Construction method of primary dilated cardiomyopathy animal model

Abstract

The invention provides a construction method of a primary dilated cardiomyopathy animal model. The method provided by the invention comprises the following steps: knocking out 11bp nucleotide sequences of 131-141 sites of a No.9 exon of an NEXN gene of an animal, so that a reading frame of the NEXN gene is changed, and generation of a truncated NEXN protein is caused, thereby constructing the animal model for the primary dilated cardiomyopathy. Based on NEXN gene mutation, the DCM mouse construction method is simple and easy to implement, and the model character is remarkable.

Description

Technical Field

[0001] The present invention relates to the technical field of constructing medical experimental models, and specifically to a method for constructing a genetic engineering mouse animal model of primary dilated cardiomyopathy (DCM). Background Art

[0002] DCM is a type of cardiomyopathy characterized by the enlargement of the left ventricle or both ventricles of the heart and accompanied by systolic dysfunction. It has many causes, and infections, immune responses, genetics, poisons, and metabolic disorders can all cause DCM. The symptoms of dilated cardiomyopathy are diverse, mainly including heart failure, arrhythmia, sudden death, etc. In the early stage, there may be no symptoms or mild symptoms, while in the late stage of the disease, there will be obvious heart failure symptoms. Patients will experience dyspnea, paroxysmal nocturnal dyspnea, and decreased exercise tolerance during activities, and may also include loss of appetite, abdominal distension, and lower limb edema. DCM causes great damage and has a high fatality rate, which is an important factor endangering human health.

[0003] The etiology of DCM is complex, and currently there is a lack of effective treatment methods in clinical practice. Therefore, simulating the pathological changes and disease development of human dilated cardiomyopathy through animal models is very important for studying the pathogenesis of dilated cardiomyopathy and drug screening. Human DCM models can be replicated in experimental animals such as mice, rats, zebrafish, and beagle dogs through biological methods [1-3] , but the breeding cost of large animals is relatively high and the surgical operation is difficult [4] , so currently the research on DCM models mainly focuses on small rodents.

[0004] Doxorubicin (DOX) can induce pathological manifestations similar to human DCM in mice and rats, and it is the most commonly used animal model in DCM research. However, the model induced by DOX cannot simulate the characteristics of primary DCM [5] . DCM caused by gene mutations is a common form of primary cardiomyopathy. The DCM animal model constructed based on gene mutations can more comprehensively reflect the essence of human cardiomyopathy. Previous primary DCM animal models were mostly constructed based on gene mutations such as MYH7, SCN5A, TTN, and LMNA [6] . The forms of gene mutations not only include base deletions and insertions, but also point mutations. Primary DCM caused by point mutations is also an important way to construct animal models [1,7] . NEXN is a key gene related to the occurrence of DCM discovered in 2009 [8] . Although there is currently a genetic engineering mouse model caused by NEXN point mutations [9] , for NEXN, the gene polymorphism determines that there are other point mutations that cause DCM

[0005] References

[0006] 1. Yang L, Sun J, Chen Z, Liu L, Sun Y, Lin J, Hu X, Zhao M, Ma Y, Lu D, Li Y, Guo Y, Dong E. The LMNA p.R541C mutation causes dilated cardiomyopathy in human and mice. Int J Cardiol 2022, 363:149 - 158.

[0007] 2. Chen Q, Zeng Y, Yang X, Wu Y, Zhang S, Huang S, Zhong Y, Chen M. Resveratrol ameliorates myocardial fibrosis by regulating Sirt1 / Smad3 deacetylation pathway in rat model with dilated cardiomyopathy. BMC Cardiovasc Disord 2022, 22(1):17.

[0008] 3. Hofeichner J, Gahr BM, Huber M, Boos A, Rottbauer W, Just S. CRISPR / Cas9 - mediated nexilin deficiency interferes with cardiac contractile function in zebrafish in vivo. Sci Rep 2023, 13(1):22679.

[0009] 4. Zhang D, Zhu JY, Zhang M, Li B. Establishment and evaluation of animal models of dilated cardiomyopathy. Laboratory Animal Science 2023, 40(4):55 - 61.

[0010] 5. Yu W, Deng D, Li Y, Ding K, Qian Q, Shi H, Luo Q, Cai J, Liu J. Cardiomyocyte - specific Tbk1 deletion aggravated chronic doxorubicin cardiotoxicity via inhibition of mitophagy. Free Radic Biol Med 2024, 222:244 - 258.

