Application of nlrp3 gene and / or its knockout reagent in construction of animal model of arterial vascular calcification

Abstract

The present application relates to the application of Nlrp3 gene and / or its knockout reagent in constructing an arterial vessel calcification animal model. The method for constructing the arterial vessel calcification animal model comprises the following steps: (1) constructing a gene editing system for targeting knockout of mouse Nlrp3 gene; (2) using the gene editing system in step (1) to perform gene editing on mouse fertilized eggs, and inserting loxP sites at both ends of the Nlrp3 gene; (3) mating the mouse obtained in step (2) with a mouse expressing Cre recombinase to obtain a mouse with Nlrp3 gene knockout, and obtaining the arterial vessel calcification animal model after culture. The present application obtains an arterial vessel calcification mouse by specifically knocking out the Nlrp3 gene of the mouse, and has important application value in the mechanism exploration and drug research and development of arterial vessel calcification diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to the application of the Nlrp3 gene and / or its knockout reagents in constructing animal models of arterial vascular calcification. Background Technology

[0002] Vascular calcification is a common pathological manifestation in atherosclerosis (As), hypertension, diabetic vascular complications, vascular injury, chronic kidney disease, and aging. It is mainly characterized by increased vascular wall stiffness and decreased compliance, easily leading to myocardial ischemia, left ventricular hypertrophy, and heart failure, and triggering thrombosis and plaque rupture. It is a significant factor contributing to the high incidence and mortality of cardiovascular and cerebrovascular diseases. The pathogenesis of vascular calcification is complex. Previous mechanistic studies have revealed the importance of vascular smooth muscle cell transdifferentiation, phosphorus and calcium environment, and matrix vesicles in the progression of vascular calcification. However, the underlying molecular mechanisms of vascular calcification still need to be elucidated. Currently, there are no effective treatments for vascular calcification; therefore, animal models of vascular calcification are crucial for studying its pathological mechanisms and developing treatment methods.

[0003] Currently, animal models of vascular calcification mainly include drug-induced vascular calcification (such as vitamin D3-induced and warfarin-induced vascular calcification), vascular calcification caused by chronic kidney disease (such as vitamin D3 combined with subtotal nephrectomy), and gene knockout vascular calcification models. For example, CN117210497A discloses the application of the Abcg4 gene in constructing a mouse model of cerebral arteriosclerosis with pulmonary enlargement. By knocking out the Abcg4 gene in mice, the aforementioned mouse model of cerebral arteriosclerosis with pulmonary enlargement was obtained. These models and methods provide important tools for studying vascular calcification, helping to deepen the understanding of its pathophysiological process and providing an experimental basis for developing new treatment methods. Developing animal models of vascular calcification with different induction mechanisms is of great significance for a deeper understanding of pathological mechanisms and the expansion of treatment methods.

[0004] In conclusion, developing novel animal models of arterial calcification is of great significance for the treatment of diseases related to vascular calcification. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of the Nlrp3 gene and / or its knockout reagent in constructing an animal model of arterial vascular calcification. By specifically knocking out the Nlrp3 gene in mice, an arterial vascular calcification mouse model was obtained, which has significant application value in exploring the mechanism of arterial vascular calcification disease and in drug discovery.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides the application of the Nlrp3 gene and / or its knockout reagent in constructing an animal model of arterial vascular calcification.

[0008] Preferably, the application specifically includes knocking out the Nlrp3 gene in experimental animals to obtain an animal model of arterial vascular calcification.

[0009] This invention employs a novel construction strategy, discovering that knocking out the Nlrp3 gene can effectively induce aortic calcium salt deposition in animals, thereby constructing an animal model of arterial calcification. This model can be used for simulating vascular calcification-related diseases, elucidating pathogenesis, and screening and testing therapeutic drugs and methods.

[0010] Secondly, the present invention provides a method for constructing an animal model of arterial vascular calcification, the method comprising the following steps:

[0011] (1) Construct a gene editing system that targets and knocks out the Nlrp3 gene in mice;

[0012] (2) Gene editing of mouse fertilized eggs was performed using the gene editing system described in step (1), and loxP sites were inserted at both ends of the Nlrp3 gene;

[0013] (3) The mice obtained in step (2) are bred with mice expressing Cre recombinase to obtain mice with Nlrp3 gene knocked out, and the animal model of arterial calcification is obtained after culturing.

[0014] The method for constructing an animal model of arterial calcification provided by this invention solves the problems of high gene off-target rate and low animal survival rate in traditional gene knockout technology, avoids the problem of gene embryo lethality, and the obtained gene-edited mouse model has important clinical translational value for exploring the mechanism of arterial calcification disease and drug development.

