Method for constructing map3k3 i441m mutation knock-in transgenic mouse model and use

Abstract

The present invention provides a method for constructing a Map3k3I441M mutation knock-in transgenic mouse model and a use. The method comprises: replacing the region from exon 1 to part of intron 2 of a mouse with a mutant gene fragment, wherein the sequence of the mutant gene fragment is as shown in SED ID NO: 1 in the sequence listing; using a BCA clone as a template to amplify a homologous arm-containing mouse genome fragment as a targeting vector; co-injecting Cas9 and gRNA as well as the targeting vector into fertilized eggs; allowing the fertilized eggs to develop to obtain positive F0 generation samples; co-housing the positive F0 generation samples for breeding to obtain F1 generation samples; performing genotyping on the F1 generation samples to screen for Map3k3I441M samples; and mating the Map3k3I441M samples from the F1 generation samples with a Cdh5-CreERT tool sample to produce F2 generation samples. A model constructed by the construction method can accurately reflect the real situation in a mouse.

Description

A method for constructing Map3k3 I441M Methods and applications of mutant knock-in transgenic mouse models

[0001] Cross-references to related applications

[0002] This application claims the rights to the patent filed on July 15, 2024, entitled "A method for constructing Map3k3". I441M The priority of Chinese patent application 202410942248.3, entitled "Methods and applications of mutation knock-in transgenic mouse models", is hereby acknowledged, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biotechnology, and more specifically, to a method for constructing Map3k3. I441M Methods and applications of mutant knock-in transgenic mouse models. Background Technology

[0004] Cavernous malformations (CCMs) are common vascular malformations of the central nervous system, consisting of densely packed, abnormally dilated, and leaking capillaries and veins, affecting up to 0.5% of the total population. CCMs can cause hemorrhage, seizures, or other neurological sequelae. Based on family history, CCMs are classified into familial and sporadic types. Of these, 78%–94% of familial CCMs and 36% of sporadic CCMs are associated with inactivation mutations in one of three CCM genes: KRIT1 (CCM1), MGC4607 (CCM2), and PDCD10 (CCM3). Recent research has reported a newly discovered Map3k3 mutation. I441M Somatic mutations account for approximately 40% of sporadic CCM patients.

[0005] Currently, studies have shown that Map3k3 can be overexpressed in mouse brain endothelial cells via adeno-associated virus (AAV)-mediated transformation. I441M The mutation can generate CCM-like lesions in the brain and spinal cord of mice. However, this AAV-mediated overexpression mouse model cannot reflect the true situation in vivo because the Map3k3 mutation is a monoallelic mutation in clinical CCM specimens, and the AAV-mediated Map3k3 mutation overexpression amplifies the mutation effect, thus making the AAV-mediated Map3k3 mutation inaccurate to reflect the true situation in vivo. Therefore, there is an urgent need for a method that can construct Map3k3... I441M The knock-in mutant transgenic mouse model can accurately reflect the real situation in vivo. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for constructing Map3k3.I441M Methods and applications of mutant knock-in transgenic mouse models.

[0007] This invention provides a method for constructing Map3k3. I441M A method for creating a mutant knock-in transgenic mouse model, the method comprising:

[0008] Exon 1 to part of intron 2 in mice were replaced with a mutant gene fragment, the sequence of which is shown as SED ID NO: 1 in the sequence listing;

[0009] Using the BCA clone as a template, a mouse genome fragment containing a homologous arm was amplified and used as a targeting vector;

[0010] Cas9 mRNA and gRNA were co-injected into fertilized eggs along with the targeting vector;

[0011] The fertilized eggs were processed to obtain positive F0 generation samples;

[0012] The positive F0 generation samples were then bred together to obtain the F1 generation samples;

[0013] Genotyping was performed on the F1 generation samples to obtain Map3k3. I441M Positive sample;

[0014] The Map3k3 I441M Positive samples were crossbred with Cdh5-CreERT tool samples to produce Map3k3 targeting endothelial cells. I441M Mutation knock-in transgenic mouse model.