[0011] 6. Jin Jiamin, Gong Qian, Zhuang Lenan. Research progress on animal models and treatment of dilated cardiomyopathy. Journal of Zhejiang University (Agriculture and Life Sciences) 2024, 50(1): 1-11.

[0012] 7. Cai ZJ, Lee YK, Lau YM, Ho JC, Lai WH, Wong NL, Huang D, Hai JJ, Ng KM, Tse HF, Siu CW. Expression of Lmna-R225X nonsense mutation results in dilated cardiomyopathy and conduction disorders (DCM-CD) in mice: Impact of exercise training. Int J Cardiol 2020, 298: 85-92.

[0013] 8. Hassel D, Dahme T, Erdmann J, Meder B, Huge A, Stoll M, Just S, Hess A, Ehlermann P, Weichenhan D, Grimmler M, Liptau H, Hetzer R, Regitz-Zagrosek V, Fischer C, Nürnberg P, Schunkert H, Katus HA, Rottbauer W. Nexilin mutations destabilize cardiac Z-disks and lead to dilated cardiomyopathy. Nat Med 2009, 15(11): 1281-1288.

[0014] 9. Liu C, Spinozzi S, Feng W, Chen ZE, Zhang L, Zhu S, Wu T, Fang X, Ouyang K, Evans SM, Chen J. Homozygous G650del nexilin variant causes cardiomyopathy in mice. JCI Insight 2020, 5(16). Summary of the Invention

[0015] In view of the defects or deficiencies of the prior art, the present invention provides a method for constructing an animal model of primary dilated cardiomyopathy.

[0016] To this end, the method provided by the present invention includes knocking out a nucleotide sequence of 11 bp at positions 131-141 of exon 9 of the NEXN gene in an animal, so that the reading frame of the NEXN gene is changed, resulting in the production of a truncated NEXN protein, thereby constructing an animal model of primary dilated cardiomyopathy.

[0017] An alternative is that the animal is a mouse.

[0018] An alternative is to specifically knock out a nucleotide sequence of 11 bp at positions 131-141 of exon 9 of the NEXN gene in an animal using the CRISPR / Cas9 technology. Further, the method includes the following steps:

[0019] (1) Design a corresponding sgRNA sequence according to exon 9 of the animal NEXN gene;

[0020] (2) Microinject a mixture of Cas9 mRNA and sgRNA into the fertilized eggs of the animal, and the obtained offspring mice after transplantation are continuously mated until homozygous animals are obtained.

[0021] An alternative is that the sgRNA sequence is as shown in SEQ ID NO.2.

[0022] An alternative is that step (1) further includes synthesizing oligos according to the sgRNA sequence, and obtaining sgRNA by in vitro transcription for microinjection.

[0023] An alternative is that step (2) includes microinjecting a mixture of Cas9 mRNA and sgRNA into the fertilized eggs of the animal. After the fertilized eggs are transplanted into the recipient animal, F0 generation animals with 11 bp knockout of exon 9 are obtained. The positive F0 generation animals are mated with wild-type animals to obtain F1 generation animals, and the F1 generation animals are intercrossed to obtain homozygous animals with 11 bp knockout of exon 9.

[0024] The present invention also provides an evaluation method for the animal model constructed by the above method. The method includes: identifying animals with a deletion of a nucleotide sequence of 11 bp at positions 131-141 of exon 9 by PCR and Sanger sequencing; detecting the heart volume, heart mass, body weight, left ventricular cavity, heart function, changes in left ventricular end-diastolic diameter, end-systolic diameter, left ventricular end-systolic anterior wall thickness, and left ventricular end-diastolic posterior wall thickness of the animals after they are born and raised for several days; among them, an animal model with an enlarged heart volume, increased heart mass, increased body weight, enlarged left ventricular cavity, decreased heart function, increased left ventricular end-diastolic diameter, increased end-systolic diameter, decreased left ventricular end-systolic anterior wall thickness, and increased left ventricular end-diastolic posterior wall thickness is an animal model of primary dilated cardiomyopathy.

[0025] The present invention solves the problems that there are few breeds and strains of primary DCM animal models at present, the stability and repeatability are poor, and the pathological manifestations and pathogenesis of human primary DCM cannot be fully reflected.

