Spontaneous early lung cancer mouse model and its construction method and application

Abstract

The present invention discloses a spontaneous early lung cancer mouse model, a construction method and an application thereof, and belongs to the technical field of animal model construction. The present invention hybridizes mice that conditionally overexpress GNGT1 with Scgb1a1‑Cre tool mice that specifically target the lungs, and the F1 generation contains the spontaneous early lung cancer mouse model. The early lung cancer mouse model of the present invention that specifically overexpresses GNGT1 in the lungs can successfully construct an early lung cancer model in a relatively short period of time, which can greatly accelerate the research progress of scientific researchers, save time and costs, and expand the range of available models for lung tumor research. The spontaneous early lung cancer mouse model provided by the present invention has high repeatability and good controllability, and is an important bridge to promote the progress of lung cancer research and ultimately its clinical transformation.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal model construction, and particularly relates to a spontaneous early lung cancer mouse model and a construction method and application thereof. Background Art

[0002] Lung cancer is one of the most malignant tumors, with high morbidity and mortality rates worldwide, severely endangering life and health. With a five-year survival rate of only 15%, early detection and treatment are crucial for improving survival in lung cancer patients. Lung cancer develops through a complex process, progressing from early atypical hyperplasia to carcinoma in situ and finally to invasive cancer, but the mechanisms underlying its development remain largely unknown. Therefore, in-depth understanding of the pathological mechanisms of lung cancer development is crucial for preventing and controlling its malignant progression and improving survival in lung cancer patients.

[0003] The GPCR superfamily, composed of G proteins and their ligands, can activate multiple secondary signaling pathways, including PI3K, MAPK, and ion channels, and participate in regulating a range of cellular biological behaviors. They play a crucial role in the development of various tumors. Aberrant expression and activation of GPCRs and their downstream signaling pathways have been shown to be important hallmarks of tumor malignancy. In recent years, numerous studies have explored models of spontaneous lung tumors, but most have focused on invasive carcinomas. Adenovirus-induced conditional mutation of Kras G12D in the lung can induce atypical adenomatous hyperplasia (ADH) in early lung cancer, but this is often accompanied by papillary adenomas. However, papillary adenomas rarely coexist with human tumors, and the formation and progression of tumors are influenced by the amount of adenovirus. Therefore, developing an animal model that can spontaneously develop early lung cancer within a short period of time is crucial for further understanding the mechanisms by which lung cancer progresses from early atypical hyperplasia to invasive carcinoma, thereby identifying biomarkers for early diagnosis and potentially important therapeutic targets. Therefore, a method for short-term generation of mice with early lung cancer is urgently needed to facilitate research into the mechanisms of lung cancer development and progression, as well as drug development targets. Summary of the Invention

[0004] The purpose of the present invention is to provide a spontaneous early lung cancer mouse model and its construction method and application. The spontaneous early lung cancer mouse model can induce the occurrence of early lung cancer in a short period of time, greatly expanding the breadth of models available for lung tumor research and saving time and cost of scientific research work.

[0005] The present invention provides a method for constructing a spontaneous early lung cancer mouse model, comprising the following steps: hybridizing mice that conditionally overexpress GNGT1 with Scgb1a1-Cre tool mice with lung-specific targeting, wherein the F1 generation contains the spontaneous early lung cancer mouse model.

[0006] Preferably, the method for constructing the conditionally overexpressing GNGT1 mouse comprises inserting an expression frame comprising the overexpressed GNGT1 gene into the Rosa26 gene site of chromosome 6 of C57BL / 6J mice to obtain the conditionally overexpressing GNGT1 mouse.

[0007] Preferably, the structure of the expression cassette includes CAG-loxp-stop-loxp-Gngt1-WPRE-pA.

[0008] Preferably, the method for constructing a lung-specifically targeted Scgb1a1-Cre tool mouse comprises site-directed knock-in of an IRES-Cre expression frame after the stop codon site of the Scgb1a1 gene.

[0009] Preferably, the hybridization includes using mice that conditionally overexpress GNGT1 as the female parent and using Scgb1a1-Cre tool mice that specifically target the lung as the male parent.

[0010] Preferably, after the hybridization, the method further comprises screening the F1 generation after the hybridization to screen mice with double heterozygous genotypes of LSL-GNGT1 and Scgb1a1-IRES-Cre;

[0011] The LSL-GNGT1 hybrid comprises a primer pair as shown in SEQ ID No. 10 and SEQ ID No. 11, and a primer pair as shown in SEQ ID No. 12 and SEQ ID No. 13, which can amplify bands;

[0012] The Scgb1a1-IRES-Cre hybrid can amplify bands using the primer pair shown in nucleotide sequences such as SEQ ID No.18 and SEQ ID No.19, and the primer pair shown in SEQ ID No.20 and SEQ ID No.21.

