Construction method and application of mouse model based on reverse insertion of HPV16 gene

Abstract

The invention discloses a construction method and application of a mouse model based on reverse insertion of an HPV16 gene, and belongs to the technical field of animal models. According to the method, a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 mediated genome editing technology is utilized, and directional knock-in of an HPV16 sequence is realized at a Klf5 gene locus of a C57BL / 6J mouse. The method comprises the following steps: (1) designing and synthesizing specific gRNA; (2) constructing a targeting carrier Donor vector; and (3) micro-injection: mixing Cas9 protein, gRNA and a targeting carrier Donor vector to prepare an RNP injection compound, micro-injecting the RNP injection compound into a mouse fertilized egg, and sending the RNP injection compound back to a pregnant mouse fallopian tube to cultivate offspring so as to generate a gene knock-in mouse. The animal model can simulate a cervical cancer precancerous lesion process caused by HPV persistent infection, and provides a reliable experimental basis for cervical cancer occurrence mechanism research and anti-HPV drug screening.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal models, and in particular relates to a method for constructing a mouse model based on reverse insertion of HPV16 gene and its application. Background Art

[0002] Cervical cancer is the most common malignant tumor in the female reproductive system, and its disease burden is particularly prominent in my country. There are more than 600,000 new cases of cervical cancer each year worldwide, of which about a quarter of new cases and deaths occur in China. The occurrence and development of cervical cancer is a complex process involving multiple stages and multi-gene mutations. There is a close and complex interaction between the host's genetic characteristics and exogenous high-risk human papillomavirus (hrHPV). Studies have shown that persistent infection with hrHPV is a direct factor in the occurrence of cervical cancer. In this process, HPV DNA fragments are non-randomly integrated into the host genome, triggering a series of molecular events, including loss of tumor suppressor gene function, increased genomic instability and cell immortalization, which are one of the key mechanisms of cervical cancer.

[0003] The evolution of cervical lesions is usually slow, and it may take decades from a normal cervix to persistent HPV infection, and then to precancerous lesions and cervical cancer. However, in this process, early HPV infection and precancerous lesions often have no obvious clinical symptoms. This feature makes it extremely difficult to obtain continuous samples from normal cervix to cancer development in clinical practice, which poses a major challenge to studying key molecular events in the early stages of cervical cancer and developing new treatments. Therefore, in order to reveal the relationship between hrHPV integration and cervical cancer occurrence, establishing cell and animal models has become a key means.

[0004] At present, the research models related to cervical lesions mainly include transplantation models, such as cell line derived xenograft models (CDX) and patient derived xenograft models (PDX). In addition, there are also lentivirus-mediated K14-HPV16 transgenic mice. However, these models have significant limitations in reproducing the HPV integration process. The integration sites of hrHPV in the host genome are dispersed, and the distribution frequency and intensity of the dominant integration sites are low. Therefore, there are certain technical difficulties and uncertainties in constructing an animal model that can reproduce the HPV integration process.

[0005] In order to successfully construct an hrHPV site-specific integration animal model that can exhibit a malignant phenotype, it is crucial to identify the dominant hrHPV integration sites in the human genome. Studies have found that the KLF5 gene is a high-frequency site for HPV integration and is already involved in the early stages of cervical cancer, driving the occurrence of cancer. Animal models are not only an important tool for studying the pathogenesis of cervical cancer and precancerous lesions, but also provide a key platform for the development of new drugs and new therapies. An ideal animal model can provide important support for clinical translational research such as disease mechanism research and drug efficacy evaluation. Summary of the invention

[0006] In view of the key role of HPV integration in the pathophysiological process of cervical diseases, this study proposes a method for constructing and applying an animal model of HPV16 sequence reversely inserted into the mouse genome to address the shortcomings of existing technologies. The realization of this technical solution will provide a powerful tool and theoretical basis for in-depth understanding of the molecular mechanism of cervical cancer and the development of new treatment strategies.

