Method for rapidly establishing ovarian cancer model based on SauriCas9

Abstract

The invention discloses a method for rapidly establishing an ovarian cancer model based on SauriCas9, and particularly discloses a recombinant plasmid for targeted knockout of Pten and Trp53 genes, and the recombinant plasmid comprises an EPI vector system. The recombinant plasmid takes an ori element as a replication start site, and sequentially comprises an sgRNA sequence of a targeted Trp53 gene and Pten controlled by double U6 promoters, a CAG promoter, a SauriCas9 nuclease expression unit, a fluorescent protein expression element, a resistance gene, an orip element and an EBNA1 protein expression element. The invention also discloses a method for rapidly establishing an ovarian cancer model based on SauriCas9, and the established ovarian cancer cell model. By adopting the method to construct the ovarian cancer cell model, the period from cell editing to animal tumor formation 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 only relies on spontaneous gene mutation or limited gene modification. The GEMM model, although it achieves precise genetic modification (such as Pten and Trp53 knockout), has a long construction cycle (usually 1-2 years), is complex to maintain and propagate, and is costly, 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 a complex mouse breeding system, 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-competent 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, complete immunity, and efficient drug screening.

[0007] The purpose of the present application is achieved by the following technical solutions: 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 OriP elements and EBNA1 protein expression elements. The recombinant plasmid takes the ori element as a replication initiation site, and sequentially comprises sgRNA sequences targeting the Trp53 gene and the Pten, 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, which are controlled by a double U6 promoter.

[0008] As some specific embodiments of the present application, the sgRNA sequence targeting the Pten gene is shown as SEQ ID NO. 1. The sgRNA sequence targeting the Trp53 gene is shown as SEQ ID NO. 2.

[0009] 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.

[0010] As some specific embodiments of the present application, the resistance gene comprises a Puromycin resistance gene or a Neomycin resistance gene, and preferably, the Puromycin resistance gene.

[0011] 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.

[0012] In a second aspect, the present application provides a method for rapidly establishing an ovarian cancer model based on SauriCas9, comprising the following steps: S1. Transfecting the recombinant plasmid of any one of the above into mouse primary MOSE cells; S2. Screening to obtain a stable cell line, which is a MEPP cell; S3. Inoculating the MEPP cell into an immunocompetent mouse to form a tumor.

[0013] 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, which are obtained after TrypLE enzyme digestion.

[0014] 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.

[0015] As some specific embodiments of the present invention, in step S2, stable cell lines are obtained by screening through resistance genes; And / or, after obtaining MEPP cells in step S2, the knockout status of Pten and Trp53 genes in the cells is verified by PCR and sequencing technologies.

[0016] As some specific embodiments of the present invention, in step S3, the MEPP cells obtained in step S2 are processed at a rate of 1×10⁻⁶. 6 ~3×10 6 A mixture of cells / μl and Matrigel was inoculated subcutaneously or into the ovarian capsule of immunized C57BL / 6 mice, rapidly forming tumors. The Matrigel included Matrigel.

[0017] In some specific embodiments, the MEPP cells obtained in step S2 are processed at a rate of 3 × 10⁻⁶. 6 When mixed with matrix gel at a concentration of cells / μl, the mixture was subcutaneously injected into immunized C57BL / 6 mice, and tumors rapidly formed within one week.

[0018] In some specific embodiments, the MEPP cells obtained in step S2 are processed at a rate of 1×10⁻⁶. 6 After being mixed with matrix gel at a concentration of cells / μl, the mixture was inoculated into the ovarian capsule of immune-intact C57BL / 6 mice, which rapidly formed tumors, metastases, and ascites within 2 months.

[0019] Thirdly, the present invention provides an ovarian cancer cell model, which is constructed using the method described in any of the preceding claims.

[0020] Fourthly, this invention provides an application of an ovarian cancer cell model in screening drugs for the treatment of ovarian cancer.

[0021] The paper "CRISPR / Cas9-mediated knockout of Trp53 and Brca2 generates an improved mouse model of high-grade ovarian serous carcinoma" used CRISPR / Cas9 technology to knock out Trp53 and Brac2 in the ID8 cell line to establish a mouse model. However, there are still some limitations and shortcomings: 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.

