Method for establishing spontaneous ovarian cancer model

Abstract

The invention discloses a method for establishing a spontaneous ovarian cancer model. The method comprises the following steps: step 1, selecting a gene most related to HGSOC; 2, verifying and evaluating the cutting efficiency of the crRNA; step 3, modifying a Cas12iMAX gene editing carrier; step 4, selecting AAV9 as a gene delivery vector; step 5, preparing an AAV9 carrier; 6, setting a research time point and an end point; and 7, establishing a mouse spontaneous ovarian cancer model production technology platform. The device has the beneficial effects that the research cost is reduced while the experiment quality is ensured. And an ideal experimental model is provided for researching the influence of a genetic background on disease development.

Description

Technical Field

[0001] The invention relates to a method for establishing a model, and in particular to a method for establishing a spontaneous ovarian cancer model. Background Art

[0002] Currently, genetically engineered mouse models (GEMMs) play a vital role in the field of cancer research. Ovarian cancer, especially high-grade serous adenocarcinoma (HGSOC), is difficult to construct using traditional genotyping mouse hybridization methods due to its complex genotype and high chromosomal instability. With the development of high-throughput sequencing and molecular biology techniques, the diagnosis and treatment of HGSOC has entered the era of precision medicine. Molecular pathological diagnosis, molecular typing, discovery of prognostic molecular markers, and early identification of drug responses provide guidance for the diagnosis and treatment of HGSOC. In addition, the development of the CRISPR / Cas system as a gene editing tool, especially the new CRISPR-Cas12i system, provides new possibilities for gene editing due to its high activity, high specificity, and wide PAM recognition range.

[0003] There are some limitations in the existing ovarian cancer model construction technology, especially in simulating the complexity and heterogeneity of HGSOC. The traditional genotype mouse hybridization method cannot effectively simulate the genotype complexity of HGSOC, which limits the in-depth understanding of the disease mechanism. At present, most of the mouse ovarian cancer models are designed and established through gene-edited mice or immunodeficient mice at home and abroad, which are quite different from human diseases. The existing HGSOC model modeling process is cumbersome, has unique requirements for mouse genotypes, and has a long tumor incubation period, which is not conducive to preclinical basic research. Therefore, it is a long way to go to establish a convenient, fast and economically low-cost genetically engineered mouse model. This study intends to use mice with normal immune function and non-specific genotypes, which are closer to human diseases and have obvious advantages. In addition, existing gene editing tools such as Cas9 and Cas12a have off-target problems, which limits their application in precision medicine. Although AAV vectors have shown potential in gene therapy, their targeting and delivery efficiency in different tissues still need to be optimized. The efficient CIRPSR tool combined with AAV vector is expected to overcome the limitations of existing technologies and provide a convenient, fast and low-cost model construction method, thereby playing an important role in the formulation of early screening strategies and targeted drug development for ovarian cancer. Max It has the advantages of high gene editing efficiency, wide range of PAM sequences and high specificity. In addition, adenovirus-associated virus (AAV) 9, as a gene delivery vector, has shown great potential in the field of gene therapy due to its tissue targeting, low immunogenicity and efficient gene delivery ability. The combined application of CRISPR tools and AAV9 will improve the delivery efficiency of gene editing tools and optimize the model construction process.

[0004] The present invention uses CRISPR / Cas12i MAX This new gene editing tool, combined with AAV9 as a gene delivery vector and in situ microinjection into the mouse ovarian bursa, aims to construct GEMMs that more accurately mimic HGSOC. Max Its features include small size, high editing efficiency, and pre-crRNA processing capabilities, making it very suitable for in vivo multi-gene editing. Max Its small size makes it more suitable for gene delivery via AAV vectors. It also has the ability to edit multiple genes simultaneously through pre-crRNA, providing an efficient solution for multiple gene editing in vivo. Summary of the invention

[0005] The main purpose of the present invention is to solve the problems that the existing HGSOC model modeling process is cumbersome, has unique requirements for mouse genotype, and has a long tumor incubation period, which is not conducive to preclinical basic research work, and to provide a method for establishing a spontaneous ovarian cancer model.