[0007] The purpose of the present invention is to solve the problem of lack of HPV integration animal models and to provide a method for constructing an animal model in which the HPV16 sequence is reversely inserted into the mouse genome and its application.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for constructing a mouse model based on reverse insertion of the HPV16 gene, comprising the following steps:

[0010] (1) Design and synthesize specific gRNA:

[0011] The gRNA includes a reverse strand sequence and a forward strand sequence; the reverse strand sequence of the gRNA is shown in SEQ ID NO.1; the forward strand sequence of the gRNA is shown in SEQ ID NO.2;

[0012] (2) Construction of Donor vector:

[0013] The nucleotide sequence of the Donor vector is shown in SEQ ID NO.12;

[0014] (3) Microinjection:

[0015] The Cas9 protein, gRNA and targeting vector Donor vector are mixed to prepare an RNP injection complex, which is microinjected into mouse fertilized eggs and sent back to the oviduct of surrogate mice to cultivate offspring to produce gene knock-in mice.

[0016] Furthermore, the NCBI ID of the Klf5 gene is: NM_009769.4.

[0017] Furthermore, the Klf5 gene (NCBI reference sequence: NM_009769.4) is located on mouse chromosome 14; the gene contains 4 exons, the start codon ATG is located in the first exon, and the stop codon TGA is located in the fourth exon (transcript: ENSMUST00000005279).

[0018] Furthermore, in the knock-in model, the "URR-E6-E7" gene cassette was reversely inserted into a position approximately 158 kb downstream of the mouse Klf5 gene.

[0019] Furthermore, the constructed targeting vector Donor vector is a targeting vector containing homology arms, and the HPV16 gene "URR-E6-E7" expression cassette is reversely inserted into the targeting vector Donor vector.

[0020] Furthermore, the URR-E6-E7 sequence in the "URR-E6-E7" expression cassette is shown in SEQ ID NO.11.

[0021] Furthermore, the primer sequences involved in the PCR identification of the gene knock-in mice include PCR primer 1 and PCR primer 2.

[0022] Furthermore, the upstream primer sequence of the PCR primer 1 is shown as SEQ ID NO.15, and the downstream primer sequence is shown as SEQ ID NO.16.

[0023] Furthermore, the upstream primer sequence of the PCR primer 2 is shown as SEQ ID NO.17, and the downstream primer sequence is shown as SEQ ID NO.18.

[0024] Furthermore, the reaction system in the PCR identification is:

[0025]

[0026] Furthermore, the reaction procedure in the PCR identification is: 94°C for 3 min; 94°C for 30 s, 60°C for 35 s, 72°C for 35 s, 35 cycles; 72°C for 5 min.

[0027] In a second aspect, the present invention also provides a mouse model based on reverse insertion of the HPV16 gene, which is constructed by the construction method described above.

[0028] In a third aspect, the present invention also provides the use of the above-mentioned mouse model in screening products for treating cervical intraepithelial neoplasia.

[0029] Furthermore, the product is a medicine.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. Fill the technical gap in the research field

[0032] This effectively solves the technical bottleneck of the lack of experimental models that can simulate the HPV DNA integration process in current research. By simulating the non-random integration of HPV DNA fragments in the host genome, the present invention provides an important tool for in-depth research on the role of HPV integration in the occurrence of cervical cancer.

[0033] 2. Promote new drug development and therapy evaluation

[0034] This animal model provides a good preclinical research platform for the development of new anti-HPV infection drugs and precision treatment methods for cervical cancer. By simulating molecular events related to viral integration in the model, the efficacy and safety of candidate drugs can be efficiently evaluated.

[0035] 3. Assisting in the study of HPV integration sites

[0036] The present invention utilizes KLF5 as a high-frequency integration site, which not only successfully reproduces the molecular mechanism driven by HPV integration, but also provides a reliable experimental basis for further exploring the dominant integration sites of HPV in the genome and their functions.

[0037] 4. Promote translational medical research

[0038] This model can deepen the understanding of the key mechanisms of cervical cancer and precancerous lesions, and at the same time provide important support for early detection of the disease, formulation of personalized treatment strategies and prognosis assessment, and has significant translational medical research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A schematic diagram of the vector construction for establishing a mouse Klf5 knock-in model in the present invention;

[0040] Figure 2 This is the result of restriction enzyme digestion verification of the targeting vector Donor vector;

[0041] Figure 3 A schematic diagram of constructing the animal model of the present invention;

[0042] Figure 4 This is the HE-stained section of the mouse model; DETAILED DESCRIPTION

[0043] In order to better illustrate the present invention, the following embodiments are listed. Obviously, the described embodiments are only a part of the present invention, not all embodiments. Based on the embodiments in the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0044] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0045] The purpose of the present invention is to solve the problem of lack of HPV site-specific integration mouse model animal model that can produce malignant phenotypes, and to provide a construction method and application of HPV site-specific integration cervical intraepithelial neoplasia animal model.

