A method for constructing a myopathy animal model, the constructed model and application thereof

Abstract

The application discloses a method for constructing a myopathy animal model, the constructed model and application of the model. The application also provides application of a reagent for knocking out or mutating a PRR14 gene or inhibiting expression of the gene in preparation of a myopathy animal model product. Specifically, compared with normal mice, total muscle mass, grip level and muscle endurance of the mice with muscle tissue-specific knockout of the PRR14 gene are all significantly reduced, the animal model provided by the application has typical characteristics of myopathy, can well simulate occurrence and development of myopathy in vivo, is an ideal animal model for basic and clinical application research of myopathy, and can be well applied to screening of drugs for treating myopathy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for constructing a myopathy animal model, the constructed model and application thereof. BACKGROUND

[0002] Myopathy refers to primary or secondary structural or functional lesions of muscle, and is a disease often manifested as persistent loss and atrophy of muscle tissue, which seriously affects the life quality and prognosis of patients. At present, common myopathies in clinical practice include dystrophic myopathy, sarcopenia, disuse atrophy, neurogenic atrophy, congenital myopathy and inflammatory myopathy. Myopathy can cause decreased muscle strength, limited activity, decreased self-care ability in daily life, easy fatigue and the like, which seriously affects the life quality and prognosis of patients.

[0003] At present, common myopathy animal models are mainly obtained by limiting food intake, which simulates muscle atrophy under malnutrition. Muscle atrophy caused by malnutrition has a very low incidence in modern society, and cannot simulate aging-related muscle atrophy. Another method for obtaining an aging-related myopathy animal model is to use an animal as a model after long-term breeding and aging. This method takes a long time, has poor consistency due to individual differences, and thus reduces the repeatability of drug screening experiments.

[0004] In summary, it is necessary to develop a myopathy animal model with small individual differences and easy to obtain, so as to simulate the occurrence and development of myopathy in vivo and be applied to basic and clinical research of myopathy.

[0005] PRR14 is a lamin protein, and current research has not found whether PRR14 is associated with the occurrence of myopathy. SUMMARY

[0006] The present application provides a method for constructing a myopathy animal model, the constructed model and application thereof, and aims to solve the problems in the prior art of preparing a myopathy animal model. The animal model provided by the present application has typical characteristics of myopathy, can well simulate the occurrence and development of myopathy in vivo, is an ideal animal model for basic and clinical application research of myopathy, and can be well applied to screening of drugs for treating myopathy.

[0007] The present application is realized by the following technical solutions:

[0008] The human PRR14 gene is located at chromosome 16p11.2, and the gene ID in NCBI is 78994; two transcripts (CDS sequences are seen in NM_001320464.3; NM_024031.5) and two encoded proteins (NP_001307393.1; NP_076936.1) have been found in GeneBank sequences.

[0009] In a first aspect, the present application provides a method for constructing a model of myopathy animal, i.e. knocking out or mutating PRR14 gene, and obtaining an animal model by inhibiting the expression of the gene.

[0010] Further, the myopathy includes, but is not limited to, at least one of dystrophic myopathy, sarcopenia, disuse atrophy, neurogenic atrophy, congenital myopathy, and inflammatory myopathy.

[0011] The above method specifically includes the following steps:

[0012] S1: selecting a parent PRR14 flox / flox The genotype PRR14 flox / + ACTA1-CreEsr1 mice are crossed with the genotype PRR14

[0013] S2: obtaining the genotype PRR14 flox / + ACTA1-CreEsr1 mice are crossed with the genotype PRR14 flox / flox mice, and the genotype PRR14 flox / flox ACTA1-CreEsr1 mice are obtained by identifying the genotype of the offspring mice;

[0014] S3: PRR14 flox / flox ACTA1-CreEsr1 mice are orally administered tamoxifen from the 7th day after birth to construct a PRR14-mKO mouse model.