[0022] 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.

[0023] Compared with the prior art, the present invention has the following beneficial effects: 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.

[0024] 2) Stability of the genetic background of the model and immune integrity: In immune-competent C57BL / 6 mice, a stable tumor was rapidly established, 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 various immune cell types (macrophages, T cells, NK cells, dendritic cells, etc.), which is more similar to the immune microenvironment of human ovarian cancer.

[0025] 3) Economic and social effects (drug screening and potential for translational application): The MEPP model is rapidly constructed, low-cost, and suitable for large-scale drug screening and high-throughput experiments, effectively reducing research costs; actual drug screening found 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 technology platform provided by the present application provides a solid foundation and prospect for the development of new drugs for the treatment of ovarian cancer, and has significant social and economic value.

[0026] 4) Compared with the method of directly knocking out Pten and Trp53 genes by genetic engineering (Comparative Example 1), the present application does not require the prior construction of complex genetically engineered mice, and avoids the defects of rapid apoptosis of primary cells after Cre virus infection and inability to expand. After EPI+SauriCas9, double gene knockout cell lines (MEPP) can be obtained by transfecting primary cells once, which can rapidly form tumors in vivo, while Comparative Example 1 cannot survive and expand after virus infection, and cannot form tumors or metastasis. Compared with the SauriCas9+ conventional vector scheme (Comparative Example 2): when using conventional vectors (without EPI), the cells rapidly undergo apoptosis after Puromycin selection, and stable cell lines cannot be obtained, and they completely lack the ability to form tumors in vivo. The EPI vector used in the present application carries the OriP / EBNA1 element, which allows SauriCas9 and sgRNA to be expressed continuously and efficiently, avoiding the toxicity of lentiviral genome integration, ensuring long-term survival, stable expansion, rapid tumor formation and metastasis of cells. BRIEF DESCRIPTION OF DRAWINGS

[0027] Other features, objects, and advantages of the present application will become more apparent from the following detailed description of non-limiting embodiments, with reference to the following drawings: Figure 1 Figure 1 is a schematic diagram of the EPI-SauriCas9-sgRNA plasmid structure constructed in Example 1; Figure 2 Figure 2 is a Sanger sequencing result graph after plasmid construction and transfection for different sgRNAs in Example 1; Figure 3Optical and fluorescent microscopy images of EPI-SauriCas9-sgRNA transfected into MOSE cells for Example 1; wherein, left panel is optical microscopy image; right panel is fluorescent microscopy image; Figure 4 Sanger sequencing results of Pten and Trp53 target site sequences of MEPP cell DNA constructed in Example 1; Figure 5 Tumorigenic and pathological features results of MEPP cells in immunocompetent C57BL / 6 mice, wherein, A is the tumor characteristics of MEPP cells subcutaneously injected with 3x10 6 Tumor characteristics of MEPP cells 2 weeks after injection; B is the H&E staining and Wt1 immunohistochemistry results of MEPP subcutaneous tumor tissue; C is the H&E staining and Wt1 immunohistochemistry results of MEPP ovarian subcapsular injection with 1x10 6 Tumor characteristics of MEPP cells 8 weeks after injection; D is the H&E staining and Wt1 immunohistochemistry results of MEPP ovarian orthotopic tissue; E is the H&E staining and Wt1 immunohistochemistry results of MEPP omentum metastatic tissue; Figure 6 RNA-seq profile of MEPP tumor single cells in Example 1, wherein, A is the unsupervised dimensionality reduction clustering plot, B is the characteristic gene expression plot of all cell clusters; Figure 7 In vivo validation of MEPP drug screening in Example 2, wherein, A is the tumor volume curve after FK228 and Thioguanine treatment, B is the Ki67 immunohistochemistry score plot, C is the TUNEL apoptosis cell staining plot; Figure 8 Cell proliferation results of genetically engineered mouse Pten flox / flox ;Trp53 flox / flox primary cells after infection, wherein, upper panel is optical microscopy image, lower panel is fluorescent microscopy image; Figure 9 Plasmid structure schematic diagram of V8-SauriCas9 constructed in Comparative Example 2; Figure 10 Cell proliferation results of Pten / Trp53 knockout using SauriCas9 + conventional vector in Comparative Example 2, wherein, upper panel is optical microscopy image, lower panel is fluorescent microscopy image. DETAILED DESCRIPTION

[0028] The application will be described in greater detail below with reference to specific embodiments. The following examples will help those skilled in the art further understand the application, but do not limit the application in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the application. These all belong to the protection scope of the application.

