A method for rapidly establishing an ovarian cancer model based on SauriCas9

Abstract

The application discloses a method for rapidly establishing an ovarian cancer model based on SauriCas9, and particularly discloses a recombinant plasmid for knocking out Pten and Trp53 genes, which comprises an EPI vector system; the recombinant plasmid takes an ori element as a replication start site, and sequentially comprises sgRNA sequences for targeting Trp53 genes and Pten controlled by a double U6 promoter, a CAG promoter, a SauriCas9 nuclease expression unit, a fluorescent protein expression element, a resistance gene, and an orip element and an EBNA1 protein expression element. The application further discloses a method for rapidly establishing an ovarian cancer model based on SauriCas9, and an ovarian cancer cell model obtained by construction. The ovarian cancer cell model is constructed by adopting the method, the period from cell editing to animal tumorigenesis is shortened, the stability and immune integrity of the genetic background of the model are ensured, and large-scale drug screening and high-throughput experiments are facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular, to a method for rapidly establishing and evaluating an ovarian cancer cell model based on SauriCas9. BACKGROUND

[0002] The existing diagnosis and treatment mode of ovarian cancer includes surgical treatment, platinum-containing chemotherapy and PARP (poly ADP-ribose polymerase) inhibitor treatment, in addition to which, the effect of treatment methods such as immunotherapy in the treatment of ovarian cancer is not ideal. Ovarian cancer is characterized by its complex mutation types, and the strong heterogeneity between different patients, different biological behaviors and different responsiveness to different treatments. Therefore, it is essential to construct an ovarian cancer model that can simulate different mutation types of patients for the development of individualized diagnosis and treatment.

[0003] The models currently used in ovarian cancer research include human organoid models, traditional mouse transplantation models (such as ID8 and its derivative strains) and genetically engineered mouse (GEMM) models. Among them, the human organoid model has been widely used in precision medicine due to its ability to retain the genetic characteristics and drug sensitivity characteristics of patient tumors; but it has the disadvantages of being unable to be cultured for a long time, lacking a complete immune and tumor microenvironment, and having a low efficiency of tumor formation in animals. Traditional mouse transplantation models, such as ID8 syngeneic transplantation cell lines, are established by spontaneous transformation in vitro, and there are large differences in genetic stability and tumor immunogenicity between them and clinical ovarian cancer. Although there have been improvements based on the deletion of Trp53 and Brca2 genes, the PTEN deletion gene background commonly found in ovarian cancer has not been covered. Although the GEMM model achieves precise knockout of Pten and Trp53, it has a long construction period, high breeding cost, complex operation and mixed genetic background, which makes it difficult to meet the needs of large-scale drug screening and rapid mechanism research.

[0004] In addition, there is currently no precedent for successfully knocking out Pten and Trp53 in mouse ovarian surface epithelial cells (MOSE) primary cells on the surface of mouse ovaries using in vitro CRISPR / Cas9 technology, and for establishing an immune-intact tumor model.

[0005] Therefore, the existing ovarian cancer models all have different degrees of technical defects: the human organoid model depends on a highly specific growth factor culture system and lacks immune and stromal cell support, resulting in the inability of the organoid to be stably passed and grown for a long time, and the animal transplantation success rate is also low; the reason is that the organoid culture system is too dependent on specific growth conditions and fails to fully simulate the real tumor microenvironment. The traditional ID8 model realizes rapid and stable in vivo tumor establishment, but because it is derived from spontaneous transformation in vitro, the genome is unstable, and it does not carry the characteristic mutations of ovarian cancer (such as Pten and Trp53 deletion), making it difficult to effectively translate drug screening results to the clinic; the root cause of this problem is that the model construction method does not undergo precise gene editing from the source, but relies on spontaneous gene mutation or limited gene modification. Although the GEMM model realizes precise genetic modification (such as Pten and Trp53 knockout), its construction cycle is too long (usually 1-2 years), the maintenance and propagation are complex and costly, and in addition, the genetic background differences caused by the crossbreeding of different background mice also cause instability of the tumor phenotype and repeatability problems of the experiment; the fundamental reason is that the GEMM model relies on embryonic level gene editing and complex mouse breeding systems, which limits the efficiency and flexibility of the experiment. Therefore, there is an urgent need for an ovarian cancer research model that is efficient and fast, has precise gene editing, and can rapidly form tumors in an immune-intact animal. SUMMARY