[0046] Example 1

[0047] The CRISPR / Cas9 technology was used to construct an animal model of cervical intraepithelial neoplasia with HPV integration at the Klf5 locus on chromosome 14. Figure 1 shown.

[0048] 1. Design and synthesize specific gRNA

[0049] The Klf5 gene (NCBI reference sequence: NM_009769.4) is located on mouse chromosome 14. The gene contains 4 exons, the start codon ATG is located in the first exon, and the stop codon TGA is located in the fourth exon.

[0050] Determine the specific target site gRNA of the gene to be knocked in Klf5 (Gene ID: 12224), find the mouse Klf5 gene DNA sequence in the mouse genome database (Transcript ID: ENSMUST00000005279.7), and design the target sequence of gRNA. The two target sequences are: reverse strand sequence: CTGTATGGGCGCTTCACAAA-TGG (SEQ ID NO: 1), forward strand sequence: GAAGCGCCCATACAGAACTC-TGG (SEQ ID NO: 2);

[0051] The complete gRNA sequence information is as follows:

[0052] Anti-chain gRNA sequence:

[0053] 5'-CTGTATTGGGCGCTTCACAAA-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT-3' (SEQ ID NO. 3);

[0054] Positive-strand gRNA sequence:

[0055] 5'-GAAGCGCCCATACAGAACTC-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT-3' (SEQ ID NO. 4);

[0056] 2. Construction of specific vector Donor vector

[0057] (1) A mouse genomic fragment containing the homology arm and the conditional knock-in gene (URR-E6-E7) was amplified from the BAC:RP23-34I24 cloning template using a high-fidelity DNA polymerase (Novozyme P515). The specific steps are as follows:

[0058] Clone template:

[0059] PCR system: Template: wild type about 400ng, plasmid about 5ng, fragment about 50ng; Primer: 4ul each; Sterile water: 40ul; Max Master Mix: 50ul; total volume: 100ul, mix well and divide into two tubes for PCR reaction.

[0060] The reaction conditions were as follows: 95°C for 5 min, 95°C for 30 s, 60°C for 30 s, 72°C at 1 kb / min (30 cycles), 72°C for 10 min, and stored at 4°C.

[0061] The primer sequences are shown in Table 1 below:

[0062] Table 1 Primer sequence list

[0063]

[0064]

[0065] (2) Donor vector assembly

[0066] The mouse genomic fragment, loxp (locus of X-over P1) and the modified PUC57 backbone (provided by Cyagen Biosciences Inc.) were assembled into a Donor vector. The specific experimental steps are as follows:

[0067] Fragment amplification (5'arm, cKO, 3'arm): Use Novozyme P515 high-fidelity enzyme, prepare a 50ul system for PCR amplification, and perform 30 cycles;

[0068] Recover gel: Perform electrophoresis on the PCR product and use Qiagen gel recovery kit (Cat. No.: 28706) to recover the gel at the position of the target product;

[0069] Ligation (backbone + fragment) / transformation: Use Novazon C115 ligase to ligate the recovered fragments, transform the assembled Donor vector into competent cells, use Takara's Stellar competent medium to transform the ligated DNA fragments into E. coli, and culture at 37°C overnight;

[0070] Bacteria inspection: Pick 16 plaques with good morphology, use Novozyme P222 Taq enzyme, prepare a 25ul system for PCR amplification; pick the plaques with the correct band, inoculate 4mL broth medium and shake for culture;

[0071] Extract plasmid from positive clones: Use alkaline lysis method (self-prepared reagent) to extract plasmid;

[0072] Enzyme digestion identification and sequencing: Select appropriate NEB endonuclease and prepare 20ul of enzyme digestion system with 600ng plasmid for enzyme digestion, and sequence the plasmid with correct enzyme digestion.