[0013] Preferably, the spontaneous early lung cancer mouse model takes less than 30 days to develop atypical hyperplasia of the lung epithelium.

[0014] The present invention also provides use of the spontaneous early lung cancer mouse model constructed using the above construction method in any of the following applications:

[0015] (1) Study the occurrence and development of lung cancer;

[0016] (2) Study the pathogenesis of lung cancer;

[0017] (3) Preparation of early detection and / or diagnosis tools for lung cancer;

[0018] (4) Develop drugs for lung cancer;

[0019] (5) Screening of drugs that are effective against lung cancer.

[0020] Beneficial effects: The present invention provides a method for constructing a spontaneous early lung cancer mouse model, comprising the following steps: hybridizing mice that conditionally overexpress GNGT1 with Scgb1a1-Cre tool mice that specifically target the lungs, and the F1 generation contains the spontaneous early lung cancer mouse model. Experiments have shown that the early lung cancer mouse model of the present invention that specifically overexpresses GNGT1 in the lungs can successfully construct an early lung cancer model in a relatively short period of time, which can greatly accelerate the research progress of scientific researchers, save time and costs, and expand the range of available models for lung tumor research. The spontaneous early lung cancer mouse model provided by the present invention has high repeatability and good controllability, and is an important bridge to promote the progress of lung cancer research and ultimately its clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the gene editing region in C57BL / 6J-LSL-GNGT1 conditional overexpression mice;

[0022] Wild type allele: wild type allele; gRNAregion: guide RNA (

[0023] gRNA) cleavage region; targeting vector: target vector; targeted allele: targeted allele; targeted allele (Stop removed): overexpressed allele after recombination (Cre recombination);

[0024] Figure 2 Schematic diagram of the in vitro transcription vector in Example 1; R26-LSL-GNGT1 DonorVector (GNGT1).Seq: R26-LSL-GNGT1 knock-in vector sequence; bp: base; 5'arm: 5' long terminal repeat; 3'arm: 3' long terminal repeat; Bgh-polyA: mammalian stop codon Bgh polyadenylation tail; cRosa-5t-r and cRosa-3t-f: non-coding gene safety sites consisting of three exons on mouse chromosome 6; WPRE: woodchuck hepatitis virus post-transcriptional regulatory element; Xhol, BgIII: restriction endonuclease cleavage sites; kozak: 3' kozak sequence; CAG-enhancer: CAG enhancer region; loxP: loxP insertion site;

[0025] Figures 3-4Schematic diagram of the tail identification strategy and results for F0 generation mice in Example 1; Wildtype allele: wild-type allele; targeted type allele: targeted allele; primer location: primer location; PCR product: PCR product; PCR sequenced region: PCR sequenced region; STOP: terminator; WPRE-polyA: woodchuck hepatitis virus post-transcriptional regulatory element-polylysine tail, exon: exon;

[0026] Figures 5-6 Schematic diagram of the tail identification strategy and results of F1 generation mice in Example 1;

[0027] Figures 7-8 This is a schematic diagram of the gene identification strategy and results of the F2 generation mice in Example 1;

[0028] Figure 9 Schematic diagram of the gene editing region in the Scgb1a1-e(IRES-Cre)1 lung-specific targeting tool mouse; Wild type allele: wild-type allele; targeting vector: targeting vector; targeted allele: targeted allele; 3'UTR: 3' untranslated region; homology arm: homologous recombination arm, coding region: coding region; uncoding region: untranslated region;

[0029] Figure 10 Schematic diagram of the in vitro transcription vector in Example 2; Amp: ampicillin resistance region; 5'arm: 5' long terminal repeat sequence; 3'arm: 3' long terminal repeat sequence; EcoRV: EcoRV restriction endonuclease; Scgb1a1-e(IRES-Cre)1DonorVector sequence: Scgb1a1-e(IRES-Cre)1 knock-in vector sequence;

[0030] Figures 11-12 Schematic diagram of the tail identification strategy and results of F0 generation mice in Example 2;

[0031] Figure 13 This is a schematic diagram of the tail identification results of F1 generation mice in Example 2;

[0032] Figures 14-15 Schematic diagram of the gene identification strategy and results of F2 generation mice in Example 2;

[0033] Figures 16-17 This is a schematic diagram of the mouse genotype identification results in Example 3;

[0034] Figure 18 Schematic diagram of the tissue of the early lung cancer mouse model obtained in Example 3;

[0035] Figure 19 Schematic diagram of the tissue of the early lung cancer mouse model obtained in Example 3. DETAILED DESCRIPTION

[0036] The present invention provides a method for constructing a spontaneous early lung cancer mouse model, comprising the following steps: hybridizing mice that conditionally overexpress GNGT1 with Scgb1a1-Cre tool mice with lung-specific targeting, wherein the F1 generation contains the spontaneous early lung cancer mouse model.