[0029] 1. The application provides a quickly established immune-intact mouse ovarian cancer model, and the specific steps are as follows: (1) Mouse ovarian surface epithelial cells (MOSE) are isolated from C57BL / 6 adult female mice, and primary MOSE cells are obtained after TrypLE enzyme digestion; (2) A plasmid expression vector containing an EPI vector system (containing OriP elements and EBNA1 protein) and SauriCas9 nuclease is constructed. The plasmid also contains sgRNA expression elements controlled by double U6 promoters, which target Pten and Trp53 genes, respectively, and contains a fluorescent protein (ZsGreen) and a Puromycin resistance gene; (3) The above-mentioned plasmid is transfected into MOSE cells by EZ trans, and 24 hours later, the transfection efficiency is observed by observing ZsGreen fluorescence. Stable transfection cell screening is carried out by Puromycin, and a stable cell strain is established within about 1 week; (4) The MOSE cells after transfection and screening are MEPP cells, and the knockout of Pten and Trp53 genes in the cells is verified by PCR and sequencing techniques; (5) The MEPP cells (3×10 6 ) are mixed with Matrigel and inoculated subcutaneously in immune-intact C57BL / 6 mice, and tumors are quickly formed within 1 week; Or, the MEPP cells (1×10 6 ) are mixed with Matrigel and inoculated into the ovarian capsule of immune-intact C57BL / 6 mice, and tumors, metastases and ascites are quickly formed within 2 months.

[0030] 2. Each technical element of the application and its specific role: MOSE primary cells: provide a stable and clear source of ovarian epithelial cells, ensuring a pure genetic background of the model; EPI vector system (containing OriP+EBNA1 expression elements): the plasmid can be stably replicated in cells with cell division, improving transfection efficiency and avoiding the safety risks of viral vectors; SauriCas9 nuclease (NNGG PAM): small volume, high editing efficiency and strong flexibility, ensuring precise and efficient knockout of Pten and Trp53 genes.

[0031] 3. Identification characteristics of biological materials: MEPP cells are typical epithelial cells, and the expression of Pax8 and Wt1 genes is significantly higher than that of mouse mesenchymal stem cells; According to scRNA-seq analysis, MEPP tumor cells can be distinguished into several subgroups, and have obvious EMT, proliferation and metastasis related gene characteristics; MEPP cells have good passage stability, and can quickly form tumors after subcutaneous or ovarian inoculation; In the tumors formed in immunocompetent mice, there are complete immune cell infiltrations (such as macrophages, T cells, NK cells, etc.), effectively simulating the immune microenvironment of human ovarian cancer.

[0032] Example 1 - Construction and verification of MEPP ovarian cancer cell model 1. Source and culture of original cells The ovaries of female C57BL / 6 mice (6-8 weeks) were taken, and Trpzyme recombinant trypsin digestion solution (derived from BasalMedia) was used for 37°C digestion for 30 minutes to obtain MOSE primary cells, which were inoculated in DMEM medium containing 10% FBS (fetal bovine serum), 100 U / mL penicillin-streptomycin, and cultured at 37°C, 5% CO2.

[0033] 2. Vector construction and gene editing method OriP elements and EBNA1 protein were obtained by PCR using epiCRISPR (addgene #135960), and were sequentially inserted into CAG promoter, SauriCas9 nuclease, ZsGreen fluorescent gene, Puromycin resistance gene, and sgRNA sequences targeting Pten and Trp53 genes controlled by double U6 promoter by Gibson assembly method to construct EPI-SauriCas9-sgRNA expression plasmid (as shown in Figure 1 ).

