Construction method and application of HPV fixed-point integration cervical intraepithelial neoplasia animal model

Abstract

The invention belongs to the technical field of animal models, and particularly relates to a construction method and application of an HPV fixed-point integration cervical intraepithelial neoplasia animal model. According to the preparation method of the HPV site-specific integration cervical intraepithelial neoplasia animal model, HPV16 gene knock-in is established on the Klf5 gene locus of a C57BL / 6J mouse through CRISPR / Cas mediated genome engineering. The method comprises the following steps: (1) determining a target sequence on a Klf5 site, and designing specific gRNA; (2) designing and constructing a knock-in vector Donor vector containing a homologous arm (Homology Arms, HAs), and inserting an HPV16 gene sequence into the knock-in vector; and (3) co-injecting the knock-in vector and the Cas9 / gRNA complex into a fertilized egg of a C57BL / 6J mouse for cultivation and birth to obtain the C57BL / 6J mouse. The animal model prepared by the method can simulate the normal-persistent HPV infection-cervical cancer precancerous lesion development stage of human cervix. And a credible animal model is provided for occurrence mechanism research and drug screening of precancerous lesions of cervical cancer.

Description

Technical Field

[0001] The embodiments of the present invention relate to the technical field of animal models, and in particular to a method for constructing and applying an HPV site-specific integrated cervical intraepithelial neoplasia animal model. Background Art

[0002] Cervical cancer is a malignant tumor that occurs in the cervix of the uterus, and its incidence ranks first among malignant tumors of the female reproductive system. The occurrence and development of cervical cancer is a multi-stage, multi-gene mutation process, and there is also a complex relationship between the host's intrinsic genetic characteristics and exogenous HPV viruses. Persistent infection with high-risk HPV (hrHPV) is a direct factor in the occurrence of cervical cancer. During the process of persistent infection with high-risk HPV, non-random integration of HPV DNA fragments into the host genome will lead to a series of molecular events including defects in tumor suppressor genes, genomic instability and cell immortalization, which is one of the key mechanisms for the occurrence of major cervical diseases.

[0003] Since cervical lesions develop slowly, lasting for more than a decade, and patients with early HPV infection and precancerous lesions clinically have no obvious symptoms, it is extremely difficult to track patients from normal cervix to persistent HPV infection to precancerous lesions and cervical cancer in a continuous sample clinically, which brings difficulties to studying the key molecular events in the early stage of cervical cancer and developing new treatments. Cell and animal models are crucial to reveal the relationship between hrHPV integration and cervical cancer. At present, cervical lesion-related models mainly include transplantation models, such as cell line derived xenograft models (CDX) and patient derived xenograft models (PDX), in addition to lentivirus-mediated K14-HPV16 transgenic mice. However, these models cannot reproduce the HPV integration process.

[0004] The integration sites of hrHPV in the human genome are relatively scattered, and the distribution frequency and intensity of the dominant integration sites in the genome are not high. Therefore, constructing an animal model of hrHPV integration has certain risks and uncertainty in effects. Determining the dominant hrHPV integration sites in the human genome is the key to successfully constructing an hrHPV site-specific integration mouse model that can produce a malignant phenotype. KLF5 is a high-frequency HPV integration site that occurs in the early stages of cervical cancer and is involved in the occurrence of HPV integration-driven cervical cancer. Animal models are an important basis for the development of new drugs and new therapies for cervical cancer and precancerous lesions. A good animal model can provide a good platform for further research on the pathogenesis of the disease and evaluation of drug efficacy and other clinical translational research.

[0005] In view of the important role that HPV integration plays in the pathophysiological process of cervical diseases, the present invention aims to improve the above-mentioned technical deficiencies of the prior art and provides a method for constructing and applying an HPV site-specific integration cervical intraepithelial neoplasia animal model. Summary of the invention

[0006] 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.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a method for constructing an HPV site-specific integration cervical intraepithelial neoplasia animal model, comprising the steps of:

[0008] ① Determine the target sequence at the Klf5 site and synthesize specific gRNA;

[0009] The gRNA includes a reverse strand sequence and a forward strand sequence; the nucleotide sequence of the reverse strand sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the forward strand sequence is shown in SEQ ID NO: 2;

[0010] ② Design and construct a knock-in vector Donor vector containing homology arms (HAs), and insert the HPV16 gene sequence "URR-E6-E7" box into the knock-in vector;

[0011] The sequence of URR-E6-E7 is as shown in SEQ ID NO: 3;

[0012] The nucleotide sequence of the Donor vector is shown in SEQ ID NO:4.

