A method for constructing and applying an animal model of sarcopenia

Abstract

This invention provides a method for constructing and applying an animal model of sarcopenia, belonging to the field of animal model construction technology. This invention uses... adora2b Targeting genes, by knocking out the aforementioned adora2b Genetic constructing of a sarcopenia animal model. This invention uses CRISPANT technology to construct a knockout gene. adora2b Zebrafish model of genes adora2b Mutant zebrafish exhibit muscle atrophy and dysfunction, showing reduced muscle fluorescence area and average intensity, increased cellular senescence, and significantly decreased expression of muscle function-related genes, indicating that the mutant zebrafish have experienced muscle degeneration and dysfunction. The present invention provides… adora2b Mutant zebrafish serve as an animal model of sarcopenia. adora2b Research on the relationship between gene mutations and the pathogenesis of sarcopenia, as well as the screening of drugs for the prevention and treatment of sarcopenia, have laid a theoretical foundation.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a method for constructing and applying an animal model of sarcopenia. Background Technology

[0002] Sarcopenia is an age-related, progressive, systemic skeletal muscle disorder characterized by a gradual decline in skeletal muscle mass, strength, and function. The development of sarcopenia results from the combined effects of genetic factors, age-related physiological changes, and external environmental factors. Studies have found that imbalances in muscle protein metabolism, satellite cell function decline, neuromuscular dysfunction, and systemic metabolic and endocrine disorders can all trigger sarcopenia. Furthermore, sedentary lifestyles, lack of exercise, insufficient nutritional intake, chronic diseases, and medication effects can all potentially accelerate the onset of sarcopenia.

[0003] The genetic regulation of sarcopenia is characterized by multiple genes and pathways, especially genes whose dysfunction directly leads to muscle loss and functional decline. For example, loss-of-function mutations in the MSTN gene result in a significant increase in muscle mass, while polymorphisms that enhance function are associated with an increased risk of muscle loss and strength decline in older adults. Polymorphisms in the promoter region of the IGF-1 gene affect its expression level; low expression genotypes are significantly associated with accelerated muscle loss and increased incidence of sarcopenia in older adults. Longevity-related polymorphisms in the FOXO3 gene can inhibit its transcriptional activity, reduce muscle protein degradation, and are associated with muscle function preservation and reduced risk of sarcopenia in older adults. Polymorphisms in the promoter region of the MuRF1 gene are associated with the rate of muscle strength decline and the severity of sarcopenia in older adults; patients with high expression genotypes experience faster muscle function decline. Meanwhile, chronic low-grade inflammation and oxidative stress are important triggers for sarcopenia, and these genes affect muscle homeostasis by regulating inflammatory responses and redox balance. Therefore, screening for genetic factors or determinants of sarcopenia has a significant promoting effect on the treatment of sarcopenia; however, animal models of sarcopenia with single-gene associations are currently lacking. Summary of the Invention

[0004] This invention provides a method for constructing and applying an animal model of sarcopenia. adora2b Targeting genes, by knocking out the aforementioned adora2b Genes can be used to construct animal models of sarcopenia.

[0005] This invention provides a method for constructing an animal model of sarcopenia, including knocking out a specific gene in the genome of a target animal. adora2b Gene.

[0006] In one specific embodiment of the present invention, the target animal includes zebrafish.

[0007] In one specific embodiment of the present invention, the knockout includes the absence of zebrafish. adora2b The bases in exon 2 of the gene.

[0008] This invention also provides a combination of sgRNAs for constructing a zebrafish model of sarcopenia, including... adora2b -sgRNA1, adora2b -sgRNA4, adora2b -sgRNA6 and adora2b -sgRNA8, wherein adora2b The nucleotide sequence of -sgRNA1 is shown in SEQ ID No. 1. adora2b The nucleotide sequence of -sgRNA4 is shown in SEQ ID No. 4. adora2b The nucleotide sequence of -sgRNA6 is shown in SEQ ID No. 6. adora2b The nucleotide sequence of -sgRNA8 is shown in SEQ ID No. 8.