[0026] The primary DCM animal model of the present invention is based on the mutation of the NEXN exon 9 sequence, has good heredity, high stability and repeatability, and significant model traits, and can be used for the research on the molecular mechanism of the occurrence and development of primary DCM and drug screening. This model can be used to study the molecular mechanism of the occurrence and development of primary DCM, prepare and screen drugs for the treatment of DCM, etc. Brief Description of the Drawings

[0027] Figure 1 It is a schematic diagram of gene editing and sgRNA sequence in the embodiment of the present invention.

[0028] Figure 2 It is the in vitro transcription result of sgRNA in the embodiment of the present invention.

[0029] Figure 3 It is the PCR detection result of the model mice in the embodiment of the present invention; among them, 1-11 are the genotype PCR detections of the F0 generation mice at birth in sequence; M is the marker.

[0030] Figure 4 It is the genotype detection result of the model mice in the embodiment of the present invention; among them, A is the analysis of the mutant genotype by Sanger sequencing; B is the amino acid sequence of the mutant Nexilin protein.

[0031] Figure 5 It is the appearance inspection of the model mice in the embodiment of the present invention.

[0032] Figure 6 It is the gross anatomy of the heart of the model mice and the heart-to-body weight ratio in the embodiment of the present invention; among them, A is the gross anatomy of the mouse heart; B is the mouse heart / body weight ratio.

[0033] Figure 7 It is the survival rate of the model mice in the embodiment of the present invention.

[0034] Figure 8 It is the detection of the Nexilin protein expression level of the model mice in the embodiment of the present invention.

[0035] Figure 9 It is the HE and Masson staining results of the model mice in the embodiment of the present invention.

[0036] Figure 10 It is the echocardiogram detection of the model mice in the embodiment of the present invention. Detailed Embodiments

[0037] Unless otherwise specified, the scientific and technical terms and related abbreviations in this article are understood according to the knowledge of those of ordinary skill in the relevant fields.

[0038] The technical solutions of the present invention will be described in detail and completely below in conjunction with specific embodiments. The described embodiments are only a part of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work fall within the scope of protection of the present invention.

[0039] Unless otherwise specified, the test conditions and operations not mentioned in the embodiments of the present invention are carried out according to the conventional methods in the art or the conditions recommended by the manufacturer.

[0040] Example 1: Design and synthesis of sgRNA sequences

[0041] (1) Design of sgRNA sequences

[0042] According to the characteristics of the NEXN gene, exon 9 was mutated to construct an animal model of dilated cardiomyopathy mice. Exon 9 consists of 198 bp nucleotides, and the sequence is shown in SEQ ID NO.1. The above sequence was uploaded to the CRISPOR and CHOPCHOP websites to analyze the potential sgRNA sequences that can be used for gene knockout. According to the analysis results, 20 bp sequences with relatively high scores on both websites and a GC content of 40%-70% were selected as candidate sgRNAs for synthesis ( Figure 1 ), and the sgRNA sequences are shown in SEQ ID NO.2.

[0043] (2) Synthesis of sgRNA

[0044] When synthesizing, a 17 bp T7 promoter sequence 5'-TAATACGACTCACTATA-3' was introduced at the 5' end of sgRNA, and a 20 bp RNA scaffold sequence was introduced at the 3' end. At the same time, according to codon preference, 3 Gs were added before sgRNA to improve the transcription efficiency. This 60 bp sequence (5'- TAATACGACTCACTATA gggagaagaaagacgacgaacagGTTTTAGAGCTAG AAATAGC-3', the underlined part is the T7 promoter sequence, the lowercase letters are the sgRNA sequences, and the uppercase letters are the scaffold sequences) was synthesized by Beijing Tsingke Biotechnology Co., Ltd. and used as the upstream primer (sgRNA-F) for sgRNA PCR amplification. In addition, according to the characteristics of the pX330 plasmid, a 20 bp sequence of 5'-AGCACCGACTCGGTGCCACT-3' was designed and synthesized as the downstream primer (sgRNA-R) for PCR amplification.

[0045] Example 2: In vitro transcription of sgRNA

[0046] (1) PCR Amplification and Purification of sgRNA Sequences

[0047] Using the pX330 plasmid as a template, PCR amplification was performed with sgRNA-F and sgRNA-R as the upstream and downstream primers. The PCR reaction system is shown in Table 1 below:

[0048] Table 1

[0049]

[0050]

[0051] The PCR reaction conditions are shown in Table 2 below:

[0052] Table 2

[0053]

[0054] The PCR product was purified using the Tiangen DNA purification kit. After equilibrating the adsorption column, the PCR product was added to the adsorption column, and the elution buffer was added twice for elution. The purified DNA was collected from the adsorption column and quantified.