[0006] The method for establishing a spontaneous ovarian cancer model provided by the present invention comprises the following steps:

[0007] Step 1: Select the genes most relevant to HGSOC. Based on the cancer genome library TCGA and PubMed literature search, 49 pathogenic genes related to human HGSOC were identified and the gene editing tool CRISPR-Cas12i was used to MAX , design and screen CRISPR-derived RNA, i.e. crRNA;

[0008] Step 2: Verify and evaluate the cleavage efficiency of crRNA on mouse ovarian cancer cell ID8;

[0009] Step 3: Modify Cas12i MAX Gene editing vector, construction of crRNA plasmid library: 49 pathogenic genes + 7 housekeeping genes, design and screen 5 crRNAs for each gene, and finally construct a library containing 280 crRNAs;

[0010] Step 4: Select AAV9 as the gene delivery vector, and use mouse ovarian bursa microinjection technology to evaluate the efficiency of AAV9 infecting mouse fallopian tubes and ovaries; design a dose concentration gradient, inject different titers of AAV9 into the ovarian bursa in situ, evaluate the infection efficiency through flow cytometry and tissue section staining results, and determine the appropriate virus injection dose;

[0011] Step 5: Prepare AAV9 vector and insert CRISPR / Cas12i MAXRNPs were packaged into AAV9 virus particles, and C57BL / 6 female mice with normal immune function and low economic cost were selected as model animals. AAV9-CRISPR / Cas12i were injected in situ into the mouse ovarian bursa. MAX RNPs were injected directly into the ovarian bursa;

[0012] Step 6: Set the research time point and endpoint, obtain mouse tissue samples, and test and evaluate the model construction process through imaging, RNA testing, serum tumor markers, etc.;

[0013] Step 7. Based on the above research, establish a technology platform for the production of spontaneous ovarian cancer models in mice.

[0014] Beneficial effects of the present invention:

[0015] The method for establishing a spontaneous ovarian cancer model provided by the present invention is intended to provide an experimental platform that is closer to human pathological characteristics to promote the research of ovarian cancer and the development of therapeutic drugs. At present, ovarian cancer models are mainly designed and established by gene-edited mice or immunodeficient mice at home and abroad, and these models are quite different from human diseases. The occurrence process of ovarian cancer is spontaneous, that is, its development is not directly affected by external factors such as infection or chemicals, but is closely related to factors such as individual genetic susceptibility, immune surveillance status and metabolic microenvironment.

[0016] The purpose of the present invention is to provide a method for establishing an ovarian cancer model using C57BL / 6 mice, which can more realistically simulate the spontaneous occurrence process of human ovarian cancer and has the following technical features:

[0017] 1. A tumor-promoting environment with normal immune function: C57BL / 6 mice have complete immune function and can simulate the role of immune surveillance in the development of human ovarian cancer, which is crucial for understanding the mechanism of tumor immune escape.

[0018] 2. Economic and cost-effectiveness: C57BL / 6 mice are easy to raise and breed, and have high experimental repeatability and result stability, thus reducing research costs while ensuring experimental quality.

[0019] 3. Clear genetic background: C57BL / 6 mice have a clear genetic background and a stable genotype, providing an ideal experimental model for studying the impact of genetic background on disease development.

[0020] 4. Representativeness of physiological functions: The metabolic system of C57BL / 6 mice is relatively complete and suitable for studying metabolic diseases, which is of great significance for exploring metabolic changes related to ovarian cancer.

[0021] 5. Broad application base: The wide application of C57BL / 6 mice in oncology, physiology, immunology, and genetics provides rich experience and data support for the construction of the ovarian cancer model of this project. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure is a flow chart of the method for establishing the model described in the present invention.

[0023] Figure 2 It is a schematic diagram of the efficient crRNA screening results and gene editing targets of trp53crRNA, brca1crRNA, ptencrRNA and Ikb1crRNA described in the present invention.

[0024] Figure 3 The anatomical structure of the mouse ovarian bursa (Bursa bursa) and the effect after injection are shown in the figure. A: Schematic diagram of the structure inside the Bursa bursa (the dotted arrow indicates the injection site) B, C: The filling of the Bursa bursa before and after injection (injection solution: 0.4% trypan blue 10 μL).