[0015] Preferably, the gene editing system includes sgRNA targeting the mouse Nlrp3 gene, Cas enzyme, and homologous recombination repair template ssDNA.

[0016] Preferably, the nucleic acid sequence of the sgRNA includes the sequences shown in SEQ ID NO.1 to SEQ ID NO.2.

[0017] Preferably, the Cas enzyme includes the Cas9 enzyme.

[0018] Preferably, the homologous recombination repair template ssDNA contains a loxP site.

[0019] Preferably, the nucleic acid sequence of the homologous recombination repair template ssDNA includes the sequence shown in SEQ ID NO.3.

[0020] SEQ ID NO. 1: TAGGTGGTATGACCGGACAGAGG.

[0021] SEQ ID NO. 2: CGTCTCAGAACGACGGAATCAGG.

[0022] SEQ ID NO. 3: ATAACTTCGTATAGCATACATTATACGAAGTTAT.

[0023] The sgRNA targeting the mouse Nlrp3 gene designed in this invention has high editing efficiency and low off-target rate.

[0024] Preferably, the initial mice used to construct the model can be C57BL / 6 mice, etc.

[0025] Preferably, the mouse expressing Cre recombinase can be a Cdh16-Cre mouse, etc.

[0026] Preferably, the method for constructing the animal model of arterial vascular calcification includes the following steps:

[0027] (1) A CRISPR-Cas9 gene editing system was prepared, comprising sgRNA, Cas9 enzyme mRNA and homologous recombination repair template ssDNA, wherein the nucleic acid sequence of the sgRNA comprises the sequence shown in SEQ ID NO.1 to SEQ ID NO.2, and the nucleic acid sequence of the homologous recombination repair template ssDNA comprises the sequence shown in SEQ ID NO.3;

[0028] (2) sgRNA, Cas9 enzyme mRNA and homologous recombination repair template ssDNA were simultaneously introduced into mouse zygotes to obtain gene recombination zygotes with loxP sites inserted at both ends of the Nlrp3 gene. The surviving zygotes after introduction were transplanted into the uterus of pseudopregnant mice to obtain F0 generation mice.

[0029] (3) The positive mice in the F0 generation were bred with wild-type mice to obtain F1 generation mice. The F1 generation heterozygous mice were then crossbred to obtain F2 generation homozygous mice and F2 generation heterozygous mice.

[0030] (4) F2 generation homozygous mice or F2 generation heterozygous mice were bred with mice expressing Cre recombinase to obtain F3 generation mice;

[0031] (5) Backcross F3 generation mice with F2 generation homozygous mice to obtain mice with Nlrp3 gene knockout;

[0032] (6) Mice with the Nlrp3 gene knocked out were cultured to obtain the animal model of arterial calcification.

[0033] Thirdly, the present invention provides the application of an animal model constructed by the method for constructing an animal model of arterial calcification as described in the second aspect in analyzing the pathogenesis of arterial calcification or in preparing diagnostic and monitoring products for arterial calcification.

[0034] Based on the animal model of arterial calcification constructed in this invention, the pathogenesis of arterial calcification can be further analyzed, or products for the diagnosis and monitoring of arterial calcification can be developed and tested.

[0035] Fourthly, the present invention provides the application of an animal model constructed by the method for constructing an animal model of arterial vascular calcification as described in the first aspect in evaluating and / or screening treatment methods or therapeutic drugs for arterial vascular calcification.

[0036] Based on the animal model of arterial calcification constructed in this invention, treatment methods or drugs for arterial calcification-related diseases can be further evaluated and / or screened. By testing the effects of the treatment methods or drugs to be screened on the animal model, effective treatment plans can be developed.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] This invention employs a novel construction strategy and a gene editing system. Knocking out the Nlrp3 gene effectively induces aortic calcium salt deposition in animals, resulting in a mouse model of arterial calcification. This model can be used to simulate vascular calcification-related diseases, elucidate the pathogenesis, and screen and test therapeutic drugs and methods, which is of great significance for the treatment of vascular calcification-related diseases. Attached Figure Description

[0039] Figure 1 This is a schematic diagram illustrating the knockout principle;

[0040] Figure 2 This is a diagram showing the genotyping results for homozygous individuals.

[0041] Figure 3 The image shows the results of Cdh16-Cre genotyping.

[0042] Figure 4 This is a diagram showing the results of gene knockout verification.