[0006] To solve the technical defects of the existing ovarian cancer research models (human organoid model, ID8 cell line model, genetically engineered mouse model), such as incomplete genetic background, low gene editing precision, long model establishment time, high cost, and lack of immune microenvironment, the purpose of the present application is to provide a method for rapidly establishing an ovarian cancer cell model based on SauriCas9, and to construct a new ovarian cancer research model that is fast, efficient, and cost-controllable. Based on mouse ovarian surface epithelial cells (MOSE), the EPI-SauriCas9 system is used to achieve precise double knockout of the Pten and Trp53 genes, to rapidly obtain an ovarian cancer research platform with a clear genetic background, stable tumor characteristics, an intact immune system, and efficient drug screening.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] In a first aspect, the present application provides a recombinant plasmid for targeted knockout of Pten and Trp53 genes, comprising an EPI vector system, wherein the EPI vector system comprises an OriP element and an EBNA1 protein expression element.

[0009] The recombinant plasmid takes the ori element as a replication initiation site, and sequentially comprises sgRNA sequences targeting the Trp53 gene and Pten controlled by a double U6 promoter, a CAG promoter, a SauriCas9 nuclease expression unit, a fluorescent protein expression element, a resistance gene, and an ori p element and an EBNA1 protein expression element.

[0010] As some specific embodiments of the present application, the sgRNA sequence targeting the Pten gene is shown in SEQ ID NO. 1.

[0011] The sgRNA sequence targeting the Trp53 gene is shown in SEQ ID NO. 2.

[0012] As some specific embodiments of the present application, the fluorescent protein comprises a green fluorescent protein or a red fluorescent protein, and optionally, the green fluorescent protein comprises a ZsGreen fluorescent protein.

[0013] As some specific embodiments of the present application, the resistance gene comprises a Puromycin resistance gene or a Neomycin resistance gene, and preferably a Puromycin resistance gene.

[0014] As some specific embodiments of the present application, the construction method of the recombinant plasmid comprises: obtaining OriP elements and EBNA1 protein by PCR using epiCRISPR, and sequentially inserting a CAG promoter, a SauriCas9 nuclease, a fluorescent protein expression element, a resistance gene, and sgRNA sequences targeting Pten and Trp53 genes controlled by a double U6 promoter by Gibson assembly.

[0015] In a second aspect, the present application provides a method for rapidly establishing an ovarian cancer model based on SauriCas9, comprising the following steps:

[0016] S1, transfecting the recombinant plasmid of any one of the above into mouse primary MOSE cells;

[0017] S2, screening to obtain a stable cell line, which is a MEPP cell;

[0018] S3, inoculating the MEPP cell into an immunocompetent mouse to form a tumor.

[0019] As some specific embodiments of the present application, in step S1, the mouse primary MOSE cells are mouse ovarian surface epithelial cells isolated from C57BL / 6 adult female mice and obtained after TrypLE enzyme digestion.

[0020] As some specific embodiments of the present application, in step S1, after the recombinant plasmid is transfected into the mouse primary MOSE cells, the transfection efficiency is determined by fluorescence intensity.

[0021] As some specific embodiments of the present application, in step S2, the stable cell strain is obtained by screening with the resistance gene.

[0022] And / or, after the MEPP cells are obtained in step S2, the Pten and Trp53 gene knockout in the cells is verified by PCR and sequencing techniques.

[0023] As some specific embodiments of the present application, in step S3, the MEPP cells obtained in step S2 are inoculated into the subcutaneous tissue or ovarian capsule of the immunocompetent C57BL / 6 mice at a concentration of 1×10 6 ~3×10 6 cells / μl mixed with the matrix glue, and the tumor is formed rapidly.

[0024] In some specific embodiments, the MEPP cells obtained in step S2 are inoculated into the subcutaneous tissue of the immunocompetent C57BL / 6 mice at a concentration of 3×10 6 cells / μl mixed with the matrix glue, and the tumor is formed rapidly within 1 week.

[0025] In some specific embodiments, the MEPP cells obtained in step S2 are inoculated into the ovarian capsule of the immunocompetent C57BL / 6 mice at a concentration of 1×10 6 cells / μl mixed with the matrix glue, and the tumor, metastasis and ascites are formed rapidly within 2 months.