[0013] ③ The knock-in vector and Cas9 / gRNA complex were co-injected into the fertilized eggs of C57BL / 6J mice to breed offspring; Cas9 mRNA, the reverse strand sequence of gRNA, the positive strand sequence of gRNA and the targeting vector Donor vector were mixed to obtain an injection complex, which was microinjected into the fertilized eggs of mice and sent back to the oviduct of the surrogate mice to breed offspring to produce gene knock-in mice.

[0014] Preferably, the Klf5 gene (NCBI reference sequence: NM_009769.4) is located on mouse chromosome 14. The gene comprises 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).

[0015] Preferably, in the knock-in model, the "URR-E6-E7" gene cassette is inserted forward into a position approximately 158 kb downstream of the mouse Klf5 gene.

[0016] Preferably, the primer sequences involved in the PCR identification of mice with successful site-directed knock-in of HPV16-E6-E7 gene include PCR Primers 1 and PCR Primers 2; PCR Primers 1 (Annealing Temperature 60.0°C):

[0017] F3: 5'-TTAAACGATTGCCTTCTACCCCAA-3' (SEQ ID NO: 5); R6: 5'-TAGCCTTTTCCCATCACATAGCTC-3' (SEQ ID NO: 6), PCR Primers2 (Annealing Temperature 60.0℃): F5: 5'-AAAGTCCTGGAGATTTGGTGCTG-3' (SEQ ID NO: 7)

[0018] R5:5'-ATGTGCCTAACAGCGGTATGTA-3'(SEQ ID NO:8)

[0019] The present invention provides an HPV site-specific integration cervical intraepithelial neoplasia animal model constructed by the above construction method for use in the study of the molecular basis of carcinogenesis caused by persistent infection of high-risk HPV.

[0020] The present invention provides an HPV site-specific integration cervical intraepithelial neoplasia animal model constructed by the above construction method for use in screening and / or testing drugs and methods for treating cervical intraepithelial neoplasia.

[0021] Compared with the prior art, the technical effects achieved by the present invention are as follows:

[0022] 1. This study identified that the hrHPV dominant integration site Klf5 is the key to successfully constructing an animal model capable of producing cervical malignant phenotypes, providing a key animal model for studying the biological function and potential impact of the HPV16 gene at the Klf5 gene site.

[0023] 2. The HPV site-specific integration animal model constructed in the present invention can produce a cervical intraepithelial neoplasia phenotype, simulate the development process of human cervical precancerous lesions, and provide an ideal animal model for subsequent studies on changes in genome structure and gene expression caused by hrHPV integration and the local microenvironment.

[0024] 3. In addition, local excision is usually used clinically to treat cervical intraepithelial neoplasia, which increases the risk of premature birth and mid-pregnancy miscarriage in women who continue to become pregnant after treatment. The construction of an animal model of cervical intraepithelial neoplasia can promote the development of new drugs for the treatment of cervical intraepithelial neoplasia, discover new treatment methods, and assist in clinical translation and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the background technology, the drawings required for use in the embodiments of the present invention or the background technology will be described below.

[0026] Figure 1 Schematic diagram of establishing a mouse Klf5 knock-in model in C57BL / 6J mice through CRISPR / cas-mediated genome engineering technology in the present invention.

[0027] Figure 2 This is the result of enzyme digestion of the PCR product of the integration fragment in the hrHPV integration model.

[0028] Figure 3 These are photos of the uterine appearance of mice in the control group and the model group in Example 1 of the present invention, microscopic photos of pathological sections stained with HE, and the results of Ki67 immunohistochemical staining. DETAILED DESCRIPTION

[0029] In order to make the technical means, creative features, objectives and effects achieved by the embodiments of the present invention easy to understand, the embodiments of the present invention are further described below in combination with diagrams and specific embodiments, but it should be understood that the protection scope of the present invention is not limited by the specific implementation methods. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0030] 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.