[0015] Specifically, the targeting vector is loxP-3*SV40 pA-loxP-partial mouse Map3k3 5'UTR-mutant human MAP3K3 CDS-P2A-EGFP-rBG pA.

[0016] Specifically, the fertilized sample is a mouse, and the Cdh5-CreERT tool sample is a Cdh5-CreERT tool mouse.

[0017] Specifically, the mutant gene fragment is the 7 bases upstream of the ATG start codon of the loxP-3*SV40 pA-loxP-mouse Map3k3 5'UTR - mutant human MAP3K3 CDS-P2A-rBG pA.

[0018] On the one hand, it provides a way to construct Map3k3 I441M The application of the mutant knock-in transgenic mouse model is characterized by the following: the application includes the construction of Map3k3. I441MMutant knock-in transgenic mouse models are used to induce CCM-like lesions.

[0019] This invention provides a method for constructing Map3k3. I441M This invention discloses a method and application for constructing a mutant knock-in transgenic mouse model, which generates CCM-like lesions in the mouse brain. Unlike previous methods using AAV-mediated mutation overexpression, this invention employs gene editing technology to replace one strand of the mouse Map3k3 gene with a version containing the p.I441M point mutation. This is consistent with the single-gene mutation of MAP3K3 discovered clinically through whole-exome sequencing, accurately reflecting the actual situation in vivo. This model can be used to induce CCM-like lesions, providing a foundation for further research on CCMs.

[0020] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures particularly pointed out in the description, claims, and drawings. Attached Figure Description

[0021] The accompanying drawings are provided to further understand the technical solutions of the present invention and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of the present invention and do not constitute a limitation on the technical solutions of the present invention.

[0022] Figure 1a is a schematic diagram of the structure of the Map3k3 gene range in a CRISPR-Cas9 knockout mouse guided by gRNA in Embodiment 1 of the present invention.

[0023] Figure 1b is a schematic diagram of the structure of the replacement vector ("loxP-3*SV40 pA-loxP-part of 5'UTR of mouse Map3k3 (7bp upstream of ATG start condon)-mutant Human MAP3K3 CDS-P2A-EGFP-rBG pA") in Embodiment 1 of the present invention.

[0024] Figure 2 shows the genotypes of mice in each group after electrophoresis provided in Embodiment 1 of the present invention.

[0025] Figure 3 is an expression diagram of the Map3k3 mutant gene in the induced knock-in mouse provided in Embodiment 1 of the present invention.

[0026] Figure 4 shows the expression of EGFP in mouse brain endothelial cells provided in Example 1 of the present invention.

[0027] Figure 5 shows the expression of Map3k3 in mouse cerebral endothelial cells according to Embodiment 1 of the present invention.I441M CCM-like lesion images were subsequently successfully generated.

[0028] Figure 6 shows the Map3k3 provided in Embodiment 1 of the present invention. iECGOF Mice and Map3k3 WT A comparative diagram of KLF4 expression in mouse brain endothelial cells.

[0029] Figure 7 shows the Map3k3 provided in Embodiment 1 of the present invention. iECGOF Mice and Map3k3 WT Statistical graph of fluorescence intensity of KLF4 expression in mouse brain endothelial cells.

[0030] Figure 8 shows the Map3k3 provided in Embodiment 1 of the present invention. iECGOF Mice and Map3k3 WT A comparative diagram of TM expression in mouse brain endothelial cells.

[0031] Figure 9 shows the Map3k3 provided in Embodiment 1 of the present invention. iECGOF Mice and Map3k3 WT Statistical graph of fluorescence intensity of TM expression in mouse brain endothelial cells.

[0032] Figure 10 shows the Map3k3 provided in Embodiment 1 of the present invention. iECGOF Mice and Map3k3 WT A comparative diagram of p-s6 expression in mouse brain endothelial cells.