[0026] In a third aspect, the present application provides an ovarian cancer cell model, which is constructed by the method of any one of the above.

[0027] In a fourth aspect, the present application provides an ovarian cancer cell model for screening drugs for treating ovarian cancer.

[0028] The paper "CRISPR / Cas9-mediated Trp53 and Brca2 knockout can generate an improved mouse model of ovarian high-grade serous carcinoma" uses CRSPR / Cas9 technology to knockout Trp53 and Brac2 in the ID8 cell line to establish a mouse model. However, there are still defects and deficiencies:

[0029] 1. Unstable cellular genetic background reduces model predictability. This paper uses the ID8 long-passaged cell line as the starting material. ID8 cells accumulate a large number of unidentified mutations and chromosomal rearrangements during continuous in vitro culture, and literature also reports low antigenicity and high CNV levels; these uncertainties affect the reproducibility of tumor lineages and drug responses. This invention uses primary MOSE cells derived from wild-type C57BL / 6 mice. Due to the limited number of culture passages and difficulty in transfection of MOSE cells, simple Trp53 / Pten knockout is insufficient to induce cancerous transformation in MOSE cells. This invention overcomes these defects by constructing specific recombinant plasmids and transfecting them into MOSE cells, successfully inducing cancerous transformation in MOSE cells. Compared to existing ID8 cell lines, the genetic background and immunogenicity of MOSE cells are closer to normal epithelium, better reflecting the development of ovarian cancer.

[0030] 2. The gene knockout method used in the paper involved two steps: first, a Trp53 knockout ID8 cell line was established, and then a Brac2 knockout cell line was further constructed using the Trp53 knockout ID8 cell line. The vector was introduced into the cells via lentiviral infection. Adding two knockout elements could result in very low infection efficiency, hence the two-step cloning method. Two rounds of single-clone screening require a total of 6-8 weeks, and repeated cloning screening in primary cells can easily lead to senescence or gene drift. Furthermore, the lentiviral infection method, after infection, results in the vector inserting into the genome, continuously producing spCas9 and gRNA, which may have significant off-target effects over the long term. In contrast, this invention relies on the EPI vector + SauriCas9. SauriCas9 is smaller than spCas9, allowing multiple knockout elements to be placed within the vector. A single transfection can achieve simultaneous and efficient double knockout of Trp53 and Pten within 48 hours. Stable strains can be obtained by Puromycin selection within ≤ 7 days, which greatly shortens the construction cycle. Furthermore, the gene knockout has a small off-target effect because the gene is lost after long-term passage by plasmid transfection.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1) Speed ​​and precision in technology: The EPI-SauriCas9 system of this invention significantly shortens the cycle from cell editing to animal tumor formation (2-8 weeks), which is several times faster than the traditional GEMM model (1-2 years); the SauriCas9 system ensures precise and efficient knockout of Pten and Trp53 genes.

[0033] 2) Stability of genetic background and immune integrity of the model: Stable tumor formation was rapidly established in immune-intact C57BL / 6 mice, successfully simulating the PTEN and TP53 double deletion genetic background commonly seen in clinical ovarian cancer; the model has a complete tumor immune microenvironment containing multiple immune cell types (macrophages, T cells, NK cells, dendritic cells, etc.), which is closer to the immune microenvironment of human ovarian cancer.

[0034] 3) Economic and social effects (potential for drug screening and translational applications): The MEPP model is rapidly constructed and inexpensive, facilitating large-scale drug screening and high-throughput experiments, effectively reducing research costs; actual drug screening revealed that FK228 and thioguanine have significant in vitro and in vivo anti-tumor effects (proving that the model has high potential for drug screening and clinical translation); the technical platform provided by this invention provides a solid foundation and promising prospects for the development of new drugs for the treatment of ovarian cancer, with significant socio-economic value.

