transgenic mouse model of lung adenocarcinoma, characterized in that the method comprises the following steps:

[0012] Step 1: Construction of a PNKD gene knock-in model mouse;

[0013] Step 2: Obtaining cKP mice;

[0014] Step 3: Mating the cKP mice in Step 2 with the PNKD gene knock-in mice in Step 1;

[0015] Step 4: Inducing genotype change with tamoxifen.

[0016] Preferably, in the method of the present invention, in Step 1, the construction of the PNKD gene knock-in model mouse is as follows:

[0017] 1) sgRNA design and vector construction: According to the design principle of the target site region, Cas9 / sgRNA is designed and the sgRNA is ligated into a plasmid vector with a T7 promoter. In vitro transcription is carried out, and the construction of the targeting vector is confirmed by enzyme digestion identification and sequencing;

[0018] 2) Injection of mouse fertilized eggs: The Cas9 / sgRNA targeting vector is microinjected into mouse fertilized eggs to construct PNKD gene knock-in mice;

[0019] 3) Genotype identification and mating of F0 generation mouse tails: Select mice with positive genotype identification results from F0 mice and mate them with wild-type mice to obtain F1 generation mice with stable genotypes;

[0020] 4) Genotype identification and expression detection of F1 generation mice: For genotype identification of F1 generation mice, RT-PCR and Southern blot techniques can be used to detect the expression of the PNKD gene. Screen out F1 generation mice with stable genotypes, and then retain them for breeding to obtain F1 generation mice with overexpressed transgenic PNKD;

[0021] Preferably, for the method of the present invention, in step two, the obtaining of cKP mice is as follows:

[0022] Mate sftpc-Cre / ERT2 mice and KrasLSL-G12D-Trp53(LSL-R172H) mice to obtain mice containing sftpc-Cre / ERT2 and KrasLSL-G12D-Trp53(LSL-R172H) genes, namely cKP mice;

[0023] Preferably, for the method of the present invention, in step three, mating the cKP mice in step two with the PNKD gene-knockin mice in step one is as follows:

[0024] Mate cKP mice and PNKD gene-knockin mice to obtain mice simultaneously having sftpc-Cre / ERT2, KrasLSL-G12D-Trp53(LSL-R172H) and PNKD genes;

[0025] Preferably, for the method of the present invention, in step four, inducing genotype change with tamoxifen is as follows:

[0026] Inject tamoxifen intraperitoneally into the mice obtained in step four, continuously inject for 5 days, once a day.

[0027] The method of the present invention is characterized in that

[0028] The sgRNA sequence used in step one is: 5’-AAGGCCGCACCCTTCTCCGGAGG-3’

[0029] During the construction process, if necessary, genotype identification can be performed on F1 generation mice, and the identification primers used are as follows:

[0030]

[0031] The method of the present invention is characterized in that

[0032] In step two, during the construction process, if necessary, genotype identification can be performed on the expression of the sftpc-Cre / ERT2 gene, and the primer sequences used are as follows:

[0033] sftpc-WT-F CATCCAACATACAGACAACGC sftpc-WT-R CTATCCTAAAAGCCCAATCCTA sftpc-cre-3-F TGCTTCACAGGGTCGGTAG sftpc-cre / ERT2-R1 GCCCAAATGTTGCTGGATAG。

[0034] The method of the present invention is characterized in that, during the construction process, if necessary, a step of gene detection can be performed on the mice obtained in step four.

[0035] The present invention further provides transgenic mice constructed by the method of the present invention.

[0036] The present invention further provides the use of the transgenic mice constructed by the method of the present invention in biomedical research.

[0037] Particularly preferably, the method of the present invention comprises the following steps:

[0038] Step 1: Construction of PNKD gene knock-in model mice:

[0039] 1) sgRNA design and vector construction: According to the design principle of the target site region, Cas9 / sgRNA is designed and the sgRNA is ligated into a plasmid vector with a T7 promoter. In vitro transcription is carried out, and the construction of the targeting vector is confirmed by restriction enzyme digestion identification and sequencing.