[0034] The constructed EPI-SauriCas9-sgRNA expression plasmid takes ori elements as the replication initiation site, and sequentially includes sgRNA sequences targeting Trp53 gene and Pten controlled by double U6 promoter, CAG promoter, SauriCas9 nuclease expression unit, fluorescent protein expression element, resistance gene, and orip and EBNA1 protein expression element. Among them, the fluorescent protein is ZsGreen fluorescent protein, and the resistance gene is Puromycin resistance gene.

[0035] In order to screen suitable sgRNA sequences targeting Pten and Trp53 genes, 3 pairs of sgRNA sequences were designed, and Hepa1-6 cell line transfection plasmid was used for verification.

[0036] The sequences of the 3 pairs of sgRNA are as follows: sgRNA-pair1: sgPten: 5'-TGGTGGGTTATGGTCTTCAA-3' (SEQ ID NO. 1); sgTrp53: 5'-TATCCGACTGTGACTCCTCC-3' (SEQ ID NO. 2).

[0037] sgRNA-pair2: sgPten: 5'-GAAACAAAAGGAGATATCAA-3' (SEQ ID NO. 3); sgTrp53: 5'-TAGATGGCCATGGCGCGGAC-3' (SEQ ID NO. 4).

[0038] sgRNA-pair3: sgPten: 5'-TGCTAACGATCTCTTTGATG-3' (SEQ ID NO. 5); sgTrp53: 5'-GACACTCGGAGGGCTTCACT-3' (SEQ ID NO. 6).

[0039] For example, the sequence of the constructed plasmid EPI-SauriCas9-sgRNA-pair1 is composed of SEQ ID NO. 7 and SEQ ID NO. 8 (totally 14972 sites). Among them, the position of the replication initiation site ori is 14128-14716, the position of 49-289 is U6 promoter, the position of 300-319 is Trp53-sgRNA1, the position of 423-663 is U6 promoter, the position of 673-692 is Pten-sgRNA1, the position of 1012-2373 is CAG promoter, the position of 2816-6100 is sauriCas9, the position of 6164-6856 is ZSgreen, the position of 6998-7594 is PuroR, the position of 8454-10244 is OriP, and the position of 10545-12471 is EBNA1.

[0040] As shown in FIG. 1, the Sanger sequencing results of the plasmids constructed for different sgRNAs in this embodiment after transfection into Hepa1-6 cells are shown. As can be seen from FIG. 1, pair3 has fewer indels, and pair1 and pair2 can effectively produce indels, so Pten / Trp53 sgRNA-pair1 and pair2 have better editing efficiency. The sgRNA-pair1 (SEQ ID NO. 1; SEQ ID NO. 2) is selected for subsequent cell experiments. Figure 2 Figure 2 As shown in FIG. 1, the Sanger sequencing results of the plasmids constructed for different sgRNAs in this embodiment after transfection into Hepa1-6 cells are shown. As can be seen from FIG. 1, pair3 has fewer indels, and pair1 and pair2 can effectively produce indels, so Pten / Trp53 sgRNA-pair1 and pair2 have better editing efficiency. The sgRNA-pair1 (SEQ ID NO. 1; SEQ ID NO. 2) is selected for subsequent cell experiments.

[0041] ​3. MOSE cell transfection and establishment of stable cell lines 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.

[0042] 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.

[0043] 4. Methods for validating in vivo tumorigenesis and tumor models 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.

[0044] 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.

[0045] 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).

[0046] As Figure 5 As shown in FIG. 1C, the MEPP cells were subcutaneously injected into the ovarian capsule of the nude mice at a dose of 1x10 6 MEPP cells can be seen significant ascites (top) and multiple peritoneal metastases (bottom) after 8 weeks. It is shown that MEPP cells not only in situ tumor rapidly, but also can simulate the common diffuse dissemination characteristics of clinical ovarian cancer.