[0037] The method for constructing a mouse that conditionally overexpresses GNGT1 according to the present invention preferably includes inserting an expression cassette containing the overexpressed GNGT1 gene into the Rosa26 gene site of chromosome 6 of the C57BL / 6J mouse to obtain the mouse that conditionally overexpresses GNGT1. The structure of the expression cassette according to the present invention includes CAG-loxp-stop-loxp-Gngt1-WPRE-pA, and the nucleotide sequence of Gngt1 in the expression cassette is preferably as shown in NM_010314.3 in the NCBI database. The mouse C57BL / 6J-GNGT1 conditional overexpression mouse (LSL-GNGT1 for short) that conditionally overexpresses GNGT1 is constructed using the method. The present invention does not specifically limit the source of the C57BL / 6J strain of mice, and it can be purchased conventionally. The LSL-GNGT1 described in the present invention is preferably purchased from Shanghai Southern Model Organisms Technology Co., Ltd., project number N1-6336, and the specific design strategy is as follows. Figure 1 shown.

[0038] The method for constructing the lung-specific targeting Scgb1a1-Cre tool mouse of the present invention preferably includes site-directed knock-in of the IRES-Cre expression cassette after the stop codon site of the Scgb1a1 gene. The lung-specific targeting Scgb1a1-Cre tool mouse (Scgb1a1-IRES-Cre) of the present invention is preferably purchased from Shanghai Model Organisms Science Co., Ltd. under the product number NM-KI-210120. The specific design strategy is as follows: Figure 9 shown.

[0039] The hybridization of the present invention preferably includes using a mouse that conditionally overexpresses GNGT1 as the female parent and a mouse that specifically targets the lung Scgb1a1-Cre as the male parent. After the hybridization, the present invention preferably further includes screening the F1 offspring to screen for mice with a double heterozygous genotype of LSL-GNGT1 and Scgb1a1-IRES-Cre; the LSL-GNGT1 heterozygosity includes using the primer pair of nucleotide sequences shown in SEQ ID No. 10 and SEQ ID No. 11, and the primer pair of SEQ ID No. 12 and SEQ ID No. 13 to amplify bands; (P1:P2)

[0040] GNGT1 forward primer P1 (SEQ ID No. 10): 5′-TCAGATTCTTTTATAGGGGACACA-3′;

[0041] GNGT1 reverse primer P2 (SEQ ID No. 11): 5′-TAAAGGCCACTCAATGCTCACTAA-3′;

[0042] GNGT1 forward primer P3 (SEQ ID No. 12): 5′-AGGAACTCAAAGGAGGCTGTG-3′;

[0043] GNGT1 reverse primer P4 (SEQ ID No. 13): 5'-AAGGAAGGTCCGCTGGATTG-3'. When the present invention uses the two pairs of primers (P1 and P2, P3 and P4) of GNGT1 for amplification, the genotype interpretation method is preferably:

[0044] Wild type: only (P1, P2) amplified a 967 bp band, (P3, P4) had no band;

[0045] Heterozygotes: (P1, P2) amplified a 967bp band, (P3, P4) also amplified a 568bp band;

[0046] Homozygotes: (P1, P2) have no band, (P3, P4) can amplify a band of 568bp.

[0047] The Scgb1a1-IRES-Cre hybrid of the present invention preferably includes primers with nucleotide sequences such as SEQ ID No. 18 and SEQ ID No. 19, and primers with SEQ ID No. 20 and SEQ ID No. 21 that can amplify bands.