[0035] 4) Compared to directly knocking out the Pten and Trp53 genes using genetic engineering methods (as in Comparative Example 1), this invention eliminates the need for pre-constructing complex genetically engineered mice and avoids the defects of rapid apoptosis and inability to proliferate after Cre virus infection of primary cells. Using EPI+SauriCas9, primary cells can obtain a dual-gene knockout cell line (MEPP) after a single transfection, rapidly forming tumors in vivo. In contrast, Comparative Example 1 cells failed to survive and proliferate after viral infection, and also failed to form tumors or metastasize. Compared to the SauriCas9+conventional vector approach (as in Comparative Example 2): when using a conventional vector (without EPI), cells rapidly undergo apoptosis after Puromycin selection, failing to obtain stable cell lines and completely lacking in vivo tumorigenesis ability. The EPI vector used in this invention, carrying the OriP / EBNA1 element, enables sustained and efficient expression of SauriCas9 and sgRNA, avoiding the toxicity of lentiviral genome integration and ensuring long-term cell survival, stable amplification, rapid tumorigenesis, and metastasis. Attached Figure Description

[0036] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0037] Figure 1 This is a schematic diagram of the EPI-SauriCas9-sgRNA plasmid structure constructed in Example 1;

[0038] Figure 2 This is a Sanger sequencing result diagram after constructing plasmids for different sgRNAs and transfecting them in Example 1;

[0039] Figure 3The images shown are optical and fluorescence micrographs of EPI-SauriCas9-sgRNA transfected into MOSE cells in Example 1; the left image is an optical micrograph, and the right image is a fluorescence micrograph.

[0040] Figure 4 This is a Sanger sequencing result of the Pten and Trp53 target site sequences of the MEPP cell DNA constructed in Example 1;

[0041] Figure 5 This image shows the tumorigenesis and pathological characteristics of MEPP cells in immune-intact C57BL / 6 mice. In this image, A represents the tumorigenesis and pathological characteristics of MEPP cells after subcutaneous injection of 3×10⁻⁶ cells. 6 Tumor feature image after 2 weeks; B shows the H&E staining and Wt1 immunohistochemical results of MEPP subcutaneous tumor tissue; C shows the results of subcapsular injection of 1×10⁻⁶ cells into the ovary. 6 Tumor feature diagram of MEPP cells after 8 weeks; D shows the H&E staining and Wt1 immunohistochemical results of MEPP ovarian orthotopic tissue; E shows the H&E staining and Wt1 immunohistochemical results of MEPP omental metastasis tissue.

[0042] Figure 6 The image shows the RNA-seq map of MEPP tumor single cells in Example 1, where A is an unsupervised dimensionality reduction clustering map and B is a map of characteristic gene expression of all cell clusters.

[0043] Figure 7 The images shown are in vivo validation diagrams of the MEPP drug screening in Example 2. In this diagram, A is the tumor volume curve after treatment with FK228 and Thioguanine, B is the Ki67 immunohistochemical score, and C is the TUNEL apoptotic cell staining diagram.

[0044] Figure 8 Pten, the genetically engineered mouse in Comparative Example 1 flox / flox ;Trp53 flox / flox + Image showing the cell proliferation results of primary cells after Cre infection, where the top image is an optical micrograph and the bottom image is a fluorescence micrograph;

[0045] Figure 9 This is a schematic diagram of the V8-SauriCas9 plasmid structure constructed in Comparative Example 2;

[0046] Figure 10 The image shows the cell proliferation results of Pten / Trp53 knockout using SauriCas9 + conventional vector in Comparative Example 2. The top image is an optical micrograph, and the bottom image is a fluorescence micrograph. Detailed Implementation

[0047] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0048] 1. This invention provides a rapidly established immune-intact mouse ovarian cancer model, the specific steps of which are as follows:

[0049] (1) Primary MOSE cells were obtained by isolating mouse ovarian surface epithelial cells (MOSE) from adult female C57BL / 6 mice and digesting them with TrypLE enzyme.

[0050] (2) Construct a plasmid expression vector containing an EPI vector system (containing OriP elements and EBNA1 protein) and SauriCas9 nuclease. The plasmid also contains sgRNA expression elements controlled by dual U6 promoters, targeting the Pten and Trp53 genes respectively, and contains fluorescent protein (ZsGreen) and Puromycin resistance genes;

[0051] (3) The above plasmids were transfected into MOSE cells via EZ trans. After 24 hours, ZsGreen fluorescence was observed to determine the transfection efficiency. Stable transfected cells were screened by Puromycin and a stable cell line was established within about 1 week.

[0052] (4) The MOSE cells after transfection and screening are MEPP cells. The knockout of Pten and Trp53 genes in the cells is verified by PCR and sequencing technology.