[0047] As Figure 5 As shown in FIG. 1D, the H&E staining results and Wt1 immunohistochemical results of the in situ tumor of MEPP after subcapsular injection, H&E can see the cell arrangement disorder, nucleus large and deep staining, nucleolus obvious, part of the cell appears nuclear mitotic figure, prompt obvious malignant tumor morphological characteristics (top). Immunohistochemical brown nuclear expression suggests Wt1 positive, consistent with the molecular marker characteristics of human serous ovarian cancer, verify the specificity of the ovarian cancer model (bottom).

[0048] As Figure 5 As shown in FIG. 1E, the H&E staining results and Wt1 immunohistochemical results of the omental metastatic tumor of MEPP after subcapsular injection, H&E can see the cell arrangement disorder, nucleus large and deep staining, nucleolus obvious, part of the cell appears nuclear mitotic figure, prompt obvious malignant tumor morphological characteristics (top). Immunohistochemical brown nuclear expression suggests Wt1 positive, consistent with the molecular marker characteristics of human serous ovarian cancer, verify the specificity of the ovarian cancer model (bottom).

[0049] 5、MEPP tumor single cell transcriptome characteristics analysis The MEPP tumor obtained by subcapsular injection of the ovary and its metastatic tumor were subjected to scRNA-seq analysis to determine the characteristics of tumor cells and immune microenvironment. It was found that the transcriptome characteristics of the MEPP model tumor were similar to those of human ovarian cancer, and the immune microenvironment was also highly similar, including rich immune cell infiltration such as T cells, NK cells, macrophages, dendritic cells, etc. The clinical relevance was verified, and the results are shown in FIG. 1F. Figure 6

[0050] As Figure 6 As shown in FIG. 1A, the unsupervised dimensionality reduction clustering of the MEPP single cell data set showed that the MEPP tumor contained tumor, stroma and various immune cell components.

[0051] As Figure 6 As shown in FIG. 1B, the cell lineage characteristic gene expression of the MEPP single cell data set verified the accuracy of the annotation of each cell cluster.

[0052] Example 2--Application of ovarian cancer model in large-scale drug screening 1、Objective: To verify that the MEPP model can be used for large-scale drug sensitivity screening and find candidate compounds for PTEN / TP53 double deletion ovarian cancer;​ 2. Take the logarithmic growth phase MEPP cells (MEPP cell strain obtained in step 3 of Example 1), trypsinize, count, and inoculate 5,000 cells / well into a 96-well plate with 100 μl of complete medium per well; 3. Use two sets of compound libraries, one containing 328 epigenetic modulators (APExBIO Cat: L1029) and the other containing 1655 natural compounds (Cat: L1039P); 4. Each compound is added at a final concentration of 5 μM, with a DMSO control group, and incubated for 72 h; 5. Add 10 μl of CCK8 reagent (Life-iLab), incubate at 37°C for 2 h, and then measure the absorbance at 450 nm; 6. A decrease in survival rate of ≥1.5-fold relative to the DMSO control group is defined as a sensitive candidate; 7. In vivo verification: in a MEPP subcutaneous tumor mouse model, FK228 (1 mg / kg) and Thioguanine (1.5 mg / kg) were administered intraperitoneally twice a week for 3 weeks, significantly inhibiting tumor volume growth (p<0.01) (as shown in Figure 7 A), and a decrease in Ki67 expression (as shown in Figure 7 B) and an increase in TUNEL-positive cells (as shown in Figure 7 C) were observed in tumor tissue IHC.

[0053] To demonstrate the unexpected synergistic effect produced by the combination of the EPI vector in the present application and SauriCas9, and to rule out the possibility that the same effect can be achieved by a single technical element, the following comparative experiment was designed.

[0054] Comparative Example 1 - MOSE primary cells from genetically engineered mice (Pten flox / flox ;Trp53 flox / flox ) Take the ovary of a C57BL / 6 Pten flox / flox ;Trp53 flox / flox genetically engineered adult female mouse, prepare MOSE primary cells according to the "source and culture of primary cells" in step 1 of Example 1, infect the MOSE primary cells with Cre recombinant lentivirus (MOI=50), and replace the culture medium after 48 h of infection; on the 7th day after infection, observe that all the cells have undergone apoptosis (as shown in Figure 8 ), and no proliferative colonies are observed in the culture dish, proving that Pten / Trp53 knockout using epithelial cells from genetically engineered mice is not sufficient to make them tumorigenic, and that MOSE relying solely on the Cre-flox system cannot make cells survive and expand.