[0055] (2) In Vitro Transcription of sgRNA

[0056] 1) Prepare the reaction system according to Table 3 below

[0057] Table 3

[0058] PCR amplification product 800 ng 5X TranscriptAid Reaction Buffer 4 μl ATP / CTP / GTP / UTP Mix 8 μl TranscriptAid Enzyme Mix 2 μl <![CDATA[DEPC H2O]]> Up to 20 μl

[0059] In vitro transcription of sgRNA was performed using the TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific TM Company) according to the instructions. The specific steps are as follows:

[0060] 2) In Vitro Transcription

[0061] After mixing the above components, incubate at 37 °C for 2 h, and collect the product for further purification.

[0062] 3) Purification of RNA Products

[0063] The in vitro transcription product of sgRNA was purified using the MEGAclear TM kit (Thermo Fisher Scientific TM Company) according to the instructions.

[0064] Add 20 μl of the transcription product to 100 μl of Elution Solution, 350 μl of Binding Solution, and 250 μl of ethanol, mix well, slowly transfer it into an elution column, centrifuge at 12,000 rpm for 30 s, and discard the centrifugate; wash twice with 500 μl of Wash Solution, add 20 μl of Wash Solution preheated at 95 °C for elution, collect the eluate, and after quantification with NanoDrop, it is the purified sgRNA. The size of the sgRNA identified by 1% agarose gel electrophoresis is about 120 bp, which is consistent with the expectation( Figure 2 ). Quantitative analysis shows that the sgRNA has OD260 / OD280 > 1.8 and OD260 / OD230 > 2.3, with a content of 1.2 μg / μl, and it is stored at -80 °C for later use.

[0065] Example 3: Microinjection and transplantation of fertilized eggs

[0066] (1) Obtaining C57BL / 6J mouse fertilized eggs

[0067] Cage together female and male mice inoculated with 5 U of PMSG and HCG. Sacrifice the mice with plugs the next day, and collect mouse fertilized egg cells from the ampulla of the fallopian tube under a stereomicroscope.

[0068] (2) Microinjection

[0069] Mix 100 ng of Cas9 mRNA (ThermoFisher Scientific TM Company) and 50 ng of sgRNA obtained in Example 2 in a ratio of 1:2 to obtain a Cas9 / sgRNA mixture. Inject the Cas9 / sgRNA mixture into the cytoplasm of C57BL / 6J mouse fertilized egg cells using an Eppendorf NK2 microinjector; continue to culture the injected fertilized egg cells at 37 °C and 5% CO2 for 24 h until the 2-cell stage.

[0070] (3) Transplantation of fertilized eggs

[0071] Transfer the 2-cell embryos to recipient ICR mice. The offspring mice are F0 generation animals for genotype identification after birth.

[0072] Example 4: Genotype identification of F0 mice

[0073] (1) Extraction of genomic DNA from F0 mice

[0074] Extract the genomic DNA of F0 generation mice using a Blood / Tissue / Cell Genomic DNA Extraction Kit (Tiangen Biochemical Technology Company). The specific method is as follows:

[0075] ① Cut the tail tissue of 7-day-old F0 mice at birth. Add 200 μl of GA solution and 20 μl of Proteinase K to the sample, and incubate overnight at 56 °C until the tissue is lysed and digested.

[0076] ② Add 200 μl of GB solution to the lysate, invert and mix well, and place at 70 °C until the solution becomes clear.

[0077] ③ Add 200 μl of absolute ethanol, shake and mix well; pour the mixture into the adsorption column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

[0078] ④ Add 500 μl of GD to the adsorption column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

[0079] ⑤ Add 600 μl of PW, centrifuge at 12000 rpm for 30 s and elute continuously twice.

[0080] ⑥ Drop 50 μl of TE eluent onto the adsorption membrane of the adsorption column, let it stand for 5 min, centrifuge at 12000 rpm for 30 s, and collect the genomic DNA for identification.

[0081] (2) Identification of F0 mouse genome

[0082] Amplify the corresponding fragment of the NEXN gene using 2×Taq MIX (Takara) and perform sequencing analysis. The specific method is as follows:

[0083] The primers required for PCR were synthesized by Beijing Tsingke Biotechnology Co., Ltd. The primers used are as follows:

[0084] Forward primer: 5’-CCTCCATATCACAGATACCCCAG-3’; Reverse primer: 5’-CCTTGCTACACTCTACCAGGTC-3’.