[0033] Figure 11 shows the Map3k3 provided in Embodiment 1 of the present invention. iECGOF Mice and Map3k3 WT Statistical graph of fluorescence intensity of p-s6 expression in mouse brain endothelial cells.

[0034] Figure 12 shows the Map3k3 provided in Embodiment 2 of the present invention. iECGOF A schematic diagram of long-term MRI observation of mouse lesions.

[0035] Figure 13 shows the Map3k3 provided in Embodiment 2 of the present invention. iECGOF T2-weighted and susceptibility-weighted imaging (SWI) of long-term MRI observation of mouse lesions.

[0036] Figure 14a shows the Map3k3 provided in Embodiment 2 of the present invention. iECGOF Graph showing the change in the number of lesions in mice.

[0037] Figure 14b shows the Map3k3 provided in Embodiment 2 of the present invention. iECGOF Graph showing the change in average lesion volume in mice.

[0038] Figure 15 shows the Map3k3 provided in Embodiment 2 of the present invention. iECGOFMice and Map3k3 WT Mouse brain endothelial cell clusters sorted by single-cell sequencing.

[0039] Figure 16 shows the Map3k3 provided in Embodiment 2 of the present invention. iECGOF Mice and Map3k3 WT Heatmap of mouse brain endothelial cell classification.

[0040] Figure 17 shows the Map3k3 provided in Embodiment 2 of the present invention. iECGOF Mice and Map3k3 WT The enrichment pathways were compared to those enriched in mice.

[0041] Figure 18 shows the Map3k3 provided in Embodiment 2 of the present invention. iECGOF Mice and Map3k3 WT A comparative diagram of p-p38 expression in mouse brain endothelial cells.

[0042] Figure 19 shows the Map3k3 provided in Embodiment 2 of the present invention. iECGOF Mice and Map3k3 WT Statistical graph of fluorescence intensity of p-p38 expression in mouse brain endothelial cells.

[0043] Figure 20 shows the Map3k3 provided in Embodiment 2 of the present invention. iECGOF Mice and Map3k3 WT A comparative diagram of cleaved-caspase3 expression in mouse brain endothelial cells.

[0044] Figure 21 shows the Map3k3 provided in Embodiment 2 of the present invention. iECGOF Mice and Map3k3 WT Statistical graph of fluorescence intensity of cleaved-caspase3 expression in mouse brain endothelial cells.

[0045] Figure 22 shows the Map3k3 provided in Embodiment 2 of the present invention. WT Pten fl / fl Map3k3 I441M and Map3k3 I441M Pten fl / fl Gross and MRI images of lesions in mice 60 days after birth.

[0046] Figure 23a shows the Map3k3 provided in Embodiment 2 of the present invention. WT Pten fl / fl Map3k3 I441M and Map3k3 I441M Pten fl / fl A statistical chart showing the number of lesions in mice 60 days after birth.

[0047] Figure 23b is a Map3k3 provided in Embodiment 2 of the present invention. WT Pten fl / fl Map3k3 I441M and Map3k3 I441M Pten fl / fl Statistical graph of the average volume of lesions in mice 60 days after birth.

[0048] Figure 24 shows a Map3k3 map for 90 days after birth provided in Embodiment 2 of the present invention. WT Pten fl / fl Map3k3 I441M and Map3k3 I441M Pten fl / fl Gross and MRI images of lesions in mice 30 days after AAV-Cre injection.

[0049] Figure 25a shows a Map3k3 map of a newborn at 90 days after birth, provided in Embodiment 2 of the present invention. WT Pten fl / fl Map3k3 I441M and Map3k3 I441M Pten fl / fl A statistical chart showing the number of lesions in mice 30 days after injection of AAV-Cre.

[0050] Figure 25b shows a Map3k3 map of a newborn at 90 days after birth, provided in Embodiment 2 of the present invention. WT Pten fl / fl Map3k3 I441M and Map3k3 I441M Pten fl / fl Statistical graph of the average volume of lesions in mice 30 days after injection of AAV-Cre.