[0073] The assembly was completed by Saiye Biotechnology Co., Ltd.

[0074] Reaction system: 100 ng of modified PUC57 backbone; 50 ng of ligation fragment; 5 μL of 2×ClonExpress Mix (Novozyme C115); add sterile water to 10 μL;

[0075] Reaction procedure: reaction temperature 50°C; hot cover temperature 105°C; reaction time 15 min;

[0076] The URR-E6-E7 sequence is shown in SEQ ID NO.11:

[0077] TGCTTG CCATGC GTGCCA AATCCC TGTTTT CCTGAC CTGCAC TGCTTG

[0078] CCAACC ATTCCA TTGTTT TTTACA CTGCAC TATGTG CAACTA CTGAAT

[0079] CACTAT GTACAT TGTGTC ATATAA AATAAA TCACTA TGCGCC AACGCC

[0080] TTACAT ACCGCT GTTAGG CACATA TTTTTG GCTTGT TTTAAC TAACCT

[0081] AATTGC ATATTT GGCATA AGGTTT AAACTT CTAAGG CCAACT AAATGT

[0082] CACCCT AGTTTA TACATG AACTGT GTAAAG GTTAGT CATACA TTGTTC

[0083] ATTTGT AAAACT GCACAT GGGTGT GTGCAA ACCGTT TTGGGT TACACA

[0084] TTTACA AGCAAC TTATAT AATAAT ACTAAA CTACAA TAATTC ATGTAT

[0085] AAAACT AAGGGC GTAACC GAAATC GGTTGA ACCGAA ACCGGT

[0086] TAGTAT AAAAGC AGACAT TTTATG CACCAA AAGAGA ACTGCA ATGTTT

[0087] CAGGAC CCACAG GAGCGA CCCAGA AAGTTA CCACAG TTATGC

[0088] ACAGAG CTGCAA ACAACT ATACAT GATATA ATATTA GAATGT GTGTAC

[0089] TGCAAG CAACAG TTACTG CGACGT GAGGTA TATGAC TTTGCT TTTCGG

[0090] GATTTA TGCATA GTATAT AGAGAT GGGAAT CCATAT GCTGTA TGTGAT

[0091] AAATGT TTAAAG TTTTAT TCTAAA ATTAGT GAGTAT AGACAT TATTGT

[0092] CONFIDENTIAL GTGTAT HELP HELP HELP AACAAA

[0093] CCGTTG TGTGAT TTGTTA ATTAGG TGTATT AACTGT CAAAAG CCACTG

[0094] TGTCCT HIGH AACAA NATION CTGGAC AAAAAG HIGH

[0095] TTCCAT AATATA AGGGGT CGGTGG ACCGGT CGATGT ATGTCT TGTTGC

[0096] NATIONAL TCATGC ACACGT AGAGAA ACCCAG CTGTAA TCATGC

[0097] ATGGAG ATACAC CTACAT TGCATG AATATA TGTTAG ATTTGC AACCAG

[0098] AGACAA CTGATC TCTACT GTTATG AGCAAT TAAATG ACAGCT CAGAGG

[0099] ATGAAGG ATGAAA TAGATG GTCCAG CTGGAC AAGCAG AACCGG

[0100] ACTAGAG CCCATT ACAATA TTGTAA CCTTTT GTTGCA AGTGTG ACTCTA

[0101] CGCTTC GGTTGT GCGTAC AAAGCA CACACG TAGACA TTCGTA CTTTGG

[0102] AAGACC TGTTAA TGGGCA CACTAG GAATTG TGTGCC CCATCT GTTCTCAGAAAC CATAA(SEQ ID NO.11);

[0103] The Donor vector sequence is shown in SEQ ID NO.12 below: wherein the double underline is the Homology arm, the underline is the Kan cassette region, the wavy line is the Amp cassette region, and the black bold part is URR-E6-E7;

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112] (3) Verify the correctness of the entire carrier

[0113] The Donor vector was verified by enzyme digestion and electrophoresis. The band size and position were correct after digestion with different enzymes. The results were as follows: Figure 2 Among them, when using NcoI enzyme, the size of the enzyme digestion is: 4.6 / 2.6 / 1.0 / 0.6kb; when using ApaLI+EcoRV enzyme, the size of the enzyme digestion is: 4.7 / 2.0 / 1.2 / 0.8kb; when using BamHI+PvuI enzyme, the size of the enzyme digestion is: 4.2 / 1.9 / 1.7 / 1.0kb; when using NotI enzyme, the size of the enzyme digestion is: 8.8kb.