[0040] The sgRNA sequence used in this step is: 5’-AAGGCCGCACCCTTCTCCGGAGG-3’

[0041] 2) Injection of mouse fertilized eggs: The Cas9 / sgRNA targeting vector is microinjected into mouse fertilized eggs to construct PNKD gene knock-in mice.

[0042] 3) Genotype identification and mating of F0 generation mouse tails: Select mice with positive genotype identification results from F0 mice and mate them with wild-type mice to obtain F1 generation mice with stable genotypes.

[0043] 4) Genotype identification and expression detection of F1 generation mice: Genotype identification is performed on F1 generation mice, and RT-PCR and Southern blot techniques can be used to detect the expression of the PNKD gene. Screen out F1 generation mice with stable genotypes, and then perform breeding for seed preservation to obtain F1 generation mice overexpressing transgenic PNKD.

[0044] The genotype identification primers in this step are as follows:

[0045]

[0046] Step 2: Mating and breeding of sftpc-Cre / ERT2 mice:

[0047] The sftpc-Cre / ERT2 mice are commercially purchased and their genotypes are identified using RT-PCR technology after purchase. The primer sequences used are as follows:

[0048] sftpc-WT-F CATCCAACATACAGACAACGC sftpc-WT-R CTATCCTAAAAGCCCAATCCTA sftpc-cre-3-F TGCTTCACAGGGTCGGTAG sftpc-cre / ERT2-R1 GCCCAAATGTTGCTGGATAG

[0049] Step 3: Obtaining cKP mice:

[0050] Mate sftpc-Cre / ERT2 mice with KrasLSL-G12D-Trp53(LSL-R172H) mice. Among them, KrasLSL-G12D-Trp53(LSL-R172H) mice have a conditional activating point mutation of the KRAS gene (KRAS-G12D) and a dominant inhibitory point mutation of the P53 gene (TP53-R172H). Through mating, mice containing the sftpc-Cre / ERT2 and KrasLSL-G12D-Trp53(LSL-R172H) genes were obtained, which are called cKP mice.

[0051] Step 4: Mate the cKP mice in Step 3 with the PNKD gene-knockin mice in Step 1:

[0052] Mate cKP mice with PNKD gene-knockin mice to obtain mice that simultaneously have the sftpc-Cre / ERT2, KrasLSL-G12D-Trp53(LSL-R172H), and PNKD genes.

[0053] Step 5: Induce genotype change with tamoxifen:

[0054] Inject tamoxifen intraperitoneally into the mice obtained in Step 4, once a day for 5 consecutive days. Tamoxifen can induce the expression of sftpc-Cre / ERT2 and activate Cre recombinase. Cre recombinase can recognize the loxp sequence, resulting in the expression of the PNKD gene, the expression of the KRAS-G12D gene, and the inactivation of P53.

[0055] Step 6: Detection:

[0056] Perform gene detection on the mice obtained in Step 5, and the results are as follows: Detect the PNKD gene in different organs of the mice. The qPCR results show that compared with the control mouse cKP mouse, the PNKD gene is highly specifically expressed in lung tissue.

[0057] The following are the explanations of the terms and phrases in the present invention:

[0058] Overexpression: Refers to the expression level of a certain gene being higher than the normal level in cells or organisms. Usually achieved by introducing exogenous DNA or increasing the copy number of the gene. Overexpression can be used to study gene function, protein function, and its effects in organisms.

[0059] Transgenic: Refers to the process of introducing foreign genes or DNA sequences into an organism. Transgenic technology usually involves introducing foreign genes into the genome of the target organism so that they can be expressed in the cells of the target organism.

[0060] Lung adenocarcinoma: A common type of lung cancer that originates from glandular cells in the lung tissue. Lung adenocarcinoma is usually not related to smoking but is related to other factors (such as environmental factors, genetic factors).

[0061] Myofibrillogenesis regulator 1 (MR1): A protein that participates in regulating the formation and organization of muscle fibers. It plays an important role in muscle development and muscle cell function.

[0062] PNKD: PNKD stands for Paroxysmal nonkinesigenic dyskinesia, a rare movement disorder disease. It is characterized by intermittent, non-movement-induced involuntary movements, including muscle spasms, muscle contortions, and limb incoordination.