[0085] The PCR reaction system is shown in Table 4:

[0086] Table 4

[0087]

[0088]

[0089] The PCR reaction conditions are shown in Table 5:

[0090] Table 5

[0091]

[0092] After the PCR product was identified by 1% agarose gel electrophoresis ( Figure 3), sequenced and analyzed for genotype by Beijing Tsingke Biotechnology Co., Ltd. using Sanger sequencing. The sequencing results showed that exon 9 of the NEXN gene in F0 mice was knocked out by 11 bp (Δ11) at nucleotides 131 - 142 nt ( Figure 4 -A); the wild-type (wt) NEXN gene encodes a Nexilin protein of 672 amino acids, while the 11-bp deletion results in a frameshift change, generating a TAA stop codon at 1171 - 1173 bp, leading to premature termination of translation and only a truncated Nexilin protein of 391 amino acids (truncated NEXN protein) can be translated ( Figure 4 -B).

[0093] Example 5: Establishment of DCM homozygous mice

[0094] Δ11 F0 mice were mated with wild-type mice to obtain F1 heterozygous mice, and the F1 heterozygous mice were intercrossed to obtain F2 mice. F2 mice of different genders were continuously intercrossed until DCM homozygous mice with the Δ11 mutation were obtained.

[0095] Example 6: Identification and evaluation of DCM homozygous mice

[0096] In this example, the appearance and gross anatomy of the heart of the DCM homozygous mice constructed by the method of Example 5 were observed; the expression level of NEXN protein was detected by Western blot; the pathological changes of the mouse heart tissue were detected by HE and Masson staining; and the cardiac function changes of the mice were detected by echocardiography.

[0097] (1) General status observation and gross anatomy

[0098] After conventional feeding, the wild mice had good survival conditions, but the DCM homozygous mice (Δ11) were thin and had sparse hair ( Figure 5 as shown). After dissection, it was found that the heart volume was significantly increased ( Figure 6 A), the heart weight ratio increased ( Figure 6 B), and they began to die 4 days after birth, and all mice died on the 6th day ( Figure 7 ).

[0099] (2) Expression level of NEXN protein in the heart tissue of model mice

[0100] Fresh heart tissue of mice was excised, ground, and lysed with an appropriate amount of RIPA lysis buffer. After protein quantification, SDS-PAGE electrophoresis was performed with a 10% separating gel, and the separated gel was transferred to a membrane at a voltage of 100 V for 1.5 h. The PVDF membrane was blocked, incubated with a 1:500 anti-NEXN antibody (Wuhan Sanying Biotechnology Co., Ltd.) overnight at 4 °C, followed by addition of the secondary antibody, eluted with TBST, and detected by ECL luminescence. The results showed that the expression of NEXN in the heart tissue of DCM homozygous mice was significantly decreased compared with that of wild-type mice.Figure 8 )。

[0101] (3) Histopathological changes in the heart tissue of model mice

[0102] Fresh hearts of mice were excised, rinsed with PBS, fixed with 4% paraformaldehyde for 48 h, embedded in paraffin, sectioned, dewaxed, and stained with HE and Masson by the conventional method. The HE- and Masson-stained sections were scanned and analyzed with 3DHISTECHs. HE staining showed that the left ventricular cavity of DCM homozygous mice was significantly enlarged, the volume increased, and the ventricular wall became thinner, which was consistent with the pathological manifestations of dilated cardiomyopathy. However, Masson staining showed that the characteristic of myocardial cell fibrosis was not obvious ( Figure 9 )。

[0103] (4) Evaluation of cardiac function in model mice

[0104] Six-day-old mice were placed on their backs, and conductive gel was applied to their chests. Echocardiography of the mouse heart was performed with a small animal photoacoustic ultrasound imaging system (Fujifilm VisualSonics LAZR-X). According to the obtained M-mode echocardiogram images, indices such as left ventricular anterior wall thickness in diastole (LVAWd), left ventricular anterior wall thickness in systole (LVAWs), left ventricular posterior wall thickness in diastole (LVPWd), left ventricular posterior wall thickness in systole (LVPWs), left ventricular internal diameter in diastole (LVIDd), left ventricular internal diameter in systole (LVIDs), left ventricular ejection fraction (EF), and left ventricular fractional shortening (FS) were calculated. The results showed that the cardiac function of DCM homozygous mice was severely impaired, with decreased LVAWd and LVAWs, increased LVIDd, LVIDs, and LVPWd, and significantly decreased LVEF and LVFS, while LVPWs did not change ( Figure 10 )。