[0015] The gene identification in step S2 is to amplify the gene fragment by PCR according to the position of the Flox region constructed by the targeting vector, and to judge the genotype of the mouse by agarose gel electrophoresis according to the length of the gene fragment and the presence or absence of the product;

[0016] The sequence of the PRR14 primer pair in the PCR reaction system is as follows:

[0017] ForwardP1, 5'-GTCTCTACTGCAAGAAAGGCTGAG-3';

[0018] ReverseP2, 5'-ATTTCTCCCCTACCTAAAGGACG-3';

[0019] The sequence of the Cre transgene primer pair is as follows:

[0020] ForwardP3, 5'-CGAGCCGAGAGTAGCAGTTGTA-3';

[0021] Reverse P4, 5'-AGGTGGACCTGATCATGGAG-3';

[0022] The PRR14 knockout primer pair is verified as follows:

[0023] Forward P5, 5'-GGAGGAACTGGCCCTCATTC-3';

[0024] Reverse P6, 5'-ATTTCTCCCCTACCTAAAGGACG-3';

[0025] In the step S3, the tamoxifen is dissolved in 2% ethanol aqueous solution, and the concentration of the tamoxifen is 0.8 mg / mL, which is used to feed the mice as daily drinking water.

[0026] In a second aspect of the present application, the myopathy animal model constructed by the above method is provided.

[0027] In a third aspect of the present application, the myopathy animal model is used in the research of myopathy.

[0028] In a fourth aspect of the present application, the myopathy animal model is used in the screening of drugs for treating myopathy.

[0029] The method comprises the following steps: applying a test candidate drug to the myopathy animal model constructed by the above method, and comparing with the myopathy cell model without the test candidate drug, wherein the test compound resulting in improvement or cure of the myopathy symptoms after application is the candidate drug for treating myopathy. The above screened candidate drug can constitute a screening library, and further cell experiments, animal experiments, and / or clinical trials can be performed on these substances to further confirm the myopathy treatment effect of the potential substances.

[0030] In a fifth aspect of the present application, the reagent for knocking out or mutating or inhibiting the PRR14 gene is used in the preparation of a myopathy animal model preparation.

[0031] In the previous research work, the inventors found that the expression amount of PRR14 in the muscle tissue of patients with various myopathies is down-regulated by mining public data sets, and therefore the inventors constructed a PRR14 skeletal muscle conditional knockout mouse model. Statistical analysis found that the body weight, muscle mass, grip strength and exercise endurance of the PRR14 skeletal muscle conditional knockout mice were significantly reduced, which suggested the correlation between the down-regulation of PRR14 gene expression and myopathy.

[0032] Since the present application adopts knockout PRR14 for induction, the constructed model is more similar to the pathogenesis in the in vivo environment, has high consistency, avoids heterogeneity caused by individual differences, and is more accurate and efficient than the previous model in drug screening.

[0033] The present application has the following beneficial effects:

[0034] (1) The present application first discloses that knockout PRR14 can induce muscle atrophy in mice, which is more similar to the pathogenesis in humans. (2) The myopathy animal model of the present application has typical characteristics of myopathy and can well simulate the occurrence and development of myopathy in vivo, and is an ideal animal model for basic and clinical application research of myopathy, thereby being well applied to screening drugs for treating myopathy.

[0035] (3) The myopathy animal model of the present application can be induced and modeled at any development stage of the animal, can accurately simulate the pathogenesis of the disease at different ages, and is conducive to the study of the mechanism of different stages of the disease, and is an ideal animal model for basic and clinical application research of myopathy. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 GWAS analysis found that PRR14 has a strong correlation with the traits of BMI and lean body mass.

[0037] Figure 2 The expression levels of PRR14 in patient samples and normal control samples in several myopathy microarray external data sets are shown in FIG. A, which shows that the expression amount of PRR14 in muscle tissue of patients with DM type 2 is decreased, FIG. B shows that the expression amount of PRR14 in muscle tissue of patients with LGMD2A is decreased, FIG. C shows that the expression amount of PRR14 in muscle tissue of patients with JDM is decreased, and FIG. D shows that the expression amount of PRR14 in muscle precursor cells of patients with progeria (HGPS) is decreased.