[0114] 3. Microinjection

[0115] The RNP injection complex is injected into the pronucleus of the fertilized egg using microinjection technology, and then the fertilized egg is transferred into the oviduct of the pseudo-pregnant mother. The fertilized egg continues to develop into an individual, and the tail of the offspring is identified 7 days after birth to obtain positive mice.

[0116] Preparation of Cas9 protein, gRNA and Donor vector injection complex (RNP injection complex): including tube 1 solution and tube 2 solution, mixing tube 1 solution and tube 2 solution to obtain RNP injection complex;

[0117] Tube 1 solution: add 0.8uL 100pmol / uL CrRNA to 5.2uL RNase-free water, then add 0.6uL 100pmol / uL TracrRNA, mix well and incubate for 5min, then add 0.2uL Cas9 protein (NEB, catalog number M0646M), mix well and incubate for 10min to obtain tube 1 solution;

[0118] Tube 2 solution: Donor vector plasmid with a final concentration of 15 ng / uL;

[0119] Among them, by artificially synthesizing crRNA (CRISPR RNA) sequence (IDT company) and tracrRNA (trans-activating crRNA) sequence (GenScript Biotech Co., Ltd.), crRNA will combine with tracrRNA to form gRNA sequence. The crRNA sequence information and tracrRNA sequence information are shown in SEQ ID NO.13 and SEQ ID NO.14 below:

[0120] CrRNA:

[0121] 5'-GAACACUAGUGCACUUAUCCGUUUUAGAGCUAUGCUGUUUUG-3' (SEQ ID NO. 13);

[0122] tracrRNA:

[0123] 5'-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCU-3' (SEQ ID NO. 14);

[0124] 4. Positive mouse identification method

[0125] (1) Extraction of genomic DNA from mouse tissues

[0126] Method 1: Kit extraction method

[0127] Mouse tail DNA was extracted using TaKaRaMiniBEST Universal Genomic DNA Extraction kit (Ver.5.0_CodeNo.9765).

[0128] Preparation:

[0129] 1) Prepare anhydrous ethanol;

[0130] 2) Before using WB, 65 mL of anhydrous ethanol needs to be added;

[0131] Note: The kit recommends 56 mL, but long-term data show that adding 65 mL will extract higher genomic purity.

[0132] 3) If Buffer GL precipitates, please heat it at 56°C to dissolve it and use it after returning to room temperature;

[0133] 4) When eluting DNA from the membrane, the DNA eluted with 0.25x TE buffer can be stored for a longer period of time (Note: TE buffer should be preheated at 56°C before use to increase the efficiency of DNA elution);

[0134] Experimental process:

[0135] 1) Add 180μL of Buffer GL, 20μL of Proteinase K (use 20μL of Proteinase K in the kit, the concentration is 20mg / mL), and 10μL of RNase to each EP tube containing a sample (2-5mm mouse tail) (Note: The mouse tail cannot be too long, generally 2-5mm; if the mouse tail is too thick, the lysis system can be increased to 300μL);

[0136] 2) Place in a 56°C oven to react overnight (usually 12 to 16 hours);

[0137] 3) The next morning, take the sample out of the oven and place it in a centrifuge. Centrifuge at 12,000 rpm for 2 minutes to remove the hair impurities to the bottom of the tube.