[0105] The above results have described in detail the embodiments of the present disclosure in combination with the accompanying drawings. The methods and means established in the present invention can be used to establish a stable and efficient genetically engineered animal model of primary DCM in mice. This model can simulate the development process and the body's change process of human dilated cardiomyopathy, and can be used for research such as screening of therapeutic drugs for primary DCM and the mechanism of disease development. However, the present disclosure is not limited to the specific details in the above embodiments. Within the technical scope of the present disclosure, various modifications and optimizations can be made to the technical solutions of the present disclosure, and these simple optimizations all fall within the protection scope of the present disclosure.

[0106] In addition, it should be noted that, among the various specific technical features described in the above specific embodiments, without conflict, they can be combined in any appropriate manner. To avoid unnecessary repetition, the present disclosure will not separately describe various possible combination methods.

[0107] In addition, any combination can be made between various different embodiments of the present disclosure, as long as it does not violate the idea of the present disclosure, it should also be regarded as the content disclosed by the present disclosure.

[0108] Sequence Listing of the Specification

[0109]

Claims

1. A method for constructing an animal model of primary dilated cardiomyopathy, characterized in that: The method includes knocking out the 11bp nucleotide sequence at positions 131-141 of exon 9 of the NEXN gene of the animal, thereby changing the NEXN gene reading frame and causing the production of truncated NEXN protein, thereby constructing an animal model of primary dilated cardiomyopathy.

2. The method for constructing an animal model of primary dilated cardiomyopathy according to claim 1, characterized in that: The animal is a mouse.

3. The method for constructing an animal model of primary dilated cardiomyopathy according to claim 1, characterized in that: CRISPR / Cas9 technology was used to specifically knock out the 11bp nucleotide sequence at positions 131-141 of exon 9 of the animal NEXN gene.

4. The method for constructing an animal model of primary dilated cardiomyopathy according to claim 3, characterized in that: The following steps are involved: (1) Design the corresponding sgRNA sequence based on exon 9 of the animal NEXN gene; (2) The mixture of Cas9 mRNA and sgRNA is microinjected into the fertilized eggs of the animals, and the offspring mice obtained after transplantation are continuously mated until homozygous animals are obtained.

5. The method for constructing an animal model of primary dilated cardiomyopathy according to claim 4, characterized in that: The sgRNA sequence is shown in SEQ ID NO.

2.

6. The method for constructing an animal model of primary dilated cardiomyopathy according to claim 4, characterized in that: Step (1) also includes synthesizing oligos according to the sgRNA sequence, and obtaining sgRNA by in vitro transcription for microinjection.

7. The method for constructing an animal model of primary dilated cardiomyopathy according to claim 4, characterized in that: Step (2) comprises: microinjecting a mixture of Cas9 mRNA and sgRNA into animal fertilized eggs, transplanting the fertilized eggs into recipient animals to obtain F0 generation exon 9 11bp knockout animals, mating positive F0 generation animals with wild-type animals to obtain F1 generation animals, and intercrossing F1 generations to obtain homozygous animals with exon 9 11bp knockout.

8. The evaluation method of the animal model constructed by any one of claims 1 to 7, characterized in that: Methods include: PCR and Sanger sequencing were used to identify animals with a nucleotide sequence deletion of 11 bp at positions 131-141 of exon 9; After the animals were born and raised for several days, the heart volume, heart mass, body weight, left ventricular cavity, heart function, left ventricular end-diastolic diameter, end-systolic diameter, left ventricular end-systolic anterior wall thickness and left ventricular end-diastolic posterior wall thickness were tested. Among them, the animal model with increased heart volume, increased heart mass, increased body weight, enlarged left ventricular cavity, decreased heart function, increased left ventricular end-diastolic diameter, increased end-systolic diameter, decreased left ventricular end-systolic anterior wall thickness and increased left ventricular end-diastolic posterior wall thickness is the primary dilated cardiomyopathy animal model.

9. Use of the animal model constructed by the method according to any one of claims 1 to 7 for drug research on primary dilated cardiomyopathy.

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