Construction method and application of nf2, wwc1 and wwc2 gene knockout mouse animal models and type ii neurofibroma primary cell lines
By simultaneously knocking out the Nf2, Wwc1, and Wwc2 genes in mouse Schwann cells, a rapidly developing neurofibroma model and primary cell line were constructed, overcoming the limitations of existing models and providing a stable and reliable tool for drug screening and research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- FUDAN UNIVERSITY
- Filing Date
- 2024-05-11
- Publication Date
- 2026-06-30
AI Technical Summary
Existing mouse models and primary tumor cell lines are insufficient for constructing rapidly developing type II neurofibromas, limiting their application in preclinical studies. Furthermore, existing treatments carry risks of side effects and nerve tissue damage.
By simultaneously and specifically knocking out the Nf2, Wwc1, and Wwc2 genes in mouse Schwann cells, and using Cre tools and tamoxifen induction, Nf2-/-; Wwc1-/-; Wwc2-/-; Plp1-CreERT2 mouse models were constructed, and subcutaneous neurofibromas spontaneously formed. Primary cell lines were then isolated and cultured.
We constructed a rapidly developing and highly stable neurofibroma model and corresponding primary cell lines to simulate human type II neurofibroma, providing an economical and reliable tool for drug screening and molecular mechanism research.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to the construction and application of Nf2, Wwc1 and Wwc2 gene knockout mouse animal models and primary cell lines for type II neurofibromatosis. Background Technology
[0002] Neurofibromatosis type 2 (NF2) is a hereditary neurological tumor syndrome primarily caused by mutations in the Neurofibromin 2 (NF2) gene on chromosome 22. However, the molecular mechanism underlying NF2 deficiency remains unclear. With an incidence of approximately 1 in 30,000 people, it is a rare disease. Current standard treatments primarily involve surgery and radiation therapy. However, surgery carries a high risk of nerve tissue damage and may result in complications such as deafness or death. Existing treatments, including bevacizumab, have shown some efficacy in treating neurofibromatosis, but long-term use is accompanied by side effects such as hypertension, fatigue, and headaches. Therefore, there is an urgent need to develop novel targeted therapies.
[0003] Animal tumor models and primary tumor cell lines are of great significance for drug development and preclinical research. Current research uses specific knockout of the Nf2 gene in Schwann cells of mice to create animal models of neurofibromatosis. However, neurofibromas caused by simple Nf2 gene deletion progress very slowly, often occurring late in the mouse's life, and have a low incidence rate. This significantly limits the application of such mouse models and corresponding primary tumor cell lines in preclinical studies. Developing rapidly developing, highly pathogenic mouse neurofibromatosis models and corresponding tumor cell lines is of great value and significance. Summary of the Invention
[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing methods for constructing and applying Nf2, Wwc1, and Wwc2 gene knockout mouse models and primary cell lines for neurofibromatosis type II.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0006] The first aspect of this invention is to provide a method for constructing an animal model of Nf2, Wwc1, and Wwc2 gene knockout mice, comprising the following steps:
[0007] Step 1: Using ES cell targeting, conditional knockout mice of Nf2, Wwc1, and Wwc2 were constructed respectively. After obtaining homozygous F1 generation mice of each gene, the mice of the three genes were crossed. After multiple generations of crosses, mice with the genotypes Nf2- / -, Wwc1- / -, and Wwc2- / - were finally obtained.
[0008] Step 2: Using the Schwann cell-specific Cre tool mouse Plp1-CreERT2, the Cre tool mouse is crossed with mice with the genotypes Nf2- / -; Wwc1- / -; Wwc2- / - to finally obtain Nf2- / -; Wwc1- / -; Wwc2- / -; Plp1-CreERT2 mice.
[0009] Furthermore, after tamoxifen-induced Plp1-Cre expression in the Nf2- / -;Wwc1- / -;Wwc2- / -;Plp1-CreERT2 mice, the three genes Nf2, Wwc1, and Wwc2 can be simultaneously knocked out in Schwann cells of mice.