[0043] Figure 5 The image shows the results of Alizarin Red staining of the aorta in the model group and the normal control group mice. Detailed Implementation

[0044] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0045] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0046] In this invention, a conditional gene knockout animal model can be achieved using the specific recombinase system Cre-LoxP. Crossing transgenic mice carrying a specific loxP site (Flox mice) with transgenic mice expressing the Cre enzyme in specific cells (Cre mice) yields gene knockout mice in specific tissues and cells. This conditional gene knockout method requires the simultaneous construction of two transgenic mouse types (Flox mice and Cre mice), and changing the Cre enzyme gene promoter allows for gene knockout under various conditions (such as drug induction, specific time, specific tissue, etc.). A knockout diagram is shown below. Figure 1 As shown.

[0047] Example 1

[0048] This embodiment provides a mouse with a specific knockout of the Nlrp3 gene. The mouse used to knock out the Nlrp3 gene is a C57BL / 6NCya background strain mouse. The preparation process includes the following steps:

[0049] (1) Prepare sgRNA, Cas9 mRNA and homologous recombination repair template ssDNA targeting the mouse Nlrp3 gene respectively. The nucleic acid sequence of sgRNA is shown in SEQ ID NO.1~SEQ ID NO.2, and the nucleic acid sequence of homologous recombination repair template ssDNA is shown in SEQ ID NO.3.

[0050] (2) sgRNA, Cas9 mRNA and homologous recombination repair template ssDNA were electroporated into mouse zygotes. After gene integration, recombinant zygotes containing loxP sites inserted into the Nlrp3 gene were obtained. The surviving zygotes after electroporation were transplanted into the uterus of pseudopregnant mice to obtain F0 generation mice.

[0051] (3) The positive mice in the F0 generation were bred with wild-type mice to obtain F1 generation mice. The F1 generation heterozygous mice were then crossbred to obtain F2 generation homozygous Flox mice and F2 generation heterozygous Flox mice.

[0052] (4) F2 generation homozygous Flox mice or F2 generation heterozygous Flox mice were bred with Cdh16-Cre mice to obtain F3 generation mice.

[0053] (5) Backcross F3 generation mice with F2 generation homozygous Flox mice to screen Nlrp3[flox / flox,Cdh16-Cre] mice and obtain mice with specific knockout of Nlrp3 gene.

[0054] Example 2

[0055] This embodiment provides a method for constructing an animal model of arterial vascular calcification, including the following steps:

[0056] Mice with the Nlrp3 gene specifically knocked out were cultured to 18-22 weeks of age and kept in an SPF-grade environment with free access to food and water. The room temperature was controlled at (21±2)℃ and the relative humidity was controlled at (50±15)%. The mice were fed for another 7 weeks until they were 25-29 weeks old, and the animal model of arterial calcification was obtained. Ordinary C57BL / 6 mice were cultured under the same conditions as a healthy control group.

[0057] Test Example 1

[0058] This test case involves PCR amplification of the genome of the animal model with arterial calcification obtained in Example 2.

[0059] Genomic DNA was extracted using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit (Ver.5.0_Code No.9765).

[0060] The PCR reaction system is shown in Table 1, and the reaction procedure is shown in Table 2.

[0061] Table 1

[0062] Components volume <![CDATA[ddH2O]]> 9.0μL upstream primer F 1.0μL Downstream primer R 1.0μL Premix Taq 12.5μL Genomic DNA 1.5μL

[0063] Primer F2: 5'-TTGTGGAGGATGGGAAGTCTAAAG-3'.

[0064] Primer R2: 5'-CTCAGATAGACACCATCGTCTCAG-3'.

[0065] Primer F3: 5'-CAGAATTTTGGGAGCCTGATACACT-3'.

[0066] Cdh16-Cre verification primers: F: 5'-GCAGATCTGGCTCTCCAAAG-3'; R: 5'-AGGCAAATTTTGGTGTACGG-3'.

[0067] Table 2

[0068]

[0069] Amplification was performed using primers F2, R2, and Cdh16-Cre to verify the primers, and genotypes were analyzed. The PCR products were then subjected to agarose gel electrophoresis. The results are as follows: Figure 2 and Figure 3As shown, WT is the wild-type control, Water represents the blank reaction control without DNA template, flox / flox, the target product size of the Cdh16-Cre homozygous genotype is 251bp, the target product size of the wild-type allele is 191bp, and the target product size of the Cre amplification is approximately 420bp.

[0070] Gene knockout was verified using primers F3 and R2. The electrophoresis results of the amplification products are as follows: Figure 4 As shown (validated on multiple model mice), the target product size is 467bp.

[0071] Based on the above results, this invention successfully constructed a mouse model with the Nlrp3 gene knocked out.