[0038] Figure 3 The construction strategy of the mouse with knockout PRR14 gene in skeletal muscle is shown in the schematic diagram.

[0039] Figure 4 The results of nucleic acid electrophoresis for genotype identification of control mice and PRR14-mKO mice are shown in the following table.

[0040] Figure 5 The appearance of PRR14-mKO mice and control mice is shown in the following table.

[0041] Figure 6 The body weight of 24-week-old PRR14-mKO mice and control mice is shown in the following table, and the mice without tamoxifen induction do not show the phenotype of body weight reduction.

[0042] Figure 7 : PRR14-mKO mice showed significantly reduced tibialis anterior (TA), quadriceps (Quad), gastrocnemius (GA) and triceps muscle mass compared to control mice. Mice without tamoxifen induction did not show the phenotype of reduced skeletal muscle mass.

[0043] Figure 8 : PRR14-mKO mice showed significantly reduced grip strength of the upper limbs compared to control mice. Mice without tamoxifen induction did not show the phenotype of reduced grip strength.

[0044] Figure 9 : PRR14-mKO mice showed significantly reduced time remaining on the rotating rod apparatus, indicating that PRR14 skeletal muscle conditional knockout mice showed significantly reduced muscle endurance. Mice without tamoxifen induction did not show the phenotype of reduced muscle endurance.

[0045] Figure 10 : PRR14-mKO mice vs. control mice gastrocnemius muscle cross-sections stained with WGA.

[0046] Figure 11 : PRR14-mKO mice vs. control mice gastrocnemius muscle fiber cross-sectional area.

[0047] Figure 12 : Transmission electron microscopy of skeletal muscle of PRR14-mKO mice vs. control mice. Inhomogeneous sarcoplasmic increase was observed in gastrocnemius muscle of PRR14-mKO mice. Sarcoplasm extended axially through sarcomeres, leading to loss of myofibrillar continuity. Irregular z-bands were also observed, suggesting myofibrillar dislocation. In addition, focal myofibrillar loss, extensive swelling of mitochondria, and disruption and fragmentation of cristae were observed.

[0048] Figure 13 : Transmission electron microscopy of skeletal muscle of PRR14-mKO mice vs. control mice. PRR14-mKO mice showed nuclear heterochromatin condensation, nuclear membrane invagination, and similar to the nuclear pathological changes of nuclear lamina disease myofibrillar cells.

[0049] Figure 14 : Transmission electron microscopy of skeletal muscle of PRR14-mKO mice vs. control mice. After 1 hour of treadmill exercise at 15 meters / minute, PRR14-mKO mice showed myofibrillar loss, splitting, reduced width, and sarcomere structure destruction and degradation in gastrocnemius muscle tissue.

[0050] Figure 15Changes of gastrocnemius muscle mass (A), hindlimb strength (B), PRR14 mRNA expression in skeletal muscle samples (C) of 4-month-old wild type male mice and control mice after tibial nerve transection for 14 days.

[0051] Figure 16 Changes of gastrocnemius muscle mass (A), hindlimb strength (B), gastrocnemius muscle fiber cross-section WGA staining cross-sectional area (C) and PRR14 mRNA expression in skeletal muscle samples (D) of 4-month-old wild type male mice and control mice after hindlimb immobilization for 7 days. DETAILED DESCRIPTION

[0052] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be clearly and completely described below with specific examples.

[0053] Example 1: Genome-wide association analysis and analysis of PRR14 mRNA expression changes in myopathy patients in public data sets.

[0054] Genome-wide association analysis was performed, and PRR14-related SNP sites were screened for significant association with BMI and lean body mass, as shown in Table 1. Figure 1 .