[0051] Figure 26 shows a Map3k3 map for 90 days after birth provided in Embodiment 2 of the present invention. I441M Long-term observation of MRI images in mice. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0053] Example 1

[0054] This invention provides a method for constructing Map3k3. I441MMethods for creating mutant knock-in transgenic mouse models include:

[0055] The mouse Map3k3 gene (NM_011947.4) is located on mouse chromosome 11. In this study, exon 1 and part of intron 2 in C57 mice were replaced with a mutant gene fragment. The specific location of exon 1 and part of intron 2 is 4.1kb from exon 1. The sequence of the mutant gene fragment is shown as SED ID NO: 1 in the sequence listing.

[0056] Using the BCA clone as a template, a mouse genome fragment containing a homologous arm was amplified and used as a targeting vector. In this embodiment, the targeting vector was loxP-3*SV40 pA-loxP-partial mouse Map3k3 5'UTR-mutant human MAP3K3 CDS-P2A-EGFP-rBG pA. The specific targeting vector is shown in Figures 1a and 1b.

[0057] gRNA (purchased from Cyagen Biosciences), a targeting vector (purchased from Cyagen Biosciences, containing the "loxP-3*SV40 pA-loxP-partial mouse Map3k3 5'UTR (7 bp upstream of the ATG start codon)-mutant human MAP3K3 CDS-P2A-EGFP-rBG pA" cassette), and Cas9 mRNA were co-injected into fertilized eggs to generate directed conditional knock-in offspring. Positive F0 generation samples were then combined to generate F1 generation samples.

[0058] Genotyping of F1 generation samples was performed using PCR amplification and nucleic acid electrophoresis. Specifically, the genotype of each mouse was determined by gel electrophoresis, and the specific method is as follows:

[0059] The TaKaRa MiniBEST Universal Genomic DNA Extraction kit (Ver.5.0_Code No.9765) was used to obtain high-purity genomic DNA, as detailed below:

[0060] a. Take tail tissue from F1 generation samples, each piece of tail tissue is 2-5 mm long. In a microcentrifuge tube, add 180 μL of Buffer GL, 20 μL of Proteinase K and 10 μL of RNase A to each piece of tail tissue.

[0061] b. Incubate the microcentrifuge tubes overnight at 56°C.

[0062] c. Centrifuge at 12,000 rpm for 2 minutes in a microcentrifuge to remove impurities, and retain the supernatant.

[0063] d. Add 200 μL of Buffer GB and 200 μL of anhydrous ethanol to the supernatant and mix thoroughly to obtain a mixture.

[0064] e. Place the centrifuge column in the collection tube. Centrifuge the mixture through the centrifuge column at 12,000 rpm for 2 minutes, and discard the eluent.

[0065] f. Add 500 μL of Buffer WA to the centrifuge column, then centrifuge at 12,000 rpm for 1 minute and discard the eluent.

[0066] g. Add 700 μL of Buffer WB to the centrifuge column, then centrifuge at 12,000 rpm for 1 minute and discard the eluent. (Note: Ensure that Buffer WB has been premixed with 100% ethanol. When adding Buffer WB, add it along the wall of the tube to wash away any residual salts.)

[0067] h. Repeat step g.

[0068] i. Place the centrifuge column in another collection tube and centrifuge at 12,000 rpm for 2 minutes, then discard the effluent.

[0069] j. Place the centrifuge column into a new 1.5 mL collection tube. Add 50–200 μL of sterile water or elution buffer to the center of the filter membrane in the centrifuge column, and allow the column to stand for 5 minutes. (Note: Heating the sterile water or elution buffer to 65°C can increase the elution volume.)