[0053] (5) MEPP cells (3×10 6 After being mixed with matrix gel, the mixture was injected subcutaneously into immune-intact C57BL / 6 mice, and tumors rapidly formed within one week.

[0054] Alternatively, MEPP cells (1×10⁻⁶) 6 After being mixed with matrix gel, the mixture was inoculated into the ovarian capsule of immune-intact C57BL / 6 mice, and tumors, metastases, and ascites rapidly formed within 2 months.

[0055] 2. The technical elements of this invention and their specific functions:

[0056] MOSE primary cells: provide a stable and well-defined source of ovarian epithelial cells, ensuring the purity of the model's genetic background;

[0057] EPI vector system (containing OriP+EBNA1 expression element): The plasmid can stably replicate with cell division in the cell, improving transfection efficiency and avoiding the safety risks of viral vectors.

[0058] SauriCas9 nuclease (NNGG PAM): Small in size, highly efficient in editing, and flexible, ensuring precise and efficient knockout of Pten and Trp53 genes.

[0059] 3. Identification characteristics of biological materials:

[0060] MEPP ​​cells exhibit typical epithelial cell morphology, and the expression of Pax8 and Wt1 genes is significantly higher than that of mouse mesenchymal stem cells.

[0061] scRNA-seq analysis revealed that MEPP tumor cells could be divided into several subpopulations, exhibiting distinct EMT, proliferation, and metastasis-related gene characteristics.

[0062] MEPP ​​cells exhibit good passage stability and can rapidly form tumors when inoculated subcutaneously or ovarianly.

[0063] The tumors formed in immune-intact mice exhibit complete immune cell infiltration (such as macrophages, T cells, NK cells, etc.), effectively mimicking the immune microenvironment of human ovarian cancer.

[0064] Example 1 – Construction and Validation of the MEPP Ovarian Cancer Cell Model

[0065] 1. Source and culture of primitive cells

[0066] Ovaries were harvested from female C57BL / 6 mice (6-8 weeks old) and digested with Trpzyme recombinant trypsin digestion solution (derived from BasalMedia) at 37°C for 30 minutes to obtain MOSE primary cells. These cells were then seeded in DMEM medium containing 10% FBS (fetal bovine serum) and 100 U / mL penicillin-streptomycin and cultured at 37°C with 5% CO2.

[0067] 2. Vector construction and gene editing methods

[0068] Using epiCRISPR (addgene #135960), the OriP element and EBNA1 protein were obtained by PCR. The CAG promoter, SauriCas9 nuclease, ZsGreen fluorescent gene, Puromycin resistance gene, and sgRNA sequences targeting Pten and Trp53 genes controlled by the double U6 promoter were sequentially inserted using the Gibson assembly method to construct the EPI-SauriCas9-sgRNA expression plasmid (e.g., Figure 1 (As shown).

[0069] The constructed EPI-SauriCas9-sgRNA expression plasmid uses the ori element as the replication initiation site and sequentially includes an sgRNA sequence targeting the Trp53 gene and Pten, controlled by a dual U6 promoter, a CAG promoter, a SauriCas9 nuclease expression unit, a fluorescent protein expression element, an antibiotic resistance gene, and orip and EBNA1 protein expression elements. The fluorescent protein is ZsGreen fluorescent protein, and the antibiotic resistance gene is Puromycin.

[0070] To screen for suitable sgRNA sequences targeting the Pten and Trp53 genes, three pairs of sgRNA sequences were designed and validated using plasmids transfected with the Hepa1-6 cell line.

[0071] The sequences of the three pairs of sgRNAs are shown below:

[0072] sgRNA-pair1:

[0073] sgPten:5'-TGGTGGGTTATGGTCTTCAA-3' (SEQ ID NO.1);

[0074] sgTrp53:5'-TATCCGACTGTGACTCCTCC-3' (SEQ ID NO. 2).

[0075] sgRNA-pair2:

[0076] sgPten:5'- GAAACAAAAGGAGATATCAA-3' (SEQ ID NO.3);

[0077] sgTrp53:5'-TAGATGGCCATGGCCGGAC-3' (SEQ ID NO. 4).

[0078] sgRNA-pair3:

[0079] sgPten:5'- TGCTAACGATCCTTTGATG-3' (SEQ ID NO.5);

[0080] sgTrp53:5'-GACACTCGGAGGGCTTCACT-3' (SEQ ID NO. 6).