[0009] The present invention also provides a method for constructing a zebrafish model of sarcopenia, comprising the following steps: injecting the above-mentioned sgRNA combination and Cas9 protein mixture into zebrafish fertilized eggs, culturing them to adult fish, and obtaining F0 generation mutant zebrafish; The F0 generation mutant zebrafish were crossed with wild-type zebrafish to obtain F1 generation embryos, and the F1 generation embryos were screened for the presence of [specific mutations]. adora2b The genetically mutated embryos were cultured into adult fish to obtain the sarcopenia zebrafish model.

[0010] In one specific embodiment of the present invention, the final concentration of the sgRNA combination in the mixture is 1500 ng / μL, and the final concentration of the Cas9 protein is 400 ng / μL.

[0011] In one specific embodiment of the present invention, the concentration ratio of each sgRNA in the sgRNA combination is 1:1:1:1.

[0012] In one specific embodiment of the present invention, the screening method includes extracting genomic DNA from fertilized eggs that have developed to 24 hpf; using the genomic DNA as a template, performing PCR amplification using upstream and downstream amplification primers to obtain PCR amplification products; and mixing the PCR amplification products with wild-type... adora2b Gene comparison; if the amplification results are different, then the fertilized egg is a valid knockout. adora2b The embryo with genes; The nucleotide sequences of the upstream amplification primers are shown in SEQ ID No. 9 and SEQ ID No. 11; the nucleotide sequences of the downstream amplification primers are shown in SEQ ID No. 10 and SEQ ID No. 12.

[0013] In one specific embodiment of the present invention, the PCR amplification program includes: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, 34 cycles; 72°C further extension for 5 min.

[0014] The present invention also provides the application of the sarcopenia zebrafish model constructed using the above method in screening drugs for the prevention and / or treatment of sarcopenia.

[0015] Beneficial effects: This invention uses adora2b Targeting genes, by knocking out the aforementioned adora2b Genetic constructing of a sarcopenia animal model. This invention uses CRISPANT technology to construct a knockout gene. adora2b Zebrafish model of genes adora2b Mutant zebrafish exhibited muscle atrophy and dysfunction, and, through the analysis of… adora2b The detection of muscle fluorescence intensity and cellular senescence in mutant zebrafish, along with the analysis of muscle-related gene expression levels, revealed that the mutant zebrafish exhibited decreased muscle fluorescence area and average intensity, increased cellular senescence, and... myod1 , Myf7 and Asog The significant decrease in the expression of muscle function-related genes indicates that the mutant zebrafish has experienced muscle function degeneration and dysfunction. The present invention provides… adora2b Mutant zebrafish serve as an animal model of sarcopenia. adora2b Research on the relationship between gene mutations and the pathogenesis of sarcopenia, as well as the screening of drugs for the prevention and treatment of sarcopenia, have laid a theoretical foundation. Attached Figure Description

[0016] Picture 1 This is an electrophoresis result of the sgRNA synthesized in vitro in step two of Example 1. The first lane from left to right is the DNA marker: from bottom to top, the markers are 100 bp, 250 bp, 500 bp, 750 bp, 1000 bp, 1500 bp, 2000 bp, 3000 bp, and 5000 bp. The second lane is... adora2b -sgRNA group (1, 3, 6, 7), lane 3 is adora2b -sgRNA (groups 2, 4, 5, 8); Picture 2 Figure A shows the sequencing results of the sgRNA synthesized in vitro in step three of Example 1; adora2b -sgRNA-1-3-6-7 group utilized adora2b -E2-F1 sequencing, the red box indicates the Indel mutation near CRISPR1; B: a dora2b The -sgRNA-1-3-6-7 group was sequenced using adora2b-E2-F2 sequencing; the red box indicates the Indel mutation near CRISPR6; C: adora2b The -sgRNA-2-4-5-8 group was sequenced using adora2b-E2-F1; the red box indicates the Indel mutation near CRISPR4; D: adora2b -sgRNA-2-4-5-8 group utilized adora2b -E2-F2 sequencing, the red box indicates the Indel mutation near CRISPR8; Picture 3 For example, the F1 generation sibling fish and two adora2b Sequence differences in mutant lines, indicated by sequences underlined in red. adora2b The CRISPR / Cas9 target in the image shows the bolded portion as the original spacer adjacent motif (PAM) sequence. Picture 4 The results are the morphological detection results of step 2 in Example 3; where A is the morphological detection results of the F1 generation sibling fish and the adora2b mutant line at 6 months of age; B is the morphological detection results of the F1 generation sibling fish and the homozygotes and heterozygotes of the adora2b mutant line at 72 hpf age. Picture 5 This is a diagram showing the staining results of β-galactosidase (β-gal) in Example 3, which also verifies the aging-related marker in the qPCR experiment. tert , p21 The level of expression; Picture 6 This is a result image of the muscle phenotype in step 5 of Example 3, and also verifies the muscle-related marker in the qPCR experiment. Myh7 , myod1 , foxo3a , Asog The level of expression of ). Detailed Implementation