[0010] The second aspect of this invention is to provide a method for constructing a primary cell line for type II neurofibroma. Nf2- / -;Wwc1- / -;Wwc2- / -;Plp1-CreERT2 mice spontaneously form subcutaneous schwannomas after being induced to express Plp1-Cre by tamoxifen for 7-8 months. The primary cell line for type II neurofibroma can be obtained by isolating and primary culturing the subcutaneous schwannomas.
[0011] A third aspect of this invention is to provide the application of the above-mentioned Nf2, Wwc1 and Wwc2 gene knockout mouse animal models in the preparation and screening of drugs for the treatment of neurofibromatosis type II.
[0012] A fourth aspect of this invention is to provide the application of the above-mentioned primary cell line for type II neurofibroma in the preparation and screening of drugs for the treatment of type II neurofibroma.
[0013] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:
[0014] This invention establishes a rapidly developing neurofibromatosis mouse model by simultaneously and specifically knocking out three genes—Nf2, Wwc1, and Wwc2—in Schwann cells. This model exhibits strong stability and stable inheritance, closely resembling human neurofibromatosis type II, and provides an economical, simple, and reliable animal model for the preparation and screening of drugs for treating neurofibromatosis type II. Furthermore, the primary tumor cell lines derived from this mouse model express specific molecular markers of human neurofibromatosis type II, which is beneficial for drug screening and molecular mechanism research in preclinical studies. Attached Figure Description
[0015] Figure 1 A represents the control group in this embodiment of the invention, consisting of Nf2, Wwc1-2, and Nf2; gross anatomical photograph of the DRG of Wwc1-2 gene knockout mice approximately 3 months after Cre. Figure 1 B represents the volume quantification results of the DRG in each gene knockout mouse. Figure 1 C shows the H&E staining, Schwann cell characteristic markers (such as SOX10, S100β), and proliferation markers (such as YAP / TAZ, Ki67) immunofluorescence staining results of DRGs from gene knockout mice. Figure 1 D shows the quantitative results of DRG proliferation signals in each gene knockout mouse.
[0016] Figure 2 The experimental results show that a primary cell line for type II neurofibroma was successfully constructed. Figure 2 A shows a gross photograph of spontaneously formed subcutaneous neurofibromas in Nf2- / -; Wwc1- / -; Wwc2- / -; Plp1-CreERT2 mice; Figure 2 B shows the immunofluorescence staining results of primary tumor cell lines isolated and cultured from this subcutaneous neurofibroma, expressing Schwann cell characteristic molecules.
[0017] Figure 3 Single-cell sequencing results showed cell types in neurofibromas of Nf2- / -; Wwc1- / -; Wwc2- / -; Plp1-CreERT2 mice, human NF1 neurofibromas, human NF2 neurofibromas, and human malignant peripheral nerve sheath tumors (MPNST). Figure 3 B shows the distribution of Schwann cells in mouse neurofibromas, human NF1 neurofibromas, human NF2 neurofibromas, and human malignant peripheral nerve sheath tumors. Figure 3 C shows the changes in Schwann cell annotation between mouse neurofibroma and different subtypes of human neurofibroma.
[0018] Preservation Information
[0019] The mouse neurofibroma type II cell line SC228 was deposited on April 16, 2024, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: C2024119. Detailed Implementation
[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.
[0021] NF2 is a multifunctional protein whose primary function is tumor suppression. This tumor-suppressive function is widely believed to be achieved through its action on the Hippo signaling pathway, a highly conserved signaling cascade that plays a crucial role in regulating organ size, tissue homeostasis, and tumorigenesis. Within this pathway, NF2 promotes the activation of LATS1 / 2 kinases, which in turn phosphorylate and inactivate the effector molecules YAP and TAZ, thus maintaining homeostasis. In the absence of NF2, LATS1 / 2 kinase activity is inhibited, preventing the phosphorylation of YAP / TAZ. Activated YAP / TAZ then enters the cell nucleus, regulating the expression of downstream target genes and promoting cell proliferation. Uncontrolled cell proliferation leads to tumor development. Recent studies have shown that YAP / TAZ activity is critical in the development of neurofibromas induced by Nf2 deficiency in mice.