[0072] Test Example 2

[0073] In this test case, the arterial calcification model mice prepared in Example 2 and the control mice were used. The aorta was taken for embedding, sectioning and alizarin red (vascular calcification) pathological staining. The staining steps are as follows:

[0074] 1. Sample fixation

[0075] Fix with 4% paraformaldehyde (fixation for 20 min), then rinse 3 times with PBS to remove fixative residue.

[0076] 2. Staining

[0077] To prepare a 1% Alizarin Red S solution, the pH is usually adjusted using deionized water with a pH of 4.2.

[0078] Immerse the sample in Alizarin Red S staining solution and stain for 20 minutes.

[0079] 3. Rinse

[0080] Rinse the sample gently with deionized water several times until there is no obvious staining residue in the background.

[0081] It can then be observed under a microscope.

[0082] Staining results as follows Figure 5 As shown, alizarin red staining of the aorta in normal C57BL / 6 mice showed no obvious calcium salt deposition, while alizarin red staining of the aorta in the arterial calcification model mice showed deep staining and obvious calcium salt deposition, indicating that the arterial calcification model mouse was successfully constructed.

[0083] In summary, this invention presents a novel construction strategy and a gene editing system. Knocking out the Nlrp3 gene effectively induces aortic calcium salt deposition in animals, resulting in a mouse model of arterial calcification. This model can be used for simulating vascular calcification-related diseases, elucidating the pathogenesis, and screening and testing therapeutic drugs and methods, which is of great significance for the treatment of vascular calcification-related diseases.

[0084] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for constructing an animal model of arterial vascular calcification, characterized in that, The method for constructing the animal model of arterial vascular calcification includes the following steps: (1) Construct a gene editing system that targets and knocks out the Nlrp3 gene in mice; (2) Gene editing of mouse fertilized eggs was performed using the gene editing system described in step (1), and loxP sites were inserted at both ends of the Nlrp3 gene; (3) The mice obtained in step (2) are bred with mice expressing Cdh16-Cre recombinase to obtain mice with specific knockout of Nlrp3 gene, and the animal model of arterial calcification is obtained after culturing. The nucleic acid sequence of sgRNA includes the sequences shown in SEQ ID NO.1 to SEQ ID NO.2; The nucleic acid sequence of the homologous recombination repair template ssDNA includes the sequence shown in SEQ ID NO.

3.

2. The method for constructing an animal model of arterial vascular calcification according to claim 1, characterized in that, The gene editing system includes sgRNA targeting the mouse Nlrp3 gene, Cas enzyme, and homologous recombination repair template ssDNA.

3. The method for constructing an animal model of arterial vascular calcification according to claim 2, characterized in that, The Cas enzyme includes the Cas9 enzyme.

4. The method for constructing an animal model of arterial vascular calcification according to claim 2, characterized in that, The homologous recombination repair template ssDNA contains a loxP site.

5. The method for constructing an animal model of arterial vascular calcification according to any one of claims 1 to 4, characterized in that, The method for constructing the animal model of arterial vascular calcification includes the following steps: (1) Preparation of CRISPR-Cas9 gene editing system, including sgRNA, Cas9 enzyme mRNA and homologous recombination repair template ssDNA; (2) sgRNA, Cas9 enzyme mRNA and homologous recombination repair template ssDNA were simultaneously introduced into mouse zygotes to obtain gene recombination zygotes with loxP sites inserted at both ends of the Nlrp3 gene. The surviving zygotes after introduction were transplanted into the uterus of pseudopregnant mice to obtain F0 generation mice. (3) The positive mice in the F0 generation were bred with wild-type mice to obtain F1 generation mice. The F1 generation heterozygous mice were then crossbred to obtain F2 generation homozygous mice and F2 generation heterozygous mice. (4) F2 generation homozygous mice or F2 generation heterozygous mice were bred with mice expressing Cdh16-Cre recombinase to obtain F3 generation mice; (5) Backcross F3 generation mice with F2 generation homozygous mice to obtain mice with specific knockout of Nlrp3 gene; (6) Mice with Nlrp3 gene knocked out were cultured to obtain the animal model of arterial calcification.

6. The application of an animal model constructed by the method for constructing an animal model of arterial calcification according to any one of claims 1-5 in analyzing the pathogenesis of arterial calcification or in preparing diagnostic and monitoring products for arterial calcification.

7. The use of an animal model constructed by the method for constructing an animal model of arterial vascular calcification according to any one of claims 1-5 in evaluating and / or screening treatment methods or therapeutic drugs for arterial vascular calcification.

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