[0017] This invention provides a method for constructing an animal model of sarcopenia, including knocking out a specific gene in the genome of a target animal. adora2b Gene.

[0018] In this invention, ADORA2B is an adenosine A2B receptor, a member of the adenosine receptor family, and a typical seven-transmembrane G protein-coupled receptor. The target animal in this invention can be zebrafish. In one embodiment, zebrafish, as a small vertebrate model organism, possesses a highly conserved genome structure and signaling pathways with humans, exhibits transparent embryonic development, and reproduces rapidly, making it suitable for large-scale genetic manipulation and drug screening. In particular, zebrafish contain… adora2b The gene can be expressed in embryos and several tissues, so zebrafish were used as the target animal to construct a sarcopenia model.

[0019] In the zebrafish genome, the adora2b The genomic DNA sequence of the gene is registered in NCBI under accession number 560100. As demonstrated in the examples, the constructed... adora2b Mutant zebrafish can be stably inherited, phenotypic observation is not time-limited, and gene screening can be performed rapidly in large quantities. This invention does not specifically limit the gene knockout method; conventional gene editing methods in the art can be used, such as the CRISPANT technology used in the examples.

[0020] The target animal of this invention includes zebrafish, and the knockout includes zebrafish lacking zebrafish. adora2b The bases in exon 2 of the gene.

[0021] This invention also provides a combination of sgRNAs for constructing a zebrafish model of sarcopenia, including... adora2b -sgRNA1, adora2b -sgRNA4, adora2b -sgRNA6 and adora2b -sgRNA8, wherein adora2b The nucleotide sequence of -sgRNA1 is shown in SEQ ID No. 1. adora2b The nucleotide sequence of -sgRNA4 is shown in SEQ ID No. 4. adora2b The nucleotide sequence of -sgRNA6 is shown in SEQ ID No. 6. adora2b The nucleotide sequence of -sgRNA8 is shown in SEQ ID No. 8.

[0022] In one embodiment of the present invention, zebrafish is used. adora2b Using exon 2 of the gene as the target site, eight sgRNAs were designed, and their nucleotide sequences are shown in Table 1. Furthermore, validation revealed that... adora2b -sgRNA1, adora2b -sgRNA4, adora2b -sgRNA6 and adora2b-sgRNA8 has the activity of guiding Cas9 to target and cleave the target genomic DNA sequence. Therefore, four active sgRNAs were used to construct an sgRNA combination for the subsequent construction of a zebrafish model of sarcopenia.

[0023] Table 1. sgRNAs and primers involved in this invention

[0024] In this invention, the method for screening for active sgRNAs includes the following steps: (1) Target design and sgRNA template construction Based on the conserved functional domain sequences of the target gene, multiple candidate targets were selected and corresponding sgRNA sequences were designed. Each candidate sgRNA sequence was fused with the T7 promoter sequence and the sgRNA backbone sequence to construct an in vitro transcription template, and the corresponding DNA template was obtained by PCR amplification.

[0025] (2) In vitro transcription to synthesize sgRNA The DNA template obtained in step (1) was transcribed in vitro using T7 RNA polymerase to synthesize the corresponding sgRNA; the transcription product was treated with DNase I to remove the DNA template, and then the sgRNA was purified by alcohol precipitation and resuspended in nuclease-free water.

[0026] (3) sgRNA quality detection The sgRNA sample obtained in step (2) was taken and its integrity was checked by agarose gel electrophoresis to confirm that the bands were clear and undegraded; at the same time, its concentration and purity were determined by NanoDrop to ensure OD 260 / 280 The ratio is between 1.8 and 2.0.