[0031] The present invention provides a method for constructing an HPV site-specific integration cervical intraepithelial neoplasia animal model, comprising the steps of:

[0032] ① Determine the target sequence at the Klf5 site and synthesize specific gRNA;

[0033] The gRNA includes a reverse strand sequence and a forward strand sequence; the nucleotide sequence of the reverse strand sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the forward strand sequence is shown in SEQ ID NO: 2;

[0034] ② Design and construct a knock-in vector Donor vector containing homology arms (HAs), and insert the HPV16 gene sequence "URR-E6-E7" box into the knock-in vector;

[0035] The sequence of URR-E6-E7 is as shown in SEQ ID NO: 3;

[0036] The nucleotide sequence of the Donor vector is shown in SEQ ID NO:4.

[0037] ③ The knock-in vector and Cas9 / gRNA complex were co-injected into the fertilized eggs of C57BL / 6J mice to breed offspring; Cas9 mRNA, the reverse strand sequence of gRNA, the positive strand sequence of gRNA and the targeting vector Donor vector were mixed to obtain an injection complex, which was microinjected into the fertilized eggs of mice and sent back to the oviduct of the surrogate mice to breed offspring to produce gene knock-in mice.

[0038] Preferably, the Klf5 gene (NCBI reference sequence: NM_009769.4) is located on mouse chromosome 14. The gene comprises 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).

[0039] Preferably, in the knock-in model, the "URR-E6-E7" gene cassette is inserted forward into a position approximately 158 kb downstream of the mouse Klf5 gene.

[0040] Preferably, the primer sequences involved in the PCR identification of mice with successful site-directed knock-in of HPV16-E6-E7 gene include PCR Primers 1 and PCR Primers 2; PCR Primers 1 (Annealing Temperature 60.0°C):

[0041] F3: 5'-TTAAACGATTGCCTTCTACCCCAA-3' (SEQ ID NO: 5); R6: 5'-TAGCCTTTTCCCATCACATAGCTC-3' (SEQ ID NO: 6), PCR Primers2 (Annealing Temperature 60.0℃): F5: 5'-AAAGTCCTGGAGATTTGGTGCTG-3' (SEQ ID NO: 7)

[0042] R5:5'-ATGTGCCTAACAGCGGTATGTA-3'(SEQ ID NO:8)

[0043] The present invention provides an HPV site-specific integration cervical intraepithelial neoplasia animal model constructed by the above construction method for use in the study of the molecular basis of carcinogenesis caused by persistent infection of high-risk HPV.

[0044] The present invention provides an HPV site-specific integration cervical intraepithelial neoplasia animal model constructed by the above construction method for use in screening and / or testing drugs and methods for treating cervical intraepithelial neoplasia.

[0045] Example 1 Construction of an animal model of cervical intraepithelial neoplasia with HPV integration at the Klf5 site on chromosome 14 using CRISPR / Cas9 technology. The HPV gene integration site of mice in the control group was at the Rosa26 gene, such as Figure 1 shown.

[0046] ①Design and synthesize specific gRNA

[0047] 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.

[0048] 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 (TranscriptID: ENSMUST00000005279.7), and design the target sequence of gRNA. The two target sequences are: reverse strand sequence: CTGTATGGGCGCTTCACAAA-TGG (SEQID NO: 1), forward strand sequence: GAAGCGCCCATACAGAACTC-TGG (SEQ ID NO: 2).

[0049] ② Method for constructing a specific vector Donor vector Step (1) Use a high-fidelity DNA polymerase (Novozyme P515) to amplify a mouse genomic fragment containing a homology arm and a conditional knock-in gene (URR-E6-E7) from the BAC: RP23-34I24 cloning template. The specific steps are as follows:

[0050] 1. Clone template:

[0051] Sample materials: Novagen P515 enzyme PCR system; template: about 400ng of wild type, about 5ng of plasmid, about 50ng of fragment; primers: 4ul each; sterile water: 40ul; enzyme: 50ul; total volume: 100ul (mix well and divide into two tubes).

[0052] 2. The reaction conditions are as follows: 95℃ for 5 min, 95℃ for 30 s, 60℃ for 30 s, 72℃ at 1 kb / min (30 cycles), 72℃ for 10 min, and stored at 4℃.