[0048] IRES-Cre forward primer P1 (SEQ ID No. 18): 5′-GGTCTTCAGTCCCCTTCAGC-3′;

[0049] IRES-Cre reverse primer P2 (SEQ ID No. 19): 5′-ACTCACTCCAGCTCCAATGC-3′;

[0050] IRES-Cre forward primer P3 (SEQ ID No. 20): 5′-GGTCTTCAGTCCCCTTCAGC-3′;

[0051] IRES-Cre reverse primer P4 (SEQ ID No. 21): 5'-AGACCCCTAGGAATGCTCGT-3'. When the present invention uses the above two pairs of primers (IRES-Cre P1 and IRES-Cre P2, IRES-Cre P3 and IRES-Cre P4) for amplification verification, the genotype determination method preferably includes:

[0052] Wild type: only (P1, P2) amplified a 672 bp band, (P3, P4) had no band;

[0053] Heterozygotes: (P1, P2) amplified a 672 bp band, (P3, P4) also amplified a 647 bp band;

[0054] Homozygotes: (P1, P2) have no band, (P3, P4) can amplify a band of 647bp.

[0055] The spontaneous early lung cancer mouse model of the present invention takes less than 30 days to produce atypical hyperplasia of the lung epithelium.

[0056] The present invention also provides a spontaneous early lung cancer mouse model constructed using the above construction method, wherein the median onset time of the spontaneous early lung cancer mouse model is less than 30 days. The spontaneous early lung cancer of the present invention preferably includes early squamous cell carcinoma of the lung combined with early adenocarcinoma.

[0057] The present invention also provides use of the spontaneous early lung cancer mouse model constructed using the above construction method in any of the following applications:

[0058] (1) Study the occurrence and development of lung cancer;

[0059] (2) Study the pathogenesis of lung cancer;

[0060] (3) Preparation of early detection and / or diagnosis tools for lung cancer;

[0061] (4) Develop drugs for lung cancer;

[0062] (5) Screening of drugs that are effective against lung cancer.

[0063] To further illustrate the present invention, the spontaneous early lung cancer mouse model provided by the present invention, its construction method and application are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0064] Example 1

[0065] 1. Construction of mice with conditional GNGT1 overexpression:

[0066] C57BL / 6J-GNGT1 conditional overexpression mice (LSL-GNGT1) were purchased from Shanghai Model Organisms Technology Co., Ltd. The specific design strategy is as follows Figure 1 The construction process provided by the company includes:

[0067] Cas9 mRNA and gRNA (specific sequence: 5'-GGGGACACACTAAGGGAGCTTGG-3', SEQ ID NO. 1) were obtained by in vitro transcription. A homologous recombination vector (donor vector) was constructed by in-fusion cloning. The vector contains a 3.3 kb 5' homology arm, CAG-loxp-stop-loxp-Gngt1-WPRE-pA, and a 3.3 kb 3' homology arm. Except for the sequence of the target gene, the structures and sequences of the remaining parts of the vector were obtained from Southern Model Organisms Company and are consistent with the sequences used in other materials sold by the company. Specific reference: Liu Y, Xun Z, Ma K, et al. Identification of a tumor immune barrier in the HCC microenvironment that determines the efficacy of immunotherapy.JHepatol.2023;78(4):770-782.doi:10.1016 / j.jhep.2023.01.011 such as Figure 2 shown.

[0068] Cas9 mRNA, gRNA and donor vector were microinjected into fertilized eggs of C57BL / 6J mice to obtain F0 generation mice. PCR amplification and sequencing were performed to identify the positive F0 generation mice. PCR identification scheme for homologous recombination positive mice: 5' arm homologous recombination positive genome and 3' arm homologous recombination positive genome amplification method for identification. Figure 3 shown.

[0069] The PCR amplification system was as follows: DNA template 1 μL, GNGT1 forward primer 0.3 μL, GNGT1 reverse primer 0.3 μL, KOD-Multi&Epi (TOYOBO, Code No: KME-101) 0.35 μL, 2× PCR Buffer 10 μL, and ddH O added to 20 μL;

[0070] The nucleotide sequence of the GNGT1 forward primer to be amplified in the 5' arm homologous recombination positive genome is shown in SEQ ID NO. 2, specifically: 5'-GGCGGGAGGTAGGTGGGGTGAGG-3';

[0071] The 5' arm homologous recombination positive genome should be amplified GNGT1. The nucleotide sequence of the GNGT1 reverse primer is shown in SEQ ID NO. 3, specifically: 5'-TGAGGGCAATCTGGGAAGGTT-3';

[0072] The nucleotide sequence of the GNGT1 forward primer to be amplified in the 3' arm homologous recombination positive genome is shown in SEQ ID NO. 4, specifically: 5'-GGGGGAGGGGAGTGTTGC-3';

[0073] The nucleotide sequence of the GNGT1 reverse primer for the 3' arm homologous recombination positive genome to be amplified is shown in SEQ ID NO. 5, specifically: 5'-TTCTTCCTGCCTGCCTTCTGTGAC-3;

[0074] The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 98°C for 20 s, annealing at 61°C for 30 s, and extension at 68°C for 3 min, for 35 cycles; holding at 68°C for 5 min, and then storing at 12°C for detection.