[0081] Taking sgRNA-pair1 as an example, the sequence of the constructed plasmid EPI-SauriCas9-sgRNA-pair1 consists of SEQ ID NO.7 and SEQ ID NO.8 (a total of 14972 positions). The replication origin site ori is located at positions 14128-14716, positions 49-289 are U6 promoter, positions 300-319 are Trp53-sgRNA1, positions 423-663 are U6 promoter, positions 673-692 are Pten-sgRNA1, positions 1012-2373 are CAG promoter, positions 2816-6100 are sauriCas9, positions 6164-6856 are ZSgreen, positions 6998-7594 are PuroR, positions 8454-10244 are OriP, and positions 10545-12471 are EBNA1.

[0082] like Figure 2 The image shown is a Sanger sequencing result diagram of Hepa1-6 cells after constructing plasmids for different sgRNAs in this embodiment. Figure 2 It was observed that pair3 produced fewer indels, while both pair1 and pair2 could effectively generate indels. Therefore, Pten / Trp53 sgRNA-pair1 and pair2 exhibited better editing efficiency. sgRNA-pair1 (SEQ ID NO.1; SEQ ID NO.2) was selected for subsequent cell experiments.

[0083] 3. MOSE cell transfection and establishment of stable cell lines

[0084] The EPI-SauriCas9-sgRNA expression plasmid obtained above was transfected into MOSE primary cells using the cell transfection reagent EZ Trans (Life-iLab). ZsGreen fluorescence was observed under a microscope 24 hours after transfection to confirm the transfection efficiency. Figure 3 ), thus obtaining successfully transfected MOSE cells.

[0085] Subsequently, 5 µg / ml Puromycin (YEASEN) was added for 7 days of selection to obtain MEPP cell lines with Pten and Trp53 knocked out. DNA was extracted from the transfected MEPP cells, and the target sequences of Pten and Trp53 were amplified by PCR. The PCR products were then subjected to Sanger sequencing and compared with wild-type sequences. The results are as follows: Figure 4 As shown, peak overlap, signal attenuation, and base misalignment immediately after the two sgRNA cleavage sites indicate the presence of an insertion / deletion (indel) event.

[0086] 4. Methods for validating in vivo tumorigenesis and tumor models

[0087] Take the obtained MEPP cells and adjust the cell concentration (3×10⁻⁶). 6 1 cell / 100 µL PBS [subcutaneous injection]; 1×10 6 MEPP ​​cells / 10 µL PBS [subcapsular injection in the ovary] were mixed with an equal volume of Matrigel and injected subcutaneously or subcapsularly in 6-8 week old immunized C57BL / 6 female mice. Tumor growth was monitored for 2-8 weeks. Results showed that MEPP cells stably and rapidly formed tumors, producing significant ascites and metastatic lesions. Histological sections and IHC staining confirmed the expression of the ovarian cancer marker Wt1 in the tumors, clarifying their ovarian cancer characteristics.

[0088] like Figure 5 As shown in Figure A, subcutaneous injection of 3×10 6 Fourteen days after MEPP cell induction, solid masses (white arrows) appeared on the dorsal side of mice. The tumors had clear borders and local skin elevation, suggesting that this model can rapidly form subcutaneous tumors within two weeks.

[0089] like Figure 5 Figure B shows the H&E staining and Wt1 immunohistochemical results of MEPP tumors induced by subcutaneous injection. H&E staining reveals disordered cell arrangement, large and deeply stained nuclei, prominent nucleoli, and mitotic figures in some cells, indicating significant malignant tumor morphological characteristics (top image). Immunohistochemical staining shows brownish-red nuclear expression, indicating Wt1 positivity, consistent with molecular markers of human serous ovarian cancer, validating the ovarian cancer specificity of this model (bottom image).

[0090] like Figure 5 As shown in C, 1×10⁻⁶ t / v was injected subcapsularly into the ovary. 6 Eight weeks after treatment, MEPP cells showed significant ascites (top image) and multiple peritoneal metastases (bottom image). This indicates that MEPP cells not only rapidly form tumors in situ, but also mimic the diffuse dissemination characteristics commonly seen in clinical ovarian cancer.