[0070] k. Elute the genomic DNA by centrifuging the column at 12,000 rpm for 2 minutes. To increase DNA yield, add the eluent and / or 50-200 μL of sterile water or elution buffer to the center of the filter membrane of the centrifuge column, let the column stand for 5 minutes, and then centrifuge at 12,000 rpm for 2 minutes to obtain the eluted genomic DNA.

[0071] 1. Quantifying genomic DNA. The eluted genomic DNA can be quantified by electrophoresis.

[0072] Genotyping was performed on the F1 generation samples to obtain Map3k3. I441M Positive sample;

[0073] The sequences of the forward primers required for genotyping identification are shown in SED ID NO: 2 in the sequence listing, specifically 5'-AGATCTGCAAGCTAATTCCTGC-3', and the sequences of the reverse primers are shown in SED ID NO: 3 in the sequence listing, specifically 5'-GTTGTCCAAATACTGTTGTCACCT-3'. The genotypes of each mouse are shown in Figure 2.

[0074] Map3k3 I441M Positive samples were crossbred with tissue-specific Cdh5-CreERT tool samples to produce F2 Cdh5-CreERT samples; Map3k3 I441M (Map3k3 IECGOF Map3k3 targeting endothelial cells was obtained. I441M Mutant knock-in transgenic mouse model;

[0075] Specifically, the Cdh5-CreERT tool sample is the Cdh5-CreERT tool mouse.

[0076] Specifically, the mutant gene fragment is the 7 bases upstream of the ATG start codon of the loxP-3*SV40 pA-loxP-mouse Map3k3 5'UTR - mutant human MAP3K3 CDS-P2A-rBG pA.

[0077] On day 1 after birth (P1), F2 generation mice were administered 4-hydroxytamoxifen (4-OHT) via intragastric injection to induce the expression of the Map3k3 mutant gene in the knock-in mice. Specifically, 40 μg of 4-OHT (Sigma Aldrich, H7904) was freshly dissolved in corn oil containing 9% ethanol, with a total volume of 50 μL, and then administered via gavage. The expression results are shown in Figure 3. High expression of EGFP in mouse brain endothelial cells confirmed successful mutant expression, as shown in Figure 4. Subsequently, MRI was used to assess whether abnormal lesions formed in the mice injected with 4-OHT. Map3k3 iECGOF The mice showed many abnormal low-signal lesions in T2-weighted sequences, which were very similar to human CCM lesions. The lesions were mainly located in the cerebellum, as shown in Figure 5.

[0078] CCM mutations induce cavernous malformations by activating the MEKK3-KLF4 pathway. Furthermore, elevated TM downstream of KLF4 has also been shown to contribute to cavernous malformation hemorrhage. To investigate the presence of these characteristics in mouse models, immunofluorescence staining of mouse endothelial cells for KLF4 and TM was performed. Specifically, paraffin-embedded tissue sections were prepared using the Opal multiplex IHC assay kit (PerkinElmer). The results showed increased expression of KLF4 and TM in mutant endothelial cells compared to the WT group, as shown in Figures 6 and 7, indicating activation of the classical MEKK3-KLF4 pathway. In addition, MAP3K3 mutations were also shown to activate the mTOR signaling pathway in endothelial cells. Previously, mTOR activity was thought to lead to phosphorylation of the ribosomal protein S6; to assess mTOR signaling activation, MAP3K3 was also investigated. iECGOF p-S6 fluorescence staining was performed on brain tissue from WT mice. Results showed that Map3k3... iECGOFIncreased p-S6 expression was observed in mice, as shown in Figure 8. These results support Map3k3 in terms of histopathological and molecular characteristics. I441M Mutation induction can induce CCM-like lesions.