[0055] Transcriptome data of myopathy patients were downloaded from the Gene Expression Omnibus (GEO) database (https: / / www.ncbi.nlm.nih.gov / geo / ) of NCBI, and the downloaded data sets included GSE45331, GSE11681, GSE3307 and GSE3860. The corrected expression of PRR14 in muscle tissue (GSE45331, GSE11681, GSE3307) or muscle precursor cells (GSE3860) of patients and control patients in each data set was extracted for analysis, and the results are shown in Table 2. Figure 2 The sources of myopathy samples include: muscle tissue of myotonic dystrophy, muscle tissue of limb girdle muscular dystrophy, muscle tissue of juvenile dermatomyositis, muscle precursor cells of progeria patients.

[0056] Example 2

[0057] The experimental animals used in this part are as follows: This part uses parent PRR14 flox / flox mice in C57BL / 6 genetic background and ACTA1-CreEsr1 male mice (purchased from SAIYE Biological Technology Co., Ltd.), SPF level, half male and half female, 12 weeks old, average weight about 20g.

[0058] This part uses Cre / LoxP system to establish PRR14 conditional gene knockout mouse model, which needs two kinds of transgenic animals, one is PRR14 transgenic mouse with LoxP sequence introduced into the targeted knockout group gene sequence, and the other is CreEsr1 transgenic mouse driven by ACTA1 promoter. Then conditional gene knockout mouse containing Cre recombinase gene and LoxP sequence in vivo is constructed by crossing.

[0059] Human PRR14 gene is located on chromosome 16p11.2, gene ID: 78994 in NCBI; two transcripts (CDS sequence see NM_001320464.3; NM_024031.5) and two coding proteins (NP_001307393.1; NP_076936.1) are currently found in GeneBank sequence. Exon targeted by Flox: Exon 1-11, see Figure 3 .

[0060] ACTA1 maintains extremely high expression in all periods of skeletal muscle differentiation and development, becoming one of the specific markers of skeletal muscle. Therefore, the CreEsr1 transgenic mouse driven by ACTA1 promoter is used in the present application, so that the expression of Cre can be specifically regulated in skeletal muscle tissue.

[0061] CreEsr1 is in an inactive state in the cytoplasm without tamoxifen (TAM, Tamoxifen) induction; after Tamoxifen induction, the metabolite 4-OHT (estrogen analogue) of Tamoxifen binds with Esr1, which can make CreEsr1 into the nucleus to play the activity of Cre recombinase, and specifically recognize LoxP sequence; when two LoxP sequences are located in the same DNA strand and have the same direction, the DNA fragment between the two LoxP sequences is knocked out.

[0062] The following is the method for establishing skeletal muscle tissue-specific PRR14 gene conditional knockout mouse (PRR14-mKO) model based on Cre / LoxP system.

[0063] Construction of PRR14-mKO mouse model

[0064] 1. Breeding and reproduction of mice

[0065] Parent PRR14 flox / flox mice are crossed with ACTA1-CreEsr1 mice to obtain PRR14 flox / + ACTA1-CreEsr1 genotype mice are crossed with PRR14 flox / flox genotype mice to obtain PRR14 flox / floxACTA1-CreEsr1 mice. During the breeding of model mice, male and female mice were caged and mated at a ratio of 1:2. PRR14-mKO mice were obtained by genotyping the offspring mice, which were induced for Cre recombinase activity by feeding with tamoxifen at a concentration of 0.8 mg / ml dissolved in 2% ethanol aqueous solution instead of pure water on a daily basis, so as to achieve PRR14 skeletal muscle tissue-specific knockout. The genotyping results and PRR14 mRNA expression levels are shown in Table 1 and Figure 1. Figure 4 .