[0063] Gene editing technology (such as CRISPR / Cas9): Gene editing technology is a method used to modify the genome of an organism. CRISPR / Cas9 is a commonly used gene editing tool that uses the guide RNA (sgRNA) and Cas9 protein of the CRISPR-Cas9 system to achieve precise editing of the genome, including gene knockout, gene knock-in, and gene modification.

[0064] PNKD gene knock-in: PNKD gene knock-in refers to introducing the PNKD gene into the genome of the target organism so that it can be expressed in the cells of the target organism.

[0065] sgRNA: sgRNA stands for single-guide RNA, an RNA molecule in the CRISPR / Cas9 system. sgRNA binds to the Cas9 protein and guides the Cas9 protein to precisely recognize and cut a specific DNA sequence in the target genome, thereby achieving genome editing.

[0066] Plasmid vector with T7 promoter: The T7 promoter is a commonly used promoter sequence that can promote the transcription process of genes. A plasmid vector is a DNA molecule used to carry foreign DNA fragments. The plasmid vector with T7 promoter is a plasmid vector containing the T7 promoter sequence and is used to express genes compatible with the T7 promoter in cells.

[0067] Targeting vector: A targeting vector refers to a vector used to guide genome editing tools (such as the CRISPR / Cas9 system) to target genomic loci. It usually contains DNA sequences complementary to the target genomic loci to enable the accurate positioning and cleavage of the guiding editing tools.

[0068] RT-PCR: RT-PCR (Reverse Transcription Polymerase Chain Reaction) is a technique used to detect and amplify RNA molecules. It first converts RNA into cDNA through reverse transcription and then amplifies the target DNA sequence using polymerase chain reaction (PCR).

[0069] Southern blot: It is a technique used to detect specific DNA sequences. It involves separating DNA samples by electrophoresis, specifically binding the DNA probe to the target DNA sequence, and then detecting the presence of the target DNA sequence through the radioactive or fluorescent labeling of the probe.

[0070] 3'-UTR of the sftpc gene: The sftpc gene encodes surfactant protein C. The 3'-UTR of the sftpc gene is located in the non-coding region of the sftpc gene and plays an important role in gene expression and regulation.

[0071] IRES-cre: IRES-cre represents Internal Ribosome Entry Site and Cre recombinase. IRES is a sequence structure that allows ribosomes to bypass the translation initiator at the 5' end during transcription, thus enabling the translation of internal promoters. Cre recombinase is a commonly used gene recombination tool for inducing gene recombination in specific cell types.

[0072] ERT2: ERT2 represents Estrogen Receptor Tamoxifen-inducible. ERT2 is a mutant of the estrogen receptor that can regulate gene expression by binding to drugs (such as the ketane compound Tamoxifen). The ERT2 system is commonly used to induce the expression or silencing of specific genes in specific tissues or at specific time points.

[0073] KrasLSL-G12D-Trp53 (LSL-R172H) mice: This is a genetically engineered mouse model used to study the occurrence and development of lung adenocarcinoma. KrasLSL-G12D and Trp53 (LSL-R172H) are two gene mutations located in the Kras and Trp53 genes respectively. These mutations enable these genes to be activated or inactivated by specific inducers in the mouse body.

[0074] Tamoxifen induction: In the study of genetically engineered mice, tamoxifen can be used as an inducer to activate or inhibit the expression of specific genes. By injecting tamoxifen into mice, the function of specific genes can be initiated or turned off, thereby studying the role of the gene in physiological or disease processes. Tamoxifen induction has important application value in the study of gene expression regulation and gene function research. Sequence interpretation, where both the sgRNA and primer sequences are synthesized, and the known sequences are purchased or synthesized.