[0079] Example 2

[0080] This invention provides a method for constructing Map3k3. I441M Applications of mutant knock-in transgenic mouse models include:

[0081] Map3k3 I441M Long-term evolution of CCM lesions in mutant mice

[0082] Map3k3 was continuously tracked using MRI from 4 weeks to 6 months after 4-OHT injection. I441M The progression of lesions induced by mutations (see Figure 13). Specific T2 parameters were: TR = 3200 ms, TE = 40 ms, FOV = 25 × 25 mm, matrix = 256 × 192, number of slices = 25, slice thickness = 0.5 mm. SWI parameters were: TR = 600 ms, TE = 8 ms, FOV = 25 × 25 mm. 2 The matrix was 256×192, with 25 layers and a layer thickness of 0.5 mm. Results showed that the number and average volume of lesions in mutant mice increased from 4 weeks to 2 months after 4-OHT injection. However, from the 3rd to 6th month after 4-OHT injection, the number of lesions remained stable, and the average lesion volume decreased slightly, but without statistical significance (see Figures 14a and 14b). Previous studies have shown that CCM lesions caused by CCM mutations progress continuously and can lead to short-term death in mice. In clinical patients, lesions with CCM gene mutations are more prone to cerebral hemorrhage, while those with Map3k3... I441M Mutated lesions rarely bleed. Studies have shown that Map3k3... I441M Mutations promoted endothelial cell apoptosis. Therefore, our goal is to further investigate whether apoptosis also affects Map3k3. I441M The same effect was produced when knocked into mice.

[0083] Map3k3 I441M Mutations promote endothelial cell apoptosis in transgenic mice

[0084] To explore the molecular mechanism by which mutations induce CCM lesions in vivo, we selected Map3k3 cells four weeks after 4-OHT injection. iECGOFSingle-cell RNA sequencing was performed on brain endothelial cells from mice (n=3) and Map3k3 WT mice (n=3). After sequencing and data processing, we further identified C11 and C22 as endothelial cell populations based on endothelial markers and isolated 973 endothelial cells, which were divided into 8 groups by supervised clustering (Figure 15). Endothelial cell populations were further classified into the following groups based on brain endothelial cell marker genes: large arteries (group 1), capillaries (groups 6 and 7), capillaries / venules (groups 0, 2, and 3), venules (group 5), and large veins (group 4) (see Figure 16). GO enrichment analysis showed that among the significantly upregulated genes, the apoptosis signaling pathway was enriched to varying degrees in each endothelial cell population of the mutant group, especially in group 4—large vein endothelial cells (see Figure 17). To validate these results, we performed immunofluorescence staining on the apoptotic pathway p-p38 (see Figure 18) and the apoptotic marker Cleaved Caspase-3 (see Figure 20) in the brain endothelial cells of mutant and wild-type mice. Consistent with the single-cell results, p-p38 (see Figure 19) and Cleaved Caspase-3 (see Figure 21) were highly expressed in the brain endothelial cells of Map3k3 mutant mice. In summary, this evidence suggests that the Map3k3 mutation enhances the p38 signaling pathway and is associated with endothelial cell apoptosis in mouse lesions.

[0085] Activation of the PI3K signaling pathway amplifies Map3k3 I441M lesions in mice

[0086] Most cases of chronic mycosis syndrome (CCM) in individuals with MAP3K3 mutations are accompanied by somatic PIK3CA mutations (such as H10447R, E542K, and E545K), and PIK3CA mutations are acquired mutations that activate the PIK3CA pathway. To investigate the impact of the PIK3CA pathway on MAP3K3 mutations, Pten was knocked down in the MAP3K3 pathway. I441M The PIK3CA pathway was activated in the knock-in mouse model.

[0087] Specifically, this includes: Map3k3 I441M Mice and Pten fl / fl Mice were mated to obtain Map3k3 I441M Pten fl / + Mice were then used to generate Map3k3 mice through self-pollination. I441M Pten fl / fl Mice. To accelerate the production of target mice, the expression of the mutant gene was induced by injection of AAV-Cre. AAV-Cre was administered via retroorbital vein injection to WT mice one day after birth. I441M Pten fl / fl Map3k3 I441M Ptenfl / fl Mice were observed by MRI 4 weeks after injection. Results showed that in WT and Pten... fl / fl No lesions were observed in the group, while in Map3k3 I441M Pten fl / fl In the mouse group (see Figure 22), the number and average volume of CCM lesions were significantly increased (see Figures 23a and 23b). These results indicate that activation of the PI3K signaling pathway can exacerbate the progression of CCM lesions.