[0066] 2. Verification of mouse model at macroscopic morphological level - photographs of mouse appearance and bone muscle mass measurement

[0067] By comparing the general appearance of 12-week-old PRR14-mKO mice and control mice, and the appearance of hind limb muscles, it was found that the PRR14-mKO mice in the experimental group were significantly smaller in size and had less muscle mass than the control mice, as shown in Figure 2. Figure 5 The body weights of 24-week-old PRR14-mKO mice and control mice were measured, and it was found that the body weight of the PRR14-mKO mice was significantly reduced, as shown in Figure 3. Figure 6 The mass of each muscle was measured, and it was found that the mass of the tibialis anterior muscle, quadriceps muscle, gastrocnemius muscle, and biceps brachii muscle of the PRR14-mKO mice was smaller than that of the control mice, and the difference was statistically significant, as shown in Figure 4. Figure 7 .

[0068] 3. Verification of mouse model at muscle function level - mouse upper limb grip strength and Rota-rod experiment

[0069] By comparing the upper limb grip strength and the time that the mice in the experimental group could balance on the Rota-rod system rotating at a speed of 30 rpm, it was found that the upper limb grip strength and muscle endurance of the PRR14-mKO mice were significantly reduced, as shown in Figure 5. Figure 8 , 9 .

[0070] 4. Verification of mouse model at tissue morphological level - WGA staining of gastrocnemius muscle cross-sections

[0071] Gastrocnemius muscles were taken from 12-week-old PRR14-mKO mice and control mice, embedded in OCT, and prepared into 8 nm frozen sections. The muscle fiber profile was shown by WGA staining, and the muscle fiber diameter was compared. The staining method was as follows: 4% paraformaldehyde was used to fix the frozen sections at room temperature for 15 minutes; PBS was used to wash three times, 5 minutes each time; 0.1% Triton-X100 and 5% BSA were dissolved in PBS and incubated at room temperature for 1 hour; PBS was used to wash three times, 5 minutes each time; 5 μg / mL WGA, Alexa Fluor 488 conjugate was added and incubated at room temperature for 1 hour; PBS was used to wash three times, 5 minutes each time; the sections were mounted with DAPI-containing mounting medium and observed under a fluorescence microscope. 594conjugate room temperature for 10 minutes; wash with PBS for three times. Take pictures under microscope, count the area of muscle fiber, as shown in Figure 10 、 11 The area of muscle fiber of PRR14-mKO mice is significantly reduced.

[0072] 5. Verification of mouse model at histomorphology level-radial transmission electron microscope of gastrocnemius muscle

[0073] Freshly dissected gastrocnemius muscle of 12-week-old PRR14-mKO mice and control mice were fixed in 2.5% glutaraldehyde. The tissue samples were cut into 1 mm 3 cubes, rinsed and exposed to 1% osmium tetroxide, dehydrated and embedded. The samples were then cut into ultrathin sections (60 nm) and the ultrastructure of gastrocnemius muscle was observed using transmission electron microscope. Compared with control mice, uneven sarcoplasmic increase was observed in gastrocnemius muscle of PRR14-mKO mice. Sarcoplasm extended axially through sarcomere, resulting in loss of myofibril continuity. Irregular z bands were also observed, suggesting the presence of myofibril dislocation. In addition, focal loss of myofibril, extensive swelling of mitochondria, destruction and fragmentation of cristae were observed, as shown in Figure 12 ; The nucleus of PRR14-mKO mouse muscle fiber was observed to have heterochromatin condensation and nuclear membrane invagination, indicating that the muscle fiber of PRR14-mKO mice was in a relatively static state similar to satellite cells, as shown in Figure 13 ; After 1 hour of treadmill exercise at 15 meters / minute, gastrocnemius muscle tissue of PRR14-mKO mice was observed to have myofibril loss, splitting, reduced width, sarcomere structure destruction and degradation, as shown in Figure 14 . This animal model of myopathy can well simulate the occurrence, development and structural changes of myopathy at the ultrastructural level.

[0074] Example 3: Exploration of the relationship between the occurrence and development of disuse atrophy and neurogenic atrophy and PRR14.