[0022] NF2-associated type II neurofibromas are typically benign tumors with limited cell proliferation. This may be related to the fact that NF2 is a relatively weak tumor suppressor gene, or it may be due to other compensatory mechanisms in the absence of NF2, thus limiting excessive cell proliferation. Evidence supporting this includes the stabilization of angiomotin family proteins in NF2-deficient cells to prevent full activation of YAP / TAZ. In NF2-deficient cells, RAC1 exhibits antiproliferative activity. These compensatory signaling pathways may serve as a mechanism to delay tumor progression, and therefore may also contribute to the benign nature of type II neurofibromas.
[0023] Releasing these compensatory NF2 deficiency signaling pathways could potentially fully unleash the activity of YAP / TAZ, thereby promoting cell proliferation and potentially creating a rapidly developing mouse model of type II nerve fibers.
[0024] The WWC1-3 protein is encoded by the WWC1-3 gene. In mice, only two genes, Wwc1 and Wwc2, exist. WWC1-3 is another important protein in the Hippo signaling pathway, comparable to NF2. WWC1-3 and NF2 regulate two independent Hippo signaling pathway modules: HPO1 and HPO2 signaling, respectively. These two independent signaling modules synergistically regulate the activity of LATS1 / 2, thereby jointly regulating the effector molecules of the Hippo signaling pathway. NF2 deficiency leads to an increase in WWC1-3. This increase has also been observed in neurofibromas following NF2 deficiency, suggesting that the increase in WWC1-3 may be another compensatory mechanism in NF2-deficient neurofibromas, which may be a major reason for the slow development of NF2-deficient neurofibromas.
[0025] Therefore, this invention specifically knocks out the three genes Nf2, Wwc1, and Wwc2 simultaneously in mouse Schwann cells to construct a rapidly developing neurofibroma model and the corresponding primary tumor cell line. The construction method includes the following steps:
[0026] Step 1: Using ES cell targeting, conditional knockout mice of Nf2, Wwc1, and Wwc2 were constructed respectively. After obtaining homozygous F1 generation mice of each gene, the mice of the three genes were crossed. After multiple generations of crosses, mice with the genotypes Nf2- / -, Wwc1- / -, and Wwc2- / - were finally obtained.
[0027] Step 2: Using the Schwann cell-specific Cre tool mouse Plp1-CreERT2, the Cre tool mouse is crossed with mice with the genotypes Nf2- / -; Wwc1- / -; Wwc2- / - to finally obtain Nf2- / -; Wwc1- / -; Wwc2- / -; Plp1-CreERT2 mice;
[0028] Step 3: After tamoxifen-induced Plp1-Cre expression in the Nf2- / -;Wwc1- / -;Wwc2- / -;Plp1-CreERT2 mice, the three genes Nf2, Wwc1 and Wwc2 can be simultaneously knocked out in Schwann cells of mice.
[0029] Step four: After 7-8 months of tamoxifen-induced Plp1-Cre expression in Nf2- / -;Wwc1- / -;Wwc2- / -;Plp1-CreERT2 mice, subcutaneous neurofibromas can be spontaneously formed. The corresponding primary tumor cell line (type II neurofibroma primary cell line) can be obtained by isolating and primary culturing the neurofibroma.
[0030] like Figure 1As shown, approximately three months after gene knockout, the DRG of Nf2 and Wwc1-2 knockout mice exhibited a certain degree of enlargement and proliferation compared to the control group. However, the DRG of mice with simultaneous knockout of both Nf2 and Wwc1-2 showed even more pronounced enlargement and proliferation than those with single gene knockout. Compared to control mice, the DRG volume of Nf2- / -, Wwc1- / -, and Wwc2- / - mice was more than ten times larger. Histological analysis revealed that the DRG of Nf2 and Wwc1-2 knockout mice exhibited a relatively normal tissue structure, while the tissue structure of the DRG of Nf2- / -, Wwc1- / -, and Wwc2- / - mice was disrupted, showing increased expression of Schwann cells (SOX10 and S100β positive) and fewer NF-positive neurons. In addition, Nf2- / -; Wwc1- / -; Wwc2- / - mice developed a phenotype of dermatofibroma in multiple locations throughout their bodies. These tumors also expressed characteristic Schwann cell molecules (SOX10 and S100β) and highly expressed proliferative characteristic molecules of YAP / TAZ and Ki67.