[0088] Adult Map3k3 I441M Mutant mice require activation of the PI3K signaling pathway to develop CCM-like lesions.

[0089] From the 3rd to the 6th month after 4-OHT injection, Map3k3 I441M The number of CCM lesions in mice remained stable, but the average lesion volume showed a decreasing trend. Mice aged 3 months are considered to be adult mice. To find Map3k3... I441M Whether the mutation was sufficient to induce pathogenesis in adult mice, we administered Map3k3 via AAV-Cre injection into the bulbar sinus at P90. I441M Mice and Map3k3 I441M Pten fl / fl Mice were then subjected to MRI scans one month after AAV-Cre injection (see Figure 24). Results showed that mice with Map3k3... I441M The mutant adult mice failed to develop CCM lesions, but in Map3k3 I441M Pten fl / fl Obvious CCM lesions were developed in mice (see Figures 25a and 25b). Map3k3 I441M Pten fl / fl Mice typically die within 1.5 months of AAV-Cre injection due to severe and fatal lesions. However, Map3k3... I441M Mice injected with AAV-Cre showed no CCM lesions within three months following 4-OHT injection (see Figure 26). This result indicates that Map3k3 is effective in adult mice. I441M Mutations alone are insufficient to produce lesions; CCM lesions can only occur when the PI3K signaling pathway is activated.

[0090] While the embodiments disclosed in this invention are as described above, the content is merely for the purpose of facilitating understanding of the invention and is not intended to limit the invention. Any person skilled in the art to which this invention pertains may make any modifications and changes to the form and details of the implementation without departing from the spirit and scope disclosed herein; however, the scope of patent protection of this invention shall still be determined by the scope defined in the appended claims.

Claims

1. A method of constructing a Map3k3 I441M a method of generating a mutant knock-in transgenic mouse model, characterized by, The method comprises: replacing the first exon to part of the second intron in the mouse with a mutant gene fragment, the sequence of the mutant gene fragment being shown as SED ID NO: 1 in the sequence table; amplifying a mouse genomic fragment containing a homologous arm as a targeting vector by taking the BCA clone as a template; injecting Cas9 mRNA and gRNA into the fertilized egg together with the targeting vector; obtaining a positive F0 sample through production of the fertilized egg; breeding the positive F0 sample in a cage to obtain an F1 sample; The F1 generation sample is subjected to genotyping identification, and Map3k3 is obtained I441M Positive sample; The Map3k3 I441M Positive samples were mated with Cdh5-CreERT2 tool samples to produce the Map3k3 I441M Mutant knock-in transgenic mouse models.

2. The method of claim 1, wherein, the targeting vector is loxP-3*SV40 pA-loxP-part mouse Map3k3 5'UTR-mutant human MAP3K3 CDS-P2A-EGFP-rBG pA.

3. The method of claim 1, wherein, The fertilized sample is a mouse, and the Cdh5-CreERT tool sample is a Cdh5-CreERT tool mouse.

4. The method of claim 1, wherein, The mutant gene fragment is loxP-3*SV40 pA-loxP-mouse Map3k3 5'UTR 7 bases upstream of the ATG start codon-mutant human MAP3K3 CDS-P2A-rBG pA.

5. A construct Map3k3 I441M Use of a mutant knock-in transgenic mouse model, characterized in that, The application comprises: the construction Map3k3 I441M Mutant knock-in transgenic mouse models were used to induce CCM-like pathology.

6. The use according to claim 1, characterized in that, The application comprises: constructing MAP3K3 I441M Mutant knock-in transgenic mouse models were used to study the effects of combined pi3k mutations on lesions.

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