[0075] Sequence 1: AAGGCCGCACCCTTCTCCGGAGG sgRNA sequence

[0076] Sequence 2: MAAVVAATALKGRGARNARVLRGILSGATANKASQNRTRALQSH SSPECKEEPEPLSPELEYIPRKRGKNPMKAVGLAWYSLYTRTWLGYLFYRQQLRRARNRYPKGHSKTQPRLFNGVKVLPIPVLSDNYSYLIIDTQAGLAVAVDPSDPRAVQASIEKERVNLVAILCTHKHWDHSGGNRDLSRRHRDCRVYGSPQDGIPYLTHPLCHQDVVSVGRLQIRALATPGHTQGHLVYLLDGEPYKGPSCLFSGDLLFLSGCGRTFEGTAETMLSSLDTVLDLGDDTLLWPGHEYAEENLGFAGVVEPENLARERKMQWVQRQRMERKSTCPSTLGEERAYNPFLRTHCLELQEALGPGPGPTSDDGCSRAQLLEELRRLKDMHKSK

[0077] Is the known sequence, PNKD amino acid sequence (Mouse)

[0078] Sequence 3: AGTCGCTCTGAGTTGTTATCAG PNKD identification primer 1

[0079] Sequence 4: TGAGCATGTCTTTAATCTACCTCGATG, PNKD identification primer 2

[0080] Sequence 5: AGTCGCTCTGAGTTGTTATCAG, PNKD identification primer 3

[0081] Sequence 6: GTCAATGGAAAGTCCCTATTGGCGT, PNKD identification primer 4, Sequence 7:

[0082]

[0083] is a known sequence, the sftpc mRNA sequence

[0084] Sequence 8: CATCCAACATACAGACAACGC, sftpc gene identification primer 1, Sequence 9: CTATCCTAAAAGCCCAATCCTA, sftpc gene identification primer 2, Sequence 10: TGCTTCACAGGGTCGGTAG, sftpc gene identification primer 3, Sequence 11: GCCCAAATGTTGCTGGATAG, sftpc gene identification primer 4, The beneficial effects brought by the technical solution of the present invention

[0085] 1. Compared with the commonly used KrasLSL-G12D-Trp53 (LSL-R172H) mouse model, the sftpc-Cre / ERT2, KrasLSL-G12D-Trp53 (LSL-R172H) and PNKD gene knock-in mouse model obtained by the present invention greatly shortens the onset time of lung adenocarcinoma mice and increases the incidence of lung adenocarcinoma mice. The experimental period is effectively shortened.

[0086] 2. And after the onset, the lung cancer tumor mass is significantly higher than that of the commonly used KrasLSL-G12D-Trp53 (LSL-R172H) mouse lung cancer model, providing a good experimental model for the targeted treatment of advanced tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 Seven months after tamoxifen induction in mice, the results of H&E staining of the lungs of transgenic mice showed that the disease severity of transgenic mice with PNKD knock-in was aggravated.

[0088] Figure 2 Compared with cKP mice, the incidence of lung cancer in transgenic mice with PNKD knock-in (cMR1-KI KP) was significantly increased and the onset time was significantly shortened.

[0089] Figure 3 Construction of PNKD gene knock-in model mice

[0090] Figure 4 、Mating and offspring screening

[0091] Figure 5 、Cas9 / sgRNA targeting vector map Specific embodiments

[0092] The present invention will be further described below through specific examples.

[0093] The products and their preparation methods of the present invention will be further elaborated below in combination with specific examples, but the present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The raw materials can be obtained from public commercial channels unless otherwise specified.

[0094] Example 1: Construction of PNKD gene knock-in model mice

[0095] As Figure 3 shown, according to the principle of homologous recombination, through gene targeting technology, a CAG promoter, a loxp-Stop-loxp regulatory element, and an EGE-PNKD-CDS-IRES-EGFP-WPRE-pA expression element were inserted at the Rosa26 locus. Subsequently, gene knock-in mice were constructed by injecting Cas9 / sgRNA into fertilized eggs. After microinjection into mouse fertilized eggs, F0 generation mice were born. The F0 generation mice with positive tail type identification were mated with wild-type C57BL / 6 mice to obtain F1 generation with stable genotypes.