[0027] (4) Injection of Cas9 protein and sgRNA complex The sgRNA verified in step (3) was mixed with Cas9 protein to form an RNP complex, which was then microinjected into zebrafish fertilized eggs at a dose of approximately 1 nL per embryo.

[0028] (5) Mutation efficiency detection Embryos that developed to 24 hpf after injection were collected, genomic DNA was extracted, the target region was amplified by PCR, and the amplification products were directly sequenced. By analyzing the sequencing peaks, the presence of Indel mutations was determined to confirm whether sgRNA successfully guided Cas9 to achieve targeted cleavage.

[0029] (6) Screening of active sgRNA Based on the mutation efficiency of each candidate sgRNA in step (5), sgRNAs that can stably induce mutations in the target gene are screened out as preferred tools for subsequent construction of mutant models. The mutation efficiency described in this invention is judged by the proportion of overlapping peaks (Indel signals) in the sequencing peak diagram. If the proportion is ≥50%, the sgRNA is determined to be active.

[0030] The present invention also provides a method for constructing a zebrafish model of sarcopenia, comprising the following steps: injecting the above-mentioned sgRNA combination and Cas9 protein mixture into zebrafish fertilized eggs, culturing them to adult fish, and obtaining F0 generation mutant zebrafish; The F0 generation mutant zebrafish were crossed with wild-type zebrafish to obtain F1 generation embryos, and the F1 generation embryos were screened for the presence of [specific mutations]. adora2b The genetically mutated embryos were cultured into adult fish to obtain the sarcopenia zebrafish model.

[0031] In the mixture of sgRNA and Cas9 protein described in this invention, the concentration of the sgRNA combination is 1500 ng / μL, and the concentration ratio of each sgRNA in the sgRNA combination is 1:1:1:1; the concentration of the Cas9 protein is 400 ng / μL.

[0032] The method for screening effective knockout embryos according to the present invention includes the following steps: taking fertilized eggs that have developed to 24 hpf and preparing genomic DNA; using the genomic DNA as a template, performing PCR amplification using upstream and downstream amplification primers to obtain PCR amplification products; and mixing the PCR amplification products with wild-type embryos. adora2b Gene comparison: if the amplification results are different, then the fertilized egg that develops to 24 hpf is an effective knockout embryo; The upstream amplification primers comprise nucleotide sequences as shown in SEQ ID No. 9 and SEQ ID No. 11; the downstream amplification primers comprise nucleotide sequences as shown in SEQ ID No. 10 and SEQ ID No. 12. This invention uses a zebrafish genotyping kit to prepare genomic DNA. As in the examples, the zebrafish genotyping kit from Nanjing Yaoshunyu Biotechnology Co., Ltd. was selected for genomic DNA extraction. Specific methods can be found in the kit's instruction manual.

[0033] The PCR amplification system described in this invention, in 40 μL volume, comprises: 20 μL of 2×Mastermix (Vazyme), 14 μL of ultrapure water, and 2 μL of forward and reverse mixing solutions. adora2b -F1 and adora2b -R1) primers (10 μM), 2 μL of forward and reverse primers ( adora2b -F2 and adora2b-R2) primer (10 μM) and 2 μL of genomic DNA; the PCR amplification program includes: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 34 cycles; 72℃ further extension for 5 min.

[0034] Using the sgRNA combination described in this invention, it is possible to obtain zebrafish adora2b Starting at 255bp in exon 2 of the gene, a 4bp segment is knocked out, denoted as -4bp. This knocked-out segment is ATCT. This invention uses CRISPANT technology to construct a knockout... adora2b Zebrafish model of genes adora2b Mutant zebrafish exhibited muscle atrophy and dysfunction, and, through the analysis of… adora2b The detection of muscle fluorescence intensity and cellular senescence in mutant zebrafish, along with the analysis of muscle-related gene expression levels, revealed that the mutant zebrafish exhibited decreased muscle fluorescence area and average intensity, increased cellular senescence, and... myod1 , Myf7 and Asog The significant decrease in the expression of muscle function-related genes indicates that the mutant zebrafish has experienced muscle function degeneration and dysfunction. The present invention provides… adora2b Mutant zebrafish serve as an animal model of sarcopenia.