[0138] 4) Pipette the supernatant into another clean EP tube (note: try not to suck up the hair and impurities at the bottom of the tube), then add 200μL of Buffer GB and 200μL of anhydrous ethanol, cover the lid and mix by inverting;

[0139] 5) After mixing, transfer the liquid to the prepared adsorption column, centrifuge at 12000 rpm for 2 min, and discard the filtrate;

[0140] 6) Add 500 μL of Buffer WA to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the filtrate;

[0141] 7) Add 700 μL of Buffer WB around the wall of the adsorption column, let it stand for 1 minute, centrifuge at 12000 rpm for 1 minute, and discard the filtrate (Note: Make sure that the specified volume of anhydrous ethanol has been added to WB; adding WB around the wall of the adsorption column helps to completely wash away the salt attached to the wall);

[0142] 8) Repeat step 7 and discard the filtrate;

[0143] 9) Empty spin: centrifuge at 12000 rpm for 2 min;

[0144] 10) Place the adsorption column in a new 1.5 mL centrifuge tube with the lid open and leave at room temperature for 10 minutes to remove residual ethanol (Note: residual ethanol will inhibit polymerase amplification);

[0145] 11) Add 50 μL of preheated 0.25x TE buffer to the center of the adsorption column membrane, cover the lid and let it stand at room temperature for 10 minutes (Note: preheated sterile water can improve elution efficiency);

[0146] 12) Centrifuge at 12000 rpm for 2 min to elute the DNA; the eluted liquid can be added to the center of the membrane again, let stand for 5 min, and then centrifuge at 12000 rpm for 2 min to obtain a higher concentration of DNA;

[0147] 13) Detect the concentration of the obtained DNA and observe the purity by electrophoresis.

[0148] Method 2: Crude Extraction

[0149] The rough cleavage method is used to crudely extract mouse tail or mouse paw genomic DNA, which is suitable for amplifying bands less than 1kb.

[0150] Preparation of Triton lysis solution (1L):

[0151] 1) Use an electronic balance to weigh 3.7 g KCl and 1.2 g Tris into a beaker;

[0152] 2) Add 300-400 mL of sterile water to dissolve;

[0153] 3) Add 1 mL of TritonX-100 to fully dissolve. If it cannot be fully dissolved, place it in a 56°C oven to dissolve (Note: TritonX-100 is an oily liquid. Rinse the pipette before aspirating. After draining the TritonX-100, extend the pipette tip into the solution and repeatedly blow and aspirate to avoid a large amount of TritonX-100 remaining on the inner wall of the pipette tip).

[0154] 4) Add 80 μL of concentrated hydrochloric acid to adjust the pH to 9.0;

[0155] 5) Then dilute to 1L with sterile water and store at room temperature for later use (Note: Do not sterilize TritonX-100 by high pressure, as it will produce a large amount of precipitation).

[0156] Lysis of samples:

[0157] 1) Take a mouse tail or mouse paw (~2mm) and put it into an EP tube, add 98μL Triton lysis buffer and 2μL 20mg / mL proteinase K (Note: the mouse tail should not exceed 3mm, otherwise the lysis will not be sufficient; if the mouse tail is too thick, the amount of lysis buffer can be increased appropriately; proteinase K needs to be careful not to freeze and thaw repeatedly, otherwise it will affect the activity);

[0158] 2) After adding lysis buffer and proteinase K, place the EP tube in a 56°C constant temperature device (oven or water bath) and run overnight;

[0159] 3) The next day, take out the sample from the thermostat, place it in a metal bath or PCR instrument, and react at 98°C for 15 minutes to inactivate proteinase K;

[0160] 4) Centrifuge the sample for 15 minutes, and the supernatant obtained can be used as a PCR template (Note: Generally, 1.5 μL of supernatant is added to a 25 μL PCR system);

[0161] (2) PCR amplification

[0162] The PCR reaction was carried out using the PCR reaction system described in Table 2 below:

[0163] Table 2 PCR reaction system

[0164]

[0165] PCR amplification reaction conditions were: 94°C for 3 min, (94°C for 30 s, 60°C for 35 s, 72°C for 35 s) × 35 cycles, 72°C for 5 min;

[0166] Primer sequences:

[0167] PCR primer 1 (annealing temperature 60°C):

[0168] F3:5'-TTAAACGATTGCCTTCTACCCCAA-3' (SEQ ID NO.15);

[0169] R4: 5'-TAGCCTTTTCCCATCACATAGCTC-3' (SEQ ID NO. 16);

[0170] Product size:1797bp

[0171] Wildtype allele: 530bp;