[0055] Comparative Example 2 - SauriCas9 + conventional vector (without EPI system) Following the method of "vector construction" in Example 1, Step 2, a control plasmid (V8-SauriCas9, as shown in Figure 9 ) containing SauriCas9 and dual U6-sgRNA (targeting Pten and Trp53) without OriP / EBNA1 elements was constructed and transfected into MOSE cells using EZ Trans, with 5 µg / mL Puromycin for selection. After 5 days, the survival cells were <10% and most cells were apoptotic (as shown in Figure 10 ), demonstrating that SauriCas9 with conventional vector was not sufficient to make MOSE cells tumorigenic.

[0056] Thus, compared with the above groups, the EPI vector used in the present application synergizes with SauriCas9, and within 2 weeks, it can form tumors with a diameter >10 mm in C57BL / 6 mice (as shown in Figure 5 A), and within 8 weeks, it can form a large amount of ascites and extensive metastasis in C57BL / 6 mice (as shown in Figure 5 C), and the histomorphology and marker expression are consistent with the characteristics of ovarian cancer, improving the tumorigenic efficiency.

[0057] The specific embodiments of the present application are described above. It should be understood that the present application is not limited to the specific embodiments described above, and various modifications or changes can be made by those skilled in the art within the scope of the claims, which do not affect the essential content of the present application.

Claims

1. A method for rapid establishment of an ovarian cancer model based on SauriCas9, characterized by, The method comprises the following steps: S1, transfecting a recombinant plasmid for targeting knockout of Pten and Trp53 genes into mouse primary MOSE cells; S2, screening to obtain a stable cell strain, i.e. MEPP cells; S3, inoculating the MEPP cells into immunocompetent mice to form tumors; In step S1, the recombinant plasmid for targeting knockout of Pten and Trp53 genes comprises an EPI vector system, wherein the EPI vector system comprises OriP elements and EBNA1 protein expression elements; The recombinant plasmid takes the ori element as a replication initiation site, and comprises, in sequence, 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.

2. The method of claim 1, wherein, The sgRNA sequence for targeting the Pten gene is shown in SEQ ID NO. 1, The sgRNA sequence for targeting the Trp53 gene is shown in SEQ ID NO.

2.

3. The method of claim 1, wherein, The fluorescent protein comprises a green fluorescent protein or a red fluorescent protein, and the green fluorescent protein comprises a ZsGreen fluorescent protein. And / or, the resistance gene comprises a Puromycin resistance gene or a Neomycin resistance gene.

4. The method of claim 1, wherein, The construction method of the recombinant plasmid comprises: obtaining OriP elements and EBNA1 protein by PCR using epiCRISPR, and inserting, in sequence, a CAG promoter, a SauriCas9 nuclease, a fluorescent protein expression element, a resistance gene, and sgRNA sequences for targeting Pten and Trp53 genes controlled by a double U6 promoter by using a Gibson assembly method.

5. The method of claim 1, wherein, In step S1, the mouse primary MOSE cells are mouse ovarian surface epithelial cells isolated from a C57BL / 6 adult female mouse and obtained after TrypLE enzyme digestion.

6. The method of claim 1, wherein, In step S2, the stable cell strain is obtained by screening through the resistance gene; After obtaining the MEPP cells, the knockout of Pten and Trp53 genes in the cells is verified by PCR and sequencing techniques.

7. The method of claim 1, wherein, In step S3, the MEPP cells obtained in step S2 are mixed with Matrigel matrix at a concentration of 1 x 10 6 3 x 10 6 cells / μl and inoculated into the subcutis or the ovarian capsule of immunocompetent C57BL / 6 mice to form tumors rapidly.

8. An ovarian cancer cell model obtained by using the method according to any one of claims 1-7.

9. Use of the ovarian cancer cell model according to claim 8 in ovarian cancer drug screening.

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