[0035] The present invention also provides the application of the sarcopenia zebrafish model constructed using the above method in screening drugs for the prevention and / or treatment of sarcopenia.

[0036] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a method for constructing and applying an animal model of sarcopenia provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0037] Unless otherwise specified, all raw materials used in the embodiments of this invention are commercially available.

[0038] Example 1 Step 1: Design of target sites for CRISPANT knockdown and PCR detection primers Search for zebrafish in the NCBI database adora2b The genomic DNA sequence of the gene (accession number 560100) was obtained, and its functional domains were analyzed to design a target for zebrafish. adora2b The target site on exon 2 of the gene was identified, and PCR amplification primers and sequencing primers were designed as shown in Table 1.

[0039] Step 2: In vitro synthesis and quality control of sgRNA (1) Primers for in vitro synthesis of sgRNA expression building blocks Based on the sgRNA sequences designed in Table 1, forward primers for synthesizing sgRNA were designed, and the specific sequences are shown in Table 2.

[0040] Table 2. Forward primers for sgRNA synthesis

[0041] Its reverse primer is the universal primer R-Common, with the sequence shown in SEQ ID No. 13: AAAAAAAGCACCGACTCGGTGCCAC.

[0042] (2) PCR amplification to synthesize sgRNA PCR reaction system (80μL): 40μL 2×Mastermix (Novizan), 37.5μL ultrapure water, 1μL forward and reverse mixing reagents. adora2b -sgRNA (1~4)-F and R-Common primers (5 μM) and 0.5 μL of pYSY-sgRNA plasmid (10 ng / μL) were respectively used.

[0043] PCR reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 7 s, 35 cycles; 72℃ extension for 5 min, and storage at 4℃.

[0044] PCR product purification (nuclease-free treatment) The PCR products were collected using a nuclease-free PCR clean-up (Axygen) kit. The recovery solvent was nuclease-free ultrapure water (Nanjing Yaoshunyu), and the recovery volume was 25 μL.

[0045] (4) In vitro transcription of sgRNA The purified template obtained in step (3) was transcribed in vitro using T7 RNA polymerase. Using an RNA in vitro transcription kit (MAXIscript T7, Ambion, USA), the following were added sequentially according to the kit instructions: 4 μL of 10×Transcription Buffer, 2 μL of 10 mM ATP, 2 μL of 10 mM CTP, 2 μL of 10 mM GTP, 2 μL of 10 mM UTP, 4 μL of T7 RNA polymerase mix, and 24 μL of template DNA. After gently mixing and centrifuging, the mixture was incubated at 37°C for 2 h.

[0046] Add 1.5 μL of DNase I (Ambion, USA) and incubate in a 37°C water bath for 15 min to remove the template.

[0047] Then, 160 μL of DEPC water was added to expand the volume to 200 μL. Simultaneously, 20 μL of nuclease-free 3M sodium acetate (pH 5.2) and 3 volumes of anhydrous ethanol (domestic analytical grade) were added, and the precipitate was incubated overnight at -80°C. After centrifugation at 12,000 g for 20 min at 4°C, the supernatant was removed. Then, 75% nuclease-free ethanol was added, and the mixture was centrifuged again at 12,000 g for 20 min at 4°C. After removing the supernatant, the precipitate was air-dried in a fume hood, resuspended in 20 μL of nuclease-free ultrapure water, and stored at -80°C for later use.

[0048] (5) sgRNA quality assessment sgRNA according to adora2b -sgRNA grouping (1, 3, 6, 7) and adora2b After equal amounts of -sgRNA groups (2, 4, 5, 8) were mixed, 0.5 μL of each group was taken for agarose gel electrophoresis (1%) to identify its integrity.

[0049] The results are as follows Picture 1 As shown in Table 1, the PCR product confirming the band was sent to a sequencing company (Beijing Ruiboxingke Biotechnology Co., Ltd.) for sequencing. The sequencing primers are listed in Table 1. adora2b -E2-F1 and adora2b -E2-F2, sequencing results are as follows Picture 2 As shown, based on the above experimental results, the aforementioned... adora2b -sgRNA1, adora2b -sgRNA4, adora2b -sgRNA6 and adora2b -sgRNA8 has the activity of guiding Cas9 to target and cleave the target genomic DNA sequence.