[0075] Select 4-month-old male wild-type C57 mice, after isoflurane inhalation anesthesia, make an incision in the middle and lower third of the femur along the direction of the ischial tuberosity and knee joint under the stereomicroscope, bluntly separate the biceps femoris, separate the branch of the sciatic nerve, cut off the tibial nerve, and suture the biceps femoris and skin layer by layer (Den). The control mice were made skin incision at the same position, the biceps femoris was separated, the tibial nerve was not cut off, and the skin was sutured layer by layer (sham). After 14 days, the mouse hind limb grip strength was measured, the gastrocnemius muscle (GA) weight was measured after dissection and weighing, the gastrocnemius muscle RNA was extracted and the PRR14 mRNA expression in gastrocnemius muscle tissue was detected by RT-PCR method, as shown in Figure 15 .

[0076] Select 4-month-old male wild-type C57 mice, the mouse hind limbs with 30° implementation of suspension 7 days (HS7D), 7 days later, measure the mouse hind limb grip strength, dissection and weighing gastrocnemius (GA) weight, section measurement gastrocnemius muscle fiber cross-sectional area, extraction gastrocnemius RNA and by RT-PCR method detection gastrocnemius tissue PRR14 mRNA expression, see Figure 16 The control group is not treated directly for the above detection.

[0077] The above examples are to illustrate the disclosed embodiments of the present application, and can not be understood as a limitation of the present application. In addition, the various modifications listed herein and the changes in the method, composition of the invention, are obvious to those skilled in the art without departing from the scope and spirit of the present application. Although the present application has been specifically described in conjunction with various specific preferred embodiments thereof, it should be understood that the present application should not be limited to these specific embodiments. In fact, various modifications as described above to those skilled in the art to obtain the invention are included within the scope of the present application.

Claims

1. A method for constructing an animal model of myopathy, characterized in that, Animal models were obtained by specifically knocking out or mutating the PRR14 gene in myofibers to suppress its expression. The myopathy mentioned includes, but is not limited to, at least one of the following: nutritional myopathy, sarcopenia, disuse atrophy, neurogenic atrophy, congenital myopathy, and inflammatory myopathy; Specifically, the steps include the following: S1: Use parental PRR14 flox / flox Mice were crossed with ACTA1-CreEsr1 mice to obtain the PRR14 genotype. flox / + ACTA1-CreEsr1 mice; S2: Obtain the genotype PRR14 flox / + ACTA1-CreEsr1 mice and genotype PRR14 flox / flox PRR14 was obtained by hybridization of mice and genotyping of the offspring mice. flox / flox ACTA1-CreEsr1 mice; S3: PRR14 flox / flox ACTA1-CreEsr1 mice were given oral tamoxifen on day 7 after birth to establish the PRR14-mKO mouse model; In step S3, tamoxifen is dissolved in a 2% ethanol aqueous solution, with a tamoxifen concentration of 0.8 mg / mL, and fed to mice as their daily drinking water.

2. The method according to claim 1, characterized in that, The gene identification in step S2 involves amplifying the gene fragment using PCR based on the location of the Flox region constructed by the targeting vector, and determining the mouse's genotype by agarose gel electrophoresis based on the gene fragment length and the presence or absence of the product.

3. The method according to claim 2, characterized in that, The PRR14 primer pair sequences in the PCR reaction system are as follows: ForwardP1,5'-GTCTCTACTGCAAGAAAGGCTGAG-3'; ReverseP2, 5′-ATTTCTCCCCTAACCTAAAGGACG-3′; The primer pair sequences for Cre transgenic structures are as follows: ForwardP3, 5'-CGAGCCGAGAGTAGCAGTTGTA-3'; ReverseP4, 5'-AGGTGGACCTGATCATGGAG-3'; The PRR14 knockout primer pair was verified as follows: ForwardP5,5'-GGAGGAACTGGCCCCTCATTC-3'; ReverseP6, 5'-ATTTCTCCCCTACCTAAAGGACG-3'.

4. The application of the myopathy animal model according to any one of claims 1-3 in the study of myopathy.

5. The use of the myopathy animal model according to any one of claims 1-3 in screening drugs for the treatment of myopathy.

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