[0053] 3. The primer sequences are as follows:

[0054]

[0055]

[0056] (2) Donor vector assembly method steps

[0057] Mouse genomic fragments, loxp (locus of X-over P1) and modified PUC57 backbone were assembled into Donor vector. The assembled Donor vector was transformed into competent cells, positive bacteria were screened, plasmids were extracted, enzyme digestion was performed, and sequencing was performed. The assembly was completed by Saiye Biotechnology Co., Ltd., and the method was as follows: Novozyme C115 ligase system: 100ng of treated backbone; 50ng of ligation fragment; 5μL of 2* enzyme mixture; add sterile water to 10μL. Novozyme C115 reaction program: reaction temperature 50℃; hot cover temperature 105℃; reaction time 15min; reaction system 10μL.

[0058] The sequence of URR-E6-E7 (SEQ ID NO: 3) is shown below:

[0059]

[0060]

[0061] The Donor vector sequence (SEQ ID NO: 4) is as follows,

[0062] The double underline is the Homology arm, the underline is the Kan cassette region, the wavy line is the Ampcassette region, and the black bold part is URR-E6-E7.

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069] (3) Verify the correctness of the entire carrier

[0070] Enzyme digestion verification of the Donor vector. The band size and position are correct after electrophoresis detection using different enzymes for digestion. The results are as follows: Figure 2 When NcoI enzyme was used, the size of the enzyme digestion was 4.6 / 2.6 / 1.0 / 0.6 kb; when ApaLI+EcoRV enzyme was used, the size of the enzyme digestion was 4.7 / 2.0 / 1.2 / 0.8 kb; when FspI+MfeI enzyme was used, the size of the enzyme digestion was 3.4 / 2.4 / 1.8 / 1.2 kb; when NotI enzyme was used, the size of the enzyme digestion was 8.8 kb.

[0071] ③Microinjection

[0072] Cas9 mRNA, gRNA and Donor vector (containing URR-E6-E7 box) were mixed by microinjection technology and injected into the pronucleus of fertilized eggs, and then the fertilized eggs were transferred into the oviduct of pseudopregnant mothers. The fertilized eggs continued to develop into individuals, and the tails of the pups were identified 7 days after birth to obtain positive mice.

[0073] Preparation of Cas9 mRNA, gRNA and Donor vector injection complex: artificially synthesized crRNA (CRISPRRNA) sequence (IDT) and tracrRNA (trans-activating crRNA) sequence (GenScript Biotech Co., Ltd.). crRNA will combine with tracrRNA to form gRNA sequence.

[0074] The sequence information is as follows:

[0075] CrRNA: 5'-GAACACUAGUGCACUUAUCCGUUUUAGAGCUAUGCUGUUUUG-3';

[0076] tracrRNA: 5'-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAGUGGCACCGAGUCCGGCU-3'

[0077] Tube 1 solution: add 0.8uL 100pmol / uL CrRNA to 5.2uL RNase-free water, then add 0.6uL 100pmol / uL TracRNA, mix and incubate for 5min, then add 0.2uL Cas9 protein (NEB, catalog number M0646M), mix and incubate for 10min to obtain Tube 1 solution; Tube 2 solution: Donor plasmid with a final concentration of 15ng / uL; mix Tube 1 solution and Tube 2 solution to obtain RNP injection complex.

[0078] ④Positive mouse identification method steps (1) Extraction of mouse tissue genomic DNA:

[0079] Kit extraction method: Use TaKaRa MiniBEST Universal Genomic DNA Extraction kit (Ver.5.0_Code No.9765) to extract mouse tail DNA.

[0080] 1. Preparation

[0081] 1) Prepare anhydrous ethanol;

[0082] 2) Before using WB, 65 mL of anhydrous ethanol needs to be added;

[0083] Note: The kit recommends 56 mL, but long-term data show that adding 65 mL will extract higher genomic purity.

[0084] 3) If Buffer GL precipitates, please heat it at 56°C to dissolve it and use it after returning to room temperature;

[0085] 4) When eluting DNA from the membrane, the DNA eluted with 0.25x TE buffer has a longer shelf life; TE buffer (Note: Preheating at 56°C before use will increase the DNA elution efficiency.)