[0096] Example 2: Construction of sftpc-Cre / ERT2 mice

[0097] The sftpc-Cre / ERT2 mice, with the strain background of C57BL / 6N, were provided by Beijing Weishang Lide Biotechnology Co., Ltd. The basic principle is that Cre / ERT2 mice are a type of mice expressing a fusion protein of a mutant ligand-binding domain of estrogen receptor (ERT) and Cre recombinase. Cre / ERT2 is inactive in the cytoplasm without tamoxifen induction; when induced by tamoxifen, Cre / ERT2 can enter the nucleus to exert Cre recombinase activity. By using different promoters, Cre enzyme expression can be specifically initiated in different tissues or cells. The sftpc-Cre / ERT2 knock-in mice used in this article carry an IRES-cre sequence in the 3'-UTR region of the sftpc gene and express Cre protein in type II alveolar epithelial cells by tamoxifen induction.

[0098] Example 3: Mating and offspring screening

[0099] (1) As Figure 4As shown, sftpc-Cre mice were crossed with KrasLSL-G12D; Trp53LSL-R172H transgenic mice to obtain the required cKP mice as control mice. Then, cMR1-KI mice were crossed with cKP transgenic mice to obtain the required cMR1-KI KP mice (targeting high expression of the MR-1 gene in type II alveolar epithelial cells). When the mice were 1 week old, their tails were taken for genotyping, and wild-type mice were promptly excluded. (2) When the mice were 1 week old, partial tail tissues were extracted, lysed to obtain genomic DNA, and after PCR amplification, agarose gel electrophoresis was performed to identify the genotypes of the mice. The specific steps are as follows.

[0100] ① DNA extraction: DNA was extracted from mouse tail tissues using a commercial DNA extraction kit.

[0101] ② PCR amplification: Polymerase chain reaction (PCR) amplification was performed using specific primers (targeting the DNA sequence of the target gene) to detect the presence of specific gene mutations or transgenic sequences.

[0102] ③ Gel electrophoresis: The PCR amplification products were loaded onto an agarose gel together with molecular weight markers for electrophoretic separation. By comparing the sizes of the PCR amplification products and the migration distances of the molecular weight markers, the presence of mutations or transgenic sequences in the target gene was determined.

[0103] Example 4: Micro-CT detection of the disease onset effect: In vivo Micro-CT scans of the lungs of mice were performed at corresponding time points after tamoxifen-induced gene expression. The mice were anesthetized with pentobarbital, and then the lungs were scanned using the InSyTe FLECT / CTTM system (TriFoil Imaging, Chatsworth, CA, USA). The scanner was set to the following parameters: tube voltage 35 kV, tube current 100 μA, resolution 10.32 μm / pixel. The scanned cross-sectional images were reconstructed into three-dimensional images using TriFoil Imaging software (TriFoil Imaging, Chatsworth, CA, USA).

[0104] Example 5: WB identification of gene expression specificity:

[0105] (1) Tissue collection: At the end of the experiment, the mice were sacrificed by CO2 asphyxiation, and the heart, liver, spleen, lungs, and kidneys of the mice were dissected and placed on ice.

[0106] (2) Tissue fragmentation: RIPA cell lysis buffer (containing 1% PMSF) was added at a 5-fold volume, and a stirrer was used to break the tissues into fine fragments.

[0107] (3) Tissue lysis: Continue to lyse on ice for 60 minutes. Every 10 minutes, pipette the cells to ensure complete lysis. Subsequently, centrifuge at 12,000 rpm for 10 minutes at 4°C. The collected supernatant is the protein extract.

[0108] (3) Protein quantification: Use a BCA protein assay kit (Beyotime Biotechnology Co., Ltd.) to measure the protein concentration according to the instructions.

[0109] (4) Protein preparation: Adjust to the same concentration with distilled water according to the obtained protein sample concentration. Subsequently, aliquot into tubes at 80 μg / tube (for approximately 3 loading times), and add 5× protein loading buffer. Boil in a 100°C metal bath for 5 - 10 minutes to completely denature the protein. After cooling to room temperature, centrifuge to spin down the liquid adhering to the wall, and store at -80°C for later use.