[0025] Figure 4 Schematic diagram of the results of AAV9-eGFP infection of mouse ovaries and oviducts of the present invention. A. AAV9 with a titer of 5E12 GC was injected into the mouse Bursa capsule at 2 μL (n=5) and 8 μL (n=5);

[0026] B. 2 μL (n=5) and 8 μL (n=5) of AAV9 with a titer of 5E13GC were injected into the mouse Bursa capsule respectively; C. AAV9-eGFP infected mouse fallopian tube, concentrated in the mucosal epithelium in the fallopian tube cavity; D. ENA can significantly improve the infection efficiency of AAV9. DETAILED DESCRIPTION

[0027] See also Figures 1 to 4 As shown:

[0028] The method for establishing a spontaneous ovarian cancer model provided by the present invention comprises the following steps:

[0029] Step 1: Select the genes most relevant to HGSOC. Based on the cancer genome library TCGA and PubMed literature search, 49 pathogenic genes related to human HGSOC were identified and the gene editing tool CRISPR-Cas12i was used to MAX , design and screen CRISPR-derived RNA, i.e. crRNA;

[0030] Step 2: Verify and evaluate the cleavage efficiency of crRNA on mouse ovarian cancer cell ID8;

[0031] Step 3: Modify Cas12i MAX Gene editing vector, construction of crRNA plasmid library: 49 pathogenic genes + 7 housekeeping genes, design and screen 5 crRNAs for each gene, and finally construct a library containing 280 crRNAs;

[0032] Step 4: Select AAV9 as the gene delivery vector, and use mouse ovarian bursa microinjection technology to evaluate the efficiency of AAV9 infecting mouse fallopian tubes and ovaries; design a dose concentration gradient, inject different titers of AAV9 into the ovarian bursa in situ, evaluate the infection efficiency through flow cytometry and tissue section staining results, and determine the appropriate virus injection dose;

[0033] Step 5: Prepare AAV9 vector and insert CRISPR / Cas12i MAX RNPs were packaged into AAV9 virus particles, and C57BL / 6 female mice with normal immune function and low economic cost were selected as model animals. AAV9-CRISPR / Cas12i were injected in situ into the mouse ovarian bursa. MAX RNPs were injected directly into the ovarian bursa;

[0034] Step 6: Set the research time point and endpoint, obtain mouse tissue samples, and test and evaluate the model construction process through imaging, RNA testing, serum tumor markers, etc.;

[0035] Step 7. Based on the above research, establish a technology platform for the production of spontaneous ovarian cancer models in mice.

[0036] The product formula and equipment in the above method are as follows:

[0037] 1. Product formula: CRISPR / Cas12i MAX RNPs: containing Cas12i Max protein and specific crRNA.

[0038] AAV9 vector: contains AAV9 virus particles and CRISPR / Cas12i MAX RNPs.

[0039] Experimental mice: Wild-type C57BL mice

[0040] 2. Equipment and Devices:

[0041] Instruments and reagents involved in the experiment:

[0042] Molecular biology laboratory equipment NEXTSEQ CN500 (Berry Genomics) and flow cytometer: LSRFortessa (BD) and other large instruments and equipment, as well as real-time fluorescence quantitative PCR instrument sets, nucleic acid quantification instrument, UV gel imaging system and PCR instrument, fully automatic hybridization instrument, cold light instrument, stereo microscope, micro syringe, ultrasound / nuclear magnetic resonance inspection equipment.

Claims

1. A method for establishing a spontaneous ovarian cancer model, characterized in that: The method comprises the following steps: Step 1: Select the genes most relevant to HGSOC. Based on the cancer genome library TCGA and PubMed literature search, 49 pathogenic genes related to human HGSOC were identified and the gene editing tool CRISPR-Cas12i was used to MAX , design and screen CRISPR-derived RNA, i.e. crRNA; Step 2: Verify and evaluate the cleavage efficiency of crRNA on mouse ovarian cancer cell ID8; Step 3: Modify Cas12i MAX Gene editing vector, construction of crRNA plasmid library: 49 pathogenic genes + 7 housekeeping genes, design and screen 5 crRNAs for each gene, and finally construct a library containing 280 crRNAs; Step 4: Select AAV9 as the gene delivery vector, and use mouse ovarian bursa microinjection technology to evaluate the efficiency of AAV9 infecting mouse fallopian tubes and ovaries; design a dose concentration gradient, inject different titers of AAV9 into the ovarian bursa in situ, evaluate the infection efficiency through flow cytometry and tissue section staining results, and determine the appropriate virus injection dose; Step 5: Prepare AAV9 vector and insert CRISPR / Cas12i MAX RNPs were packaged into AAV9 virus particles, and C57BL / 6 female mice with normal immune function and low economic cost were selected as model animals. AAV9-CRISPR / Cas12i were injected in situ into the mouse ovarian bursa. MAX RNPs were injected directly into the ovarian bursa; Step 6: Set the research time point and endpoint, obtain mouse tissue samples, and test and evaluate the model construction process through imaging, RNA testing, serum tumor markers, etc.; Step 7. Based on the above research, establish a technology platform for the production of spontaneous ovarian cancer models in mice.

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