[0091] like Figure 5 Figure D shows the H&E staining results and Wt1 immunohistochemical results of the in situ tumor after subcapsular injection of MEPP. H&E reveals disordered cell arrangement, large and deeply stained nuclei, prominent nucleoli, and mitotic figures in some cells, indicating significant malignant tumor morphology (top image). Immunohistochemical staining of brownish-red nuclei suggests Wt1 positivity, consistent with molecular markers of human serous ovarian cancer, validating the ovarian cancer specificity of this model (bottom image).

[0092] like Figure 5Figure E shows the H&E staining results and Wt1 immunohistochemical results of omental metastatic tumors after subcapsular injection of MEPP. H&E reveals disordered cell arrangement, large and deeply stained nuclei, prominent nucleoli, and mitotic figures in some cells, indicating significant malignant tumor morphological characteristics (top image). Immunohistochemical brown nuclear expression suggests Wt1 positivity, consistent with molecular markers of human serous ovarian cancer, validating the ovarian cancer specificity of this model (bottom image).

[0093] 5. Single-cell transcriptome characterization of MEPP tumors

[0094] scRNA-seq analysis was performed on MEPP tumors and their metastases obtained through subcapsular injection into the ovary to clarify the characteristics of tumor cells and the immune microenvironment. The transcriptomic features of the MEPP model tumors were found to be similar to those of human ovarian cancer, and the immune microenvironment was also highly similar, including abundant infiltration of immune cells such as T cells, NK cells, macrophages, and dendritic cells, validating its clinical relevance. The results are as follows: Figure 6 As shown.

[0095] like Figure 6 As shown in Figure A, this is an unsupervised dimensionality reduction clustering of the MEPP single-cell dataset. The results show that MEPP tumors contain tumor, stroma, and various immune cell components.

[0096] like Figure 6 As shown in Figure B, the characteristic gene expression of cell lineages in the MEPP single-cell dataset is presented, which verifies the accuracy of the annotation of each cell cluster.

[0097] Example 2 – Application of Ovarian Cancer Model in Large-Scale Drug Screening

[0098] 1. Objective: To validate the MEPP model for large-scale drug sensitivity screening and to discover candidate compounds for PTEN / TP53 double-deleted ovarian cancer;

[0099] 2. Take MEPP cells in the logarithmic growth phase (MEPP cell line obtained in step 3 of Example 1), digest with trypsin, count them, and seed them in 96-well plates at 5,000 cells / well, with 100 µl of complete culture medium added to each well;

[0100] 3. Two compound libraries were used, containing 328 epigenetic regulators (APExBIO Cat:L1029) and 1655 natural compounds (Cat:L1039P);

[0101] 4. All compounds were added at a final concentration of 5 µM, with a DMSO control group included, and incubated for 72 h.

[0102] 5. Add 10 µl of CCK8 reagent (Life-iLab), incubate at 37℃ for 2 h, and then measure the absorbance at 450 nm;

[0103] 6. A survival rate reduction of ≥1.5 times compared to the DMSO control group is defined as a sensitive candidate;

[0104] 7. In vivo validation: In the MEPP subcutaneous tumorigenesis mouse model, intraperitoneal administration of FK228 (1 mg / kg) and Thioguanine (1.5 mg / kg) twice weekly for 3 weeks significantly inhibited tumor volume growth (p<0.01). Figure 7 As shown in Figure A), and decreased Ki67 expression was observed in IHC of tumor tissue (as shown in Figure A). Figure 7 As shown in Figure B), an increase in TUNEL-positive cells (such as...). Figure 7 (As shown in C).

[0105] To demonstrate the unexpected synergistic effect of combining the EPI carrier with SauriCas9 in this invention, and to prove the possibility that the same effect can be achieved by eliminating a single technical component, the following comparative experiment was designed.

[0106] Comparative Example 1 – Primary MOSE cells derived from genetically engineered mice (Pten) flox / flox ;Trp53 flox / flox )

[0107] Take C57BL / 6 Pten flox / flox ;Trp53 flox / flox Genetically engineered adult female mouse ovaries were used to prepare primary MOSE cells according to step 1 of Example 1, "Source and Culture of Primitive Cells". These cells were then infected with Cre recombinant lentivirus (MOI=50), and the medium was changed 48 h after infection. On day 7 post-infection, all cells were observed to have undergone apoptosis (e.g., ...). Figure 8 As shown in the figure, no proliferation colonies were observed in the culture dish, proving that Pten / Trp53 knockout using epithelial cells from genetically engineered mice is insufficient to enable them to form tumors, and MOSE relying solely on the Cre-flox system cannot enable cells to survive and expand.