[0110] To ensure specific overexpression of the corresponding gene in lung epithelial cells, at 12 weeks of age, mice were sacrificed by CO2 asphyxiation. The heart, liver, spleen, lungs, and kidneys of the mice were dissected, and whole protein lysates were extracted. The expression of the PNKD gene was detected by WB to ensure specific high expression in the lungs.

[0111] 1. Construction of sftpc-Cre / ERT2 mice: Ensure that the IRES-cre sequence is accurately inserted into the 3'-UTR region of the sftpc gene to achieve specific expression of Cre recombinase in lung epithelial cells.

[0112] 2. Construction of PNKD knock-in mice: Design highly active Cas9 / sgRNA sequences and construct the corresponding plasmid vectors, and then microinject them into mouse fertilized eggs to construct PNKD gene knock-in mice.

[0113] 3. Genotype identification: Use reliable RT-PCR technology to identify the genotype of the mice to confirm whether the desired transgenic mice have been successfully constructed.

[0114] 4. Mating and offspring screening: Conduct correct mating and offspring screening to ensure obtaining mice with the desired genes simultaneously.

[0115] The above are only the preferred embodiments of the present invention. It should be noted that the embodiments of the present invention are not limited by the above examples. Without departing from the principle of the present invention, several changes, modifications, substitutions, combinations, and simplifications should all be equivalent replacement methods, and these should also be regarded as the protection scope of the present invention.

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Claims

1. A method for constructing a transgenic mouse model of lung adenocarcinoma with overexpression of MR-1, characterized in that, The method includes the following steps: Step 1: Construction of PNKD gene knock-in model mice; Step 2: Obtaining of cKP mice; Step 3: Mating the cKP mice in Step 2 with the PNKD gene knock-in mice in Step 1; Step 4: Inducing genotype change with tamoxifen; Among them, for Step 1, the construction of PNKD gene knock-in model mice is as follows: 1) sgRNA design and vector construction: According to the design principle of the target site region, Cas9 / sgRNA is designed and the sgRNA is ligated into a plasmid vector with a T7 promoter for in vitro transcription. The construction of the targeting vector is confirmed by restriction enzyme digestion identification and sequencing; 2) Injection into mouse fertilized eggs: The Cas9 / sgRNA targeting vector is microinjected into mouse fertilized eggs to construct PNKD gene knock-in mice; 3) Genotype identification and mating of F0 generation mouse tails: Select mice with positive genotype identification results from F0 mice and mate them with wild-type mice to obtain F1 generation mice with stable genotypes; 4) Genotype identification and expression detection of F1 generation mice: Genotype identification is performed on F1 generation mice, and RT-PCR and Southern blot techniques can be used to detect the expression of the PNKD gene. F1 generation mice with stable genotypes are screened out and then reserved for breeding to obtain F1 generation mice overexpressing transgenic PNKD; Step 2: The obtaining of cKP mice is as follows: Mate sftpc-Cre / ERT2 mice and KrasLSL-G12D-Trp53 (LSL-R172H) mice to obtain mice containing sftpc-Cre / ERT2 and KrasLSL-G12D-Trp53 (LSL-R172H) genes, namely cKP mice; Step 3: The mating of the cKP mice in Step 2 with the PNKD gene knock-in mice in Step 1 is as follows: Mate cKP mice and PNKD gene knock-in mice to obtain mice simultaneously having sftpc-Cre / ERT2, KrasLSL-G12D-Trp53 (LSL-R172H) and PNKD genes; Step 4: The inducing of genotype change with tamoxifen is as follows: Intraperitoneally inject tamoxifen into the mice obtained in Step 3 for 5 consecutive days, once a day.

2. The method according to claim 1, wherein The sgRNA sequence used in Step 1 is: 5’-AAGGCCGCACCCTTCTCCGGAGG-3’ During the construction process, genotype identification can be performed on F1 generation mice, and the identification primers used are as follows: 。 3. The method according to claim 1, wherein Step 2: During the construction process, genotype identification of sftpc-Cre / ERT2 gene expression can be performed, and the primer sequences used are as follows: 。 4. The method according to claim 1, wherein During the construction process, gene detection steps can be performed on the mice obtained in Step 4.

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