[0075] The expected results are: a 3.6kb fragment should be amplified from the 5' arm homologous recombination positive genome and a 5.2kb fragment should be amplified from the negative genome; a 3.6kb fragment should be amplified from the 3' arm homologous recombination positive genome and a 6.5kb fragment should be amplified from the negative genome. Figure 4 shown.

[0076] According to the above PCR test, the F0 generation mice with GNGT1 gene knocked in were obtained. Since the cleavage rate of the fertilized egg is very fast in the early stage, the F0 generation mice obtained are chimeras and may not have the ability of stable inheritance. They need to be passaged to obtain F1 generation mice with stable inheritance.

[0077] 2. Obtaining F1 generation positive mice:

[0078] Take the F0 generation female positive mice obtained in step 1 and mate with wild-type C57BL / 6J male mice to breed F1 generation mice. Genotype identification is carried out by PCR identification and sequencing. PCR identification scheme for F1 generation positive mice: double-arm homologous recombination positive identification, i.e., identification by 5' arm homologous recombination positive genome amplification and 3' arm homologous recombination positive genome amplification. Figure 5 shown.

[0079] The PCR amplification system was as follows: DNA template 1 μL, GNGT1 forward primer 0.3 μL, GNGT1 reverse primer 0.3 μL, KOD-Multi&Epi (TOYOBO, Code No: KME-101) 0.35 μL, 2xPCR Buffer 10 μL, and ddH O added to 20 μL;

[0080] The nucleotide sequence of the GNGT1 forward primer to be amplified in the 5' arm homologous recombination positive genome is shown in SEQ ID NO. 6, specifically: 5'-TTATGGAGGGGAGGACTGGG-3';

[0081] The 5' arm homologous recombination positive genome should be amplified GNGT1. The nucleotide sequence of the GNGT1 reverse primer is shown in SEQ ID NO. 7, specifically: 5'-TGAGGGCAATCTGGGAAGGTT-3';

[0082] The nucleotide sequence of the GNGT1 forward primer to be amplified in the 3' arm homologous recombination positive genome is shown in SEQ ID NO.8, specifically: 5'-GGGGGAGGGGAGTGTTGC-3';

[0083] The nucleotide sequence of the GNGT1 reverse primer for the 3' arm homologous recombination positive genome to be amplified is shown in SEQ ID NO. 9, specifically: 5'-TTCTTCCTGCCTGCCTTCTGTGAC-3';

[0084] The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 98°C for 20 s, annealing at 61°C for 20 s, and extension at 68°C for 3 min, for 35 cycles; holding at 68°C for 5 min, and then storing at 12°C for detection.

[0085] The expected results are: a 5.0 kb fragment should be amplified from the 5' arm homologous recombination positive genome and a 6.6 kb fragment should be amplified from the negative genome; a 3.6 kb fragment should be amplified from the 3' arm homologous recombination positive genome and a 6.5 kb fragment should be amplified from the negative genome. Figure 6 In the subsequent mouse mating and breeding process, the mouse genotype can be identified by short-fragment PCR.

[0086] The method for identifying mouse genotypes by short-fragment PCR is as follows: mice are numbered and their tails are cut 14 days after birth. The tails are required to be 0.5 cm in length and can be stored in a -20°C refrigerator for future use.

[0087] Reagents:

[0088] Rat tail lysis buffer: 10 ml of 1 M Tris HCL (pH = 8.0), 4 ml of 0.5 M EDTA, 400 ml of 1 M NaCl, 100 ml of 10% SDS, and 486 ml of ultrapure water.

[0089] Proteinase K solution (PK): 20 mg / ml, store at -20°C;

[0090] TE buffer: 1 M Tris HCL (PH=8.0) 5 ml, 0.5 M EDTA 1 ml, ultrapure water 494 ml.

[0091] Experimental process:

[0092] Add 300 μl of lysis buffer and 5 μl of PK to each rat tail sample and digest overnight at 55°C. After overnight digestion, add 600 μl of -20°C pre-cooled anhydrous ethanol to each digested sample and invert until a flocculent precipitate is clearly visible. Stop inverting and centrifuge at 12,000 rpm for 5 minutes.

[0093] After centrifugation, pour out the upper liquid and place the centrifuge tube vertically on a piece of flat paper. After 10 minutes, change the upright tube to another position, press down lightly, and wait until it is completely dry. At this time, the precipitate becomes transparent and the extracted DNA is of good quality.