[0031] like Figure 2 As shown, spontaneous formation of subcutaneous neurofibromas was observed in mice with simultaneous knockout of Nf2 and Wwc1-2 approximately 7-8 months after gene knockout. Primary cell lines of neurofibromas were obtained by isolating and culturing these subcutaneous tumors. These primary cell lines specifically expressed characteristic marker molecules of human type II neurofibromas, such as SOX10, S100β, and YAP / TAZ.
[0032] Subsequently, tumors from this mouse model were extracted for single-cell transcriptome sequencing analysis. The single-cell RNA sequencing data of these mouse tumors were then integrated and compared with those of different subtypes of human neurofibroma, including human NF1 neurofibroma, human NF2 neurofibroma, and human malignant peripheral nerve sheath tumor (MPNST). These four types of neurofibroma share most cell types, such as immune cells and endothelial cells. Figure 3As shown, Schwann cells, Myelinating SCs and a transitional cell population (Trans SCs) are present in all four types of neurofibromas, but cells from different neurofibromas exhibit their own subtype-specific characteristics. For example, MPNST cells are mostly mesenchymal-like cells (MES SCs), while the predominant cells in NF1 and NF2 tumors are NF1-like SCs and NF2-like SCs, respectively. For Schwann cells from Nf2- / -, Wwc1- / -, and Wwc2- / - mouse neurofibromas, Hippo SCs and Peg3 SCs clustered in the NF2-like group, rather than the NF1-like or MES group; Ccnd1 SCs clustered in the Trans group, indicating that these cells have the potential to transition into advanced tumor cells. Overall, the distribution of Schwann cells in Nf2- / -, Wwc1- / -, and Wwc2- / - mouse neurofibromas highly overlaps with the distribution in human NF2-associated neurofibromas. Dual indicators, including cellular composition and transcriptome analysis, showed that Nf2- / -;Wwc1- / -;Wwc2- / - mouse neurofibromas are highly similar to human NF2 neurofibromas. Therefore, this invention demonstrates the successful construction of a mouse model that accurately mimics human type II neurofibroma.
[0033] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for constructing an Nf2, Wwc1, and Wwc2 gene knockout mouse model, characterized in that, Includes the following steps: Step 1: Construct conditional knockout mice of Nf2, Wwc1, and Wwc2 respectively, obtain homozygous F1 generation mice for each gene, and then cross the mice of the three genes. After multiple generations of crosses, the final result is a mouse with the genotype Nf2. - / - ;Wwc1 - / - ;Wwc2 - / - Mice; Step two: Using the Schwann cell-specific Cre tool mouse Plp1-CreERT2, the Cre tool mouse was bred with the Nf2 genotype. - / - ;Wwc1 - / - ;Wwc2 - / - Mice were hybridized to obtain Nf2. - / - ;Wwc1 - / - ;Wwc2 - / - ;Plp1-CreERT2 mice; The Nf2 - / - ;Wwc1 - / - ;Wwc2 - / - After plp1-Cre expression was induced by tamoxifen in plp1-Cre mice, the three genes Nf2, Wwc1 and Wwc2 could be knocked out simultaneously in Schwann cells of mice.
2. The construction method according to claim 1, characterized in that, Conditional knockout mice of Nf2, Wwc1, and Wwc2 were constructed by targeting ES cells.
3. A method for constructing a primary cell line for type II neurofibroma, characterized in that, Nf2 obtained using the construction method described in claim 1 - / - ;Wwc1 - / - Wwc2 - / - ;Plp1-CreERT2 mice spontaneously form subcutaneous schwannomas after 7-8 months of tamoxifen-induced Plp1-Cre expression. Primary cell lines of type II neurofibroma can be obtained by isolating and culturing the subcutaneous schwannomas.
4. The application of Nf2, Wwc1 and Wwc2 gene knockout mouse models in the preparation and screening of drugs for the treatment of neurofibromatosis type II, wherein the Nf2, Wwc1 and Wwc2 gene knockout mouse models are constructed by the construction method described in any one of claims 1-2.
5. The application of primary cell lines for type II neurofibroma in the preparation and screening of drugs for the treatment of type II neurofibroma, wherein the primary cell lines for type II neurofibroma are constructed by the construction method described in claim 3.