[0086] 2. Experimental Procedure

[0087] 1) Add 180μL Buffer GL, 20μL Proteinase K (use 20μL of Proteinase K in the kit, the concentration is 20mg / mL), and 10μL RNase to each EP tube containing sample (2-5mm rat tail); (Note: The rat tail cannot be too long, generally 2-5mm; if the rat tail is too thick, the lysis system can be increased to 300μL.)

[0088] 2) Place in a 56°C oven to react overnight (usually 12 to 16 hours);

[0089] 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.

[0090] 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), add 200μL Buffer GB and 200μL anhydrous ethanol, cover the lid and mix by inverting;

[0091] 5) After mixing, transfer the liquid to the prepared adsorption column, centrifuge at 12000 rpm for 2 min, and discard the filtrate;

[0092] 6) Add 500 μL Buffer WA to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the filtrate;

[0093] 7) Add 700 μL Buffer WB around the wall of the adsorption column, let it stand for 1 minute, and then centrifuge it at 12,000 rpm for 1 minute; discard the filtrate; (Note: Make sure that the specified volume of anhydrous ethanol has been added to the WB; adding WB around the wall of the adsorption column will help to completely wash away the salt adhering to the wall.)

[0094] 8) Repeat step 7 and discard the filtrate;

[0095] 9) Empty spin: centrifuge at 12000 rpm for 2 min;

[0096] 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);

[0097] 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);

[0098] 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;

[0099] 13) Detect the concentration of the obtained DNA and observe the purity by electrophoresis.

[0100] Crude extraction method: Use the rough cleavage method to crudely extract mouse tail or mouse paw genomic DNA, which is suitable for amplifying bands less than 1kb.

[0101] 1. Preparation of Triton lysis solution (1L):

[0102] 1) Use an electronic balance to weigh the following items into a beaker: KCl: 3.7 g; Tris: 1.2 g;

[0103] 2) Add 300-400 mL of sterile water to dissolve;

[0104] 3) Add 1mL TritonX-100 to fully dissolve. If it cannot be fully dissolved, put it in a 56℃ 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 blow and aspirate repeatedly to avoid a large amount of TritonX-100 remaining on the inner wall of the pipette tip.)

[0105] 4) Add 80 μL of concentrated hydrochloric acid to adjust the pH to 9.0;

[0106] 5) Then dilute to 1L with sterile water and store at room temperature for later use.

[0107] (Note: Do not autoclave TritonX-100, as autoclaving will produce a large amount of precipitation.)

[0108] 2. Sample lysis:

[0109] 1) Take the mouse tail or mouse paw (~2mm) and put it into EP tube, add 98μL lysis solution and 2μL 20mg / mL proteinase K; (Note: 1 The mouse tail should not exceed 3mm, otherwise the lysis will not be sufficient; 2 If the mouse tail is too thick, you can increase the amount of lysis solution; 3 Be careful not to freeze and thaw proteinase K repeatedly, otherwise it will affect the activity.)

[0110] 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;

[0111] 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;

[0112] 4) Centrifuge the sample for 15 minutes, and the supernatant can be used as a PCR template. (Note: Generally, 1.5 μL of supernatant is added to a 25 μL PCR system.)

[0113] (2) PCR amplification:

[0114] 1. PCR reaction:

[0115] Component dosage

[0116] Mouse tail genomic DNA 1μL

[0117] Forward primer (10μM) 1μL

[0118] Reverse primer (10 μM) 1 μL

[0119] Premix Tag Polymerase 12.5μL

[0120] ddH2O 9.5μL

[0121] Total 25 μL

[0122] 2. Amplification conditions: 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.

[0123] 3. Primer sequence:

[0124] PCR Primers1(Annealing Temperature 60.0℃):

[0125] F3:5'-TTAAACGATTGCCTTCTACCCCAA-3'

[0126] R6:5'-TAGCCTTTTCCCATCACATAGCTC-3'

[0127] Product size:1797bp Wildtype allele:530bp

[0128] PCR Primers2(Annealing Temperature 60.0℃):

[0129] F5:5'-AAAGTCCTGGAGATTTGGTGCTG-3'

[0130] R5:5'-ATGTGCCTAACAGCGGTATGTA-3'

[0131] Product size:373bp

[0132] (3) Gel electrophoresis:

[0133] Run the PCR product through gel electrophoresis, using agarose or acrylamide gel to separate DNA fragments of different sizes. Observe the electrophoresis results to determine whether there is a PCR product of the expected size.