[0094] Add 200 μl of TE buffer to the centrifuge tube and shake up and down to fully dissolve the DNA to obtain a DNA template; take 3 μl of the DNA template and perform PCR in a 25 μl reaction system.

[0095] The PCR amplification system was as follows: 3 μL DNA template, 0.5 μL forward primer F, 0.5 μL reverse primer R, 12.5 μL Taq enzyme and premix, and ddH₂O to 25 μL. GNGT1 gene identification was performed using two primer pairs, each of which was amplified separately. The primer pairs used are as follows:

[0096] The nucleotide sequence of the GNGT1 forward primer P1 is shown in SEQ ID No. 10, specifically: 5′-TCAGATTCTTTTATAGGGGACACA-3′;

[0097] The nucleotide sequence of the GNGT1 reverse primer P2 is shown in SEQ ID No. 11, specifically: 5′-TAAAGGCCACTCAATGCTCACTAA-3′;

[0098] The nucleotide sequence of the GNGT1 forward primer P3 is shown in SEQ ID No. 12, specifically: 5′-AGGAACTCAAAGGAGGCTGTG-3′;

[0099] The nucleotide sequence of the GNGT1 reverse primer P4 is shown in SEQ ID No. 13, specifically: 5'-AAGGAAGGTCCGCTGGATTG-3'. Figure 7 shown.

[0100] The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min; holding at 72°C for 5 min, and then storing at 12°C until detection;

[0101] Electrophoresis: Prepare a 1% agarose gel according to the target band, dissolve 1g agarose in 100ml TAE buffer, heat in a microwave until the agarose melts, add 10μl nucleic acid dye (EB substitute), pour into the electrophoresis template, insert the comb, and wait for condensation before use; add an appropriate amount of DNA to the agarose gel, add 10μl to the small well and 20μl to the large well, and select an appropriate DNA marker according to the size of the target band and add it to the gel. After running the gel at 120V voltage, use a gel imaging system to observe and take pictures to obtain a genotype identification result report, such as Figure 8 The specific genotype interpretation method is as follows:

[0102] Wild type: only (P1, P2) amplified a 967 bp band, (P3, P4) had no band;

[0103] Heterozygotes: (P1, P2) amplified a 967bp band, (P3, P4) also amplified a 568bp band;

[0104] Homozygotes: (P1, P2) have no band, (P3, P4) can amplify a band of 568bp.

[0105] Example 2

[0106] 1. Construction of lung-specifically targeted Scgb1a1-Cre mice

[0107] The lung-specific targeting Scgb1a1-Cre tool mouse (Scgb1a1-IRES-Cre) was purchased from Shanghai Model Organisms Technology Co., Ltd. under the catalog number NM-KI-210120. The specific design strategy is as follows: Figure 9 The construction process provided by the company includes: obtaining Cas9 mRNA and gRNA by in vitro transcription; constructing a homologous recombination vector (donor vector) by the In-Fusion cloning method, which contains a 3.0kb 5' homology arm, IRES-Cre and a 3.0kb 3' homology arm. Figure 10 shown.

[0108] Cas9 mRNA, gRNA and donor vector were microinjected into fertilized eggs of C57BL / 6J mice to obtain F0 generation mice. PCR identification scheme for homologous recombination positive mice: double-arm homologous recombination positive identification, that is, identification by amplification of 5' arm homologous recombination positive genome and 3' arm homologous recombination positive genome. Figure 11 shown.

[0109] The PCR amplification system was as follows: DNA template 1 μL, forward primer 0.5 μL, reverse primer 0.5 μL, 2.5 mM dNTP 2 μL, GXL DNA Polymerase 0.8 μL, GXL PCR Buffer 2 μL, and ddH2O was added to 20 μL.

[0110] The nucleotide sequence of the 5'-arm homologous recombination positive genome amplification IRES-Cre forward primer is shown in SEQ ID NO. 14, specifically: 5'-TAAGATGCTAAGAGATTTTGATACAGTCAC-3';

[0111] The nucleotide sequence of the 5'-arm homologous recombination positive genome amplification IRES-Cre reverse primer is shown in SEQ ID NO. 15, specifically: 5'-ACGGCAATATGGTGGAAAATAACATATAGA-3';

[0112] The nucleotide sequence of the 3'-arm homologous recombination positive genome amplification IRES-Cre forward primer is shown in SEQ ID NO. 16, specifically: 5'-ACAACCATGGTGCCCAAGAA-3';

[0113] The nucleotide sequence of the 3'-arm homologous recombination positive genome amplification IRES-Cre reverse primer is shown in SEQ ID NO. 17, specifically: 5'-TCCCCAGGTTCCTCAGAGTT-3';

[0114] The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 98°C for 15 s, annealing at 63°C for 15 s, and extension at 68°C for 3 min, for 35 cycles; holding at 68°C for 5 min, and then storing at 12°C for detection.