[0050] Example 2 Step 1: Microinjection of zebrafish fertilized eggs Zebrafish fertilized eggs were collected using conventional methods, and the sgRNA obtained in Example 1 was processed according to... adora2b -sgRNA grouping (1, 3, 6, 7) and adora2b The sgRNA group (2, 4, 5, 8) is used, and the preferred concentration ratio of each sgRNA in the mixture of sgRNA and Cas9 protein is 1:1:1:1. The sgRNA group (final concentration approximately 1500 ng / μL) and Cas protein (final concentration 400 ng / μL) are mixed and microinjected into zebrafish fertilized eggs at a volume of 1 nL per embryo.

[0051] Step 2: Confirmation of the efficiency of sgRNA-guided Cas9 targeting and cleavage of the target genomic DNA sequence. (1) Preparation of target genomic DNA template When the injected embryos developed to 24 hpf, five embryos from each of the two groups were divided into three tubes to prepare genomic DNA templates (a total of 15 embryo samples). The genomic DNA templates were prepared using the "Zebrafish Genotyping Kit" (purchased from Nanjing Yaoshunyu Biotechnology Co., Ltd.). The specific reaction conditions were: 65℃ for 30 min, 95℃ for 5 min, 16℃ for 1 min, and 4℃.

[0052] (2) PCR detection Using the primer pairs in Table 1, and with the genomic DNA obtained in step (1) as a template, PCR amplification was performed. The specific reaction system and reaction procedure are as follows: PCR reaction system (40 μL): 20 μL 2×Mastermix (Vazyme), 14 μL ultrapure water, 2 μL forward and reverse mixing solutions. adora2b -E2-F1 and adora2b -E2-R1) primers (10 μM), 2 μL of forward and reverse primers ( adora2b -E2-F2 and adora2b -E2-R2) primers (10 μM) and 2 μL of genomic DNA template prepared by (1).

[0053] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 34 cycles; 72℃ final extension for 5 min, 4℃ for 5 min.

[0054] (3) Detection and sequencing of amplification products The PCR products with confirmed band sizes were sent to a sequencing company for sequencing, and the sequencing primers were used as the basis for sequencing. adora2b -F1, adora2b The sequencing results of -F2 confirmed the above. adora2b -sgRNA1, adora2b -sgRNA4, adora2b -sgRNA6 and adora2b -sgRNA8 has the activity of guiding Cas9 to target and cleave the target genomic DNA sequence.

[0055] Step 3: Breeding and rearing of candidate F1 mutant zebrafish The heritable G0 embryos were used for genetic modification to obtain F1 embryos. These F1 embryos were then raised to sexual maturity using standard methods, and genotyping was performed to screen for gene-edited mutants. The specific steps are as follows: (1) Preparation of genomic DNA templates for candidate F1 mutants a. Organizing samples Take 24 F1 adult fish, cut off part of the tail fin tissue, and put them into 200μL PCR tubes in sequence, and then store them on ice.

[0056] b. Genomic template DNA preparation Add 10 μL of Nanjing Yaoshunyu's YSY buffer to a 200 μL PCR tube containing caudal fin tissue. After rapid centrifugation, place the PCR tube in a PCR instrument and perform the following reaction: 65℃ for 30 min, 95℃ for 5 min, 16℃ for 1 min, and 4℃.

[0057] (2) PCR amplification of genomic DNA fragments Using the primers in Table 1 ( adora2b Using the genomic DNA obtained in step b as a template, PCR amplification was performed. The specific reaction system and procedure are as follows: PCR reaction system (30 μL): 20 μL 2×Mastermix, 14 μL ultrapure water, 2 μL adora2b -E2-F1 (5μM), 2μL adora2b -E2-R1 (5 μM) and 2 μL of genomic DNA template obtained from step (1).

[0058] The PCR reaction conditions were: 95℃ for 3 min; 95℃ for 15 s, 56℃ for 15 s, 72℃ for 7 s, 35 cycles; 72℃ for 5 min, 4℃.