[0134] ⑤ Detailed phenotypic analysis of HPV site-specific knock-in mice

[0135] like Figure 3As shown, the positive knock-in mice (HPV16-14 E2.2) were subjected to subsequent phenotypic analysis, including morphology (left), histopathology (middle) and immunohistochemical Ki67 staining (right) to study the effects of HPV16 gene knock-in on the physiological or pathological processes of mice. Compared with the control group integrated mice (HPV16-Rosa26), the uterine appearance and cervical tissue pathology section microscopic results of the Klf5 site HPV gene forward knock-in mice were different.

[0136] In summary, compared with the control mice, the cervix of the Klf5 site HPV integrated mice was enlarged, and the nuclei under the HE staining image under the microscope had certain atypia, distortion, and wrinkles, and the nuclear membrane was irregular, showing cervical intraepithelial neoplasia, and immunohistochemical Ki67 staining was positive. These results show that the dominant hrHPV integration site Klf5 is the key to successfully constructing an hrHPV site-specific integration mouse model that can produce a malignant phenotype, and provides an important biological model for studying the biological function and potential impact of the HPV16 gene on the mouse Klf5 gene site. The HPV site-specific integration animal model constructed by the present invention can produce a cervical intraepithelial neoplasia phenotype and simulate the process of occurrence and development of precancerous lesions of the human cervix.

[0137] The above is only a specific implementation of the present invention, but the protection scope of the embodiments of the present invention is not limited thereto. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. Therefore, the protection scope of the embodiments of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for constructing an animal model of HPV site-specific integration of cervical intraepithelial neoplasia, characterized in that: Includes steps: ① Determine the target sequence at the Klf5 site and design specific gRNA; The gRNA includes a reverse strand sequence and a forward strand sequence; the nucleotide sequence of the reverse strand sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the forward strand sequence is shown in SEQ ID NO: 2; ② Design and construct a knock-in vector Donor vector containing homology arms (HAs), and insert the HPV16 gene sequence "URR-E6-E7" box into the knock-in vector; The sequence of URR-E6-E7 is as shown in SEQ ID NO: 3; The nucleotide sequence of the Donor vector is shown in SEQ ID NO:

4. ③ The knock-in vector and Cas9 / gRNA complex were co-injected into the fertilized eggs of C57BL / 6J mice to breed offspring; Cas9 mRNA, the reverse strand sequence of gRNA, the positive strand sequence of gRNA and the targeting vector Donor vector were mixed to obtain an injection complex, which was microinjected into the fertilized eggs of mice and sent back to the oviduct of the surrogate mice to breed offspring to produce gene knock-in mice.

2. The method according to claim 1, characterized in that 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).

3. The method according to claim 1, characterized in that In the knock-in model, the "URR-E6-E7" gene cassette was inserted forward into the position approximately 158 kb downstream of the mouse Klf5 gene.

4. The method according to claim 1, characterized in that The primer sequences involved in the mouse PCR identification of the successful site-directed knock-in of HPV16-E6-E7 gene include PCR Primers1 and PCR Primers2; PCR Primers1 (AnnealingTemperature 60.0°C): F3: 5'-TTAAACGATTGCCTTCTACCCCAA-3' (SEQ ID NO: 5); R6: 5'-TAGCCTTTTCCCATCACATAGCTC-3' (SEQ ID NO: 6), PCR Primers2 (Annealing Temperature 60.0°C): F5: 5'-AAAGTCCTGGAGATTTGGTGCTG-3' (SEQ ID NO: 7) R5: 5'-ATGTGCCTAACAGCGGTATGTA-3' (SEQ ID NO: 8).

5. The HPV site-specific integration cervical intraepithelial neoplasia animal model obtained by the method according to any one of claims 1 to 4 is used in the study of the molecular basis of carcinogenesis caused by persistent infection with high-risk HPV.

6. Use of the HPV site-specific integration cervical intraepithelial neoplasia animal model obtained by the method according to any one of claims 1 to 4 in screening and / or testing drugs and methods for treating cervical intraepithelial neoplasia.

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