[0172] PCR primer 2 (annealing temperature 60°C):

[0173] F5:5'-GAGAAAGTCCTGGAGATTTGGTGC-3' (SEQ ID NO. 17);

[0174] R3: 5'-GAGGAGGATGAAATAGATGGTCCAG-3' (SEQ ID NO. 18);

[0175] Product size:407bp

[0176] WT: one band with 530bp;

[0177] Among them, PCR primer 1 was used to identify the WT band, and PCR primer 2 was used to identify the positive target band;

[0178] (3) Gel electrophoresis

[0179] The PCR products were subjected to gel electrophoresis, using agarose or acrylamide gel to separate DNA fragments of different sizes. The electrophoresis results were observed to determine whether there was a PCR product of the expected size. After the PCR products were separated by gel electrophoresis, UV imaging showed a clear single band at the 407bp position, which was consistent with the expected product size, indicating that the PCR amplification was successful and no obvious nonspecific bands or primer dimers were detected.

[0180] 5. Phenotypic Analysis of HPV Site-directed Knock-in Mice

[0181] like Figure 3 As shown, this study conducted a systematic histopathological evaluation of HPV16-14 E2.2-positive knock-in mice to reveal the mechanism by which site-specific integration of the HPV16 gene affects the pathological process of mammals.

[0182] Histological analysis showed that the cervical tissue of mice with HPV gene reversely knocked into the Klf5 locus showed typical precancerous lesions. Histological analysis of hematoxylin-eosin staining showed that the nuclei of cervical epithelial cells showed significant atypia, including nuclear membrane wrinkling, abnormal chromatin distribution, and cell polarity disorder, which are pathological signs of cervical intraepithelial neoplasia (CIN).

[0183] This study successfully constructed an hrHPV site-specific integration animal model that stably reproduces the characteristics of human cervical precancerous lesions. This model systematically reproduces the pathological development trajectory of cervical intraepithelial neoplasia by accurately simulating the integration process of HPV16 at the Klf5 site. It not only provides a quantifiable research platform for analyzing the biological effects of HPV oncogenes at specific integration sites, but also lays an experimental foundation for in-depth exploration of the molecular mechanism of the transformation of cervical precancerous lesions to invasive cancer, and has important application value in the field of translational medicine research on gynecological tumors.

[0184] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing a mouse model based on reverse insertion of HPV16 gene, characterized in that: The following steps are involved: (1) Design and synthesize specific gRNA: The gRNA includes a reverse strand sequence and a forward strand sequence; the reverse strand sequence of the gRNA is shown in SEQ ID NO.1; the forward strand sequence of the gRNA is shown in SEQ ID NO.2; (2) Construction of Donor vector: The nucleotide sequence of the Donor vector is shown in SEQ ID NO.12; (3) Microinjection: The Cas9 protein, gRNA and targeting vector Donor vector are mixed to prepare an RNP injection complex, which is microinjected into mouse fertilized eggs and sent back to the oviduct of surrogate mice to cultivate offspring to produce gene knock-in mice.

2. The construction method according to claim 1, characterized in that: The primer sequences involved in the PCR identification of the gene knock-in mice include PCR primer 1 and PCR primer 2.

3. The construction method according to claim 2, characterized in that: The upstream primer sequence of the PCR primer 1 is shown as SEQ ID NO.15, and the downstream primer sequence is shown as SEQ ID NO.

16.

4. The construction method according to claim 2, characterized in that: The upstream primer sequence of the PCR primer 2 is shown in SEQ ID NO.17, and the downstream primer sequence is shown in SEQ ID NO.

18.

5. The construction method according to claim 2, characterized in that: The reaction system in the PCR identification is:

6. The construction method according to claim 2, characterized in that: The reaction procedure in the PCR identification is: 94°C for 3 min; 94°C for 30 s, 60°C for 35 s, 72°C for 35 s, 35 cycles; 72°C for 5 min.

7. A mouse model based on reverse insertion of HPV16 gene, characterized in that: Obtained by the construction method according to any one of claims 1 to 6.

8. Use of the mouse model according to claim 7 in screening products for treating cervical intraepithelial neoplasia.

9. The use according to claim 8, characterized in that: The product described is a drug.

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