[0108] Comparative Example 2 – SauriCas9 + conventional vector (without EPI system)

[0109] Following the method described in step 2 of Example 1, a control plasmid (V8-SauriCas9, e.g., V8-SauriCas9) containing SauriCas9 and dual U6-sgRNA (targeting Pten, Trp53) without the OriP / EBNA1 element was constructed. Figure 9As shown), MOSE cells were transfected with EZ Trans, and 5 µg / mL Puromycin was added for selection. After 5 days, the cell survival rate was <10%, and most cells underwent apoptosis (as shown). Figure 10 As shown in the figure, it proves that using a conventional vector combined with SauriCas9 is insufficient to enable MOSE cells to have tumorigenic ability.

[0110] Therefore, comparing the above groups, the EPI vector used in this invention, in synergy with SauriCas9, can induce subcutaneous tumors with a diameter >10 mm in C57BL / 6 mice within 2 weeks (e.g., Figure 5 As shown in Figure A), a large amount of ascites can be formed in the peritoneal cavity of C57BL / 6 mice within 8 weeks and widely metastasize (e.g., Figure 5 As shown in Figure C), the tissue morphology and biomarker expression are consistent with the characteristics of ovarian cancer, which improves the tumor formation efficiency.

[0111] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for rapidly establishing an ovarian cancer model based on SauriCas9, characterized in that, Includes the following steps: S1. Transfect recombinant plasmids targeting the knockout of Pten and Trp53 genes into primary mouse MOSE cells; S2. Stable cell lines are obtained through screening, which are MEPP cells; S3. MEPP cells were inoculated into immune-intact mice to form tumors; In step S1, the recombinant plasmid that targets and knocks out the Pten and Trp53 genes includes an EPI vector system, which includes an OriP element and an EBNA1 protein expression element. The recombinant plasmid uses the ori element as the replication initiation site and includes, in sequence, an sgRNA sequence targeting the Trp53 gene and Pten, controlled by the dual U6 promoter, a CAG promoter, a SauriCas9 nuclease expression unit, a fluorescent protein expression element, an antibiotic resistance gene, and an orip element and an EBNA1 protein expression element.

2. The method according to claim 1, characterized in that, The sgRNA sequence targeting the Pten gene is shown in SEQ ID NO.

1. The sgRNA sequence targeting the Trp53 gene is shown in SEQ ID NO.

2.

3. The method according to claim 1, characterized in that, The fluorescent protein includes green fluorescent protein or red fluorescent protein, and the green fluorescent protein includes ZsGreen fluorescent protein; And / or, the resistance gene includes the Puromycin resistance gene or the neomycin resistance gene.

4. The method according to claim 1, characterized in that, The method for constructing the recombinant plasmid includes: using epiCRISPR, PCR to obtain the OriP element and EBNA1 protein, and sequentially inserting the CAG promoter, SauriCas9 nuclease, fluorescent protein expression element, resistance gene, and sgRNA sequence targeting the Pten and Trp53 genes controlled by the dual U6 promoter using the Gibson assembly method.

5. The method according to claim 1, characterized in that, In step S1, the primary mouse MOSE cells are mouse ovarian surface epithelial cells isolated from adult female C57BL / 6 mice and obtained by digestion with TrypLE enzyme.

6. The method according to claim 1, characterized in that, In step S2, stable cell lines are obtained by screening using resistance genes; After obtaining MEPP cells, the knockout of Pten and Trp53 genes in the cells was verified by PCR and sequencing technology.

7. The method according to claim 1, characterized in that, In step S3, the MEPP cells obtained in step S2 are processed at a rate of 1×10⁻⁶. 6 ~3×10 6 When mixed with Matrigel at a concentration of cells / μl, the mixture was inoculated subcutaneously or into the ovarian capsule of immunized C57BL / 6 mice to rapidly form tumors; the Matrigel included Matrigel.

8. An ovarian cancer cell model, constructed using the method described in any one of claims 1-7.

9. The application of the ovarian cancer cell model as described in claim 8 in ovarian cancer drug screening.

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