[0115] The expected results are: a 3.9kb fragment should be amplified from the 5' arm homologous recombination positive genome, and no fragment should be amplified from the negative genome; a 4.4kb fragment should be amplified from the 3' arm homologous recombination positive genome, and no fragment should be amplified from the negative genome. Figure 12 shown.

[0116] According to the above PCR detection, F0 generation mice with IRES-Cre gene knock-in were obtained, and the mice were passaged to obtain F1 generation mice with stable inheritance.

[0117] 2. Mating the F0 generation IRES-Cre female mice obtained in step 1 with wild-type C57BL / 6J male mice to obtain F1 generation mice. Genotypes were then confirmed by PCR and sequencing. The PCR identification protocol and implementation process for F1 generation mice were the same as for F0 generation mice.

[0118] The expected results are: a 3.9kb fragment should be amplified from the 5' arm homologous recombination positive genome, and no fragment should be amplified from the negative genome; a 4.4kb fragment should be amplified from the 3' arm homologous recombination positive genome, and no fragment should be amplified from the negative genome. Figure 13 shown.

[0119] The method for subsequent tail identification of F2 generation mice is the same as that in Example 1.

[0120] The PCR amplification system was as follows: 3 μL DNA template, 0.5 μL forward primer F, 0.5 μL reverse primer R, 12.5 μL Taq enzyme and reaction premix, and ddH2O to 25 μL. IRES-Cre gene identification was performed using two pairs of primers for amplification. The primer pairs used are as follows:

[0121] The nucleotide sequence of the IRES-Cre forward primer P1 is shown in SEQ ID No. 18, specifically: 5′-GGTCTTCAGTCCCCTTCAGC-3′;

[0122] The nucleotide sequence of the IRES-Cre reverse primer P2 is shown in SEQ ID No. 19, specifically: 5′-ACTCACTCCAGCTCCAATGC-3′;

[0123] The nucleotide sequence of the IRES-Cre forward primer P3 is shown in SEQ ID No. 20, specifically: 5′-GGTCTTCAGTCCCCTTCAGC-3′;

[0124] The nucleotide sequence of the IRES-Cre reverse primer P4 is shown in SEQ ID No. 21, specifically: 5'-AGACCCCTAGGAATGCTCGT-3'. Figure 14 shown.

[0125] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min, for 35 cycles; holding at 72°C for 5 min, and then storing at 12°C until detection;

[0126] Electrophoresis: Prepare a 1% agarose gel according to the target band, dissolve 1g agarose in 100ml TAE buffer, heat in a microwave until the agarose melts, add 10μl nucleic acid dye (EB substitute), pour into the electrophoresis template, insert the comb, and wait for condensation before use; add an appropriate amount of DNA to the agarose gel, add 10μl to the small well and 20μl to the large well, and select an appropriate DNA marker according to the size of the target band and add it to the gel. After running the gel at 120V voltage, use a gel imaging system to observe and take pictures to obtain a genotype identification result report, such as Figure 15 The specific genotype interpretation method is as follows:

[0127] Wild type: only (P1, P2) amplified a 672 bp band, (P3, P4) had no band;

[0128] Heterozygotes: (P1, P2) amplified a 672 bp band, (P3, P4) also amplified a 647 bp band;

[0129] Homozygotes: (P1, P2) have no band, (P3, P4) can amplify a band of 647bp.

[0130] Example 3

[0131] Two LSL-GNGT1 female mice obtained in Example 1 were mated with Scgb1a1-IRES-Cre homozygous male mice obtained in Example 2. Thirteen F1 pups were obtained after 3 weeks of mating. The tails of the pups were identified according to the method in Example 1. The results were as follows: Figures 16-17 shown.

[0132] Depend on Figures 16-17The results showed that four mice were double heterozygous for LSL-GNGT1 and Scgb1a1-IRES-Cre, with a positive rate of 30.77% and a theoretical positive rate of 50.00%. Nine of the 13 F1 mice were heterozygous for Scgb1a1-IRES-Cre. The double heterozygous LSL-GNGT1 and Scgb1a1-IRES-Cre mice are the spontaneous early-stage lung cancer mouse model described in this invention, also known as GNGT1-CRE mice.

[0133] Example 4

[0134] Ten LSL-GNGT1; Scgb1a1-IRES-Cre double heterozygous genotype mice were constructed using the methods of Examples 1 to 3. After the genotype was confirmed by tail identification, the mice were routinely raised in an SPF animal room and observed once a week within 4 weeks of age. The health status of the mice was observed every two days after 4 weeks of age. The mice were sacrificed at 4 weeks of age, 8 weeks of age, 12 weeks of age, and 16 weeks of age, respectively. The lung tissues were preserved in formalin and subsequently paraffin-embedded and sectioned for HE staining or immunohistochemistry experiments (such as Figure 18 Shown are histological light microscopic images of early lung cancer lesions in 4-week-old mice, with early alveolar epithelial lesions on the left and early bronchial epithelial lesions on the right).

[0135] Depend on Figure 18 The mouse model constructed in this invention can spontaneously and rapidly develop early-stage lung tumors. These lung tumors exhibit early lesions in both the alveolar and bronchial epithelium. These early lesions represent the two most common pathological types of lung cancer encountered in clinical practice: lung adenocarcinoma and squamous cell carcinoma. The pathological characteristics of this mouse model closely match these characteristics, making it clinically representative and suitable for laboratory research. Furthermore, this model is a world first and has not been previously reported.

[0136] Comparative Example 1

[0137] The conditionally overexpressed LSL-GNGT1 heterozygous mice constructed in Example 1 and the Scgb1a1-Cre heterozygous mice constructed in Example 2 were genotyped accurately by the tail identification method in Example 1 using wild-type mice. They were then routinely raised in an SPF animal room and observed once a week within 4 weeks of age. The health status of the mice was observed every two days after 4 weeks of age. The mice were sacrificed at 4 weeks of age, 8 weeks of age, 12 weeks of age, and 16 weeks of age, respectively. The lung tissues were preserved in formalin and subsequently paraffin-embedded and sectioned for HE staining or immunohistochemistry experiments to determine whether early lung cancer changes occurred. None of the mice showed morphological characteristics of early lung cancer, such as Figure 19 shown.

[0138] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for constructing a spontaneous early lung cancer mouse model, characterized in that: The following steps are involved: Mice that conditionally overexpress GNGT1 were crossed with lung-specifically targeted Scgb1a1-Cre mice, and the F1 generation contained the spontaneous early lung cancer mouse model. After the hybridization, the method further includes screening the F1 generation after the hybridization to screen mice with double heterozygous genotypes of LSL-GNGT1 and Scgb1a1-IRES-Cre; The LSL-GNGT1 hybrid comprises a primer pair as shown in SEQ ID No. 10 and SEQ ID No. 11, and a primer pair as shown in SEQ ID No. 12 and SEQ ID No. 13, which can amplify bands; The Scgb1a1-IRES-Cre hybrid can amplify bands using the primer pair shown in nucleotide sequences such as SEQ ID No.18 and SEQ ID No.19, and the primer pair shown in SEQ ID No.20 and SEQ ID No.

21.

2. The construction method according to claim 1, characterized in that: The method for constructing the conditionally overexpressing GNGT1 mouse comprises inserting an expression frame containing the overexpressed GNGT1 gene into the Rosa26 gene site of chromosome 6 of the C57BL / 6J mouse to obtain the conditionally overexpressing GNGT1 mouse.

3. The construction method according to claim 2, characterized in that: The structure of the expression cassette includes CAG-loxp-stop-loxp-Gngt1-WPRE-pA.

4. The construction method according to claim 1, characterized in that: The method for constructing a lung-specifically targeted Scgb1a1-Cre tool mouse includes site-specifically knocking in an IRES-Cre expression frame after the stop codon site of the Scgb1a1 gene.

5. The construction method according to claim 1, characterized in that: The hybridization includes using mice that conditionally overexpress GNGT1 as the female parent and using Scgb1a1-Cre tool mice that specifically target the lungs as the male parent.

6. The construction method according to claim 1, characterized in that: The spontaneous early lung cancer mouse model takes less than 30 days to produce atypical hyperplasia of the lung epithelium.

7. Use of the spontaneous early lung cancer mouse model constructed by the construction method according to any one of claims 1 to 6 in any of the following applications; (1) Study the occurrence and development of lung cancer; (2) Study the pathogenesis of lung cancer; (3) Develop early diagnosis tools for lung cancer; (4) Develop drugs for lung cancer; (5) Screening for drugs that are effective against lung cancer.

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