[0059] (3) Carry adora2b Screening and identification of F1 mutants of mutated alleles The PCR products obtained above were sent to a commercial company for sequencing. Sequencing primers were used. adora2b -E2-F1 / adora2b -E2-R1, the sequence obtained directly from the image is shown in Table 3: the specific sequence sequencing results of the mutation are shown in Table 4.

[0060] Table 3 Mutation types of mutants

[0061] Table 4. Partial genome sequences and predicted protein sequences

[0062] As shown in Table 4, compared to the wild type, the mutant F1 generation zebrafish exhibited superior characteristics. adora2b A frameshift mutation of -4bp occurs at 255bp in exon 2 of the gene, resulting in a mutant zebrafish denoted as adora2b Mutant. Wild-type sibling fish and adora2b The partial structure and sequence of the (-4bp) mutant show the target site of the sgRNA, such as... Picture 3 As shown.

[0063] Example 3 1. Materials and Methods The wild-type zebrafish strain used was the Tuebingen strain (abbreviated as TU, originating from Streisinger Lab), and the mutant was the adora2b (-4bp) mutant obtained in Example 2; The above-mentioned zebrafish were raised in accordance with the standard protocol guidelines of the Organization for Economic Cooperation and Development (OECD): I. Daily Feeding of Zebrafish: Zebrafish are raised in an automated circulating water system (using 15g of high-grade sea salt crystals added to 80L of pure water, stirred well; seawater salinity 28%, conductivity 450-550μS), with the water temperature controlled at (28.5±1)℃. They are placed in an indoor environment with controllable photoperiod, and their life cycle is regulated using artificial light, alternating between 14 hours of illumination and 10 hours of darkness. The feed consists of 24-hour-hatched brine shrimp, fed once daily at noon. The high-grade sea salt crystals were purchased from Tianjin Zhongyan Marine Biological Science Co., Ltd.

[0064] II. Zebrafish Mating and Egg Collection: Adult male and female zebrafish were kept separately. The night before the experiment, males and females were transferred to a mating box at a 2:2 ratio and separated by a partition. The partition was removed the following morning, and the males chased the females before collecting the embryos at 12:00 PM. After cleaning the embryos with zebrafish culture solution, the eggs were aspirated using a dropper and transferred to a petri dish. The embryos were cultured at a constant temperature of 28.5℃, and the developmental stage was observed under a microscope. The appropriate developmental stage was selected according to the experimental requirements. The zebrafish were then raised and bred.

[0065] 2. Morphological assessment The morphology of zebrafish juveniles from step 1 was observed under a stereomicroscope. Daily monitoring was conducted, and to quantify the growth and development of the juveniles, the body length was defined as the distance from the center of one zebrafish eye to the tip of the tail bud. After obtaining morphological images, the body length was measured and quantitatively analyzed using ImageJ software. The results are as follows: Picture 4 As shown, observation was performed using a stereomicroscope. adora2b Mutant zebrafish juveniles were found to have significantly slower development, shorter body length, and delayed yolk sac absorption compared to wild-type juveniles.

[0066] 3. β-galactosidase (β-gal) staining Cell senescence was detected using the β-galactosidase staining kit from Beyotime Biotechnology Co., Ltd. Embryos were collected after 72 hours of treatment, washed three times with PBS, and fixed with β-galactosidase staining fixative for 30 minutes. The staining intensity was finally quantified using ImageJ. The staining results are shown below. Picture 5 As shown, the increased staining area suggests that the mutant zebrafish exhibits increased cellular senescence.

[0067] 4. Muscle phenotype Will adora2b The 120-hour embryos of the mutant zebrafish hybrid muscle fluorescence transgenic fish CZ12 Tg(-1.9mylz2:EGFP, purchased from the National Zebrafish Resource Center, originating from Yonghua Sun Lab) and the 120-hour embryos of its self-crossed offspring were compared and analyzed. Images were acquired and observed using a fluorescence microscope. ImageJ software was used to quantitatively detect fluorescence intensity and area parameters. Simultaneously, qPCR technology was used to experimentally analyze the expression levels of muscle-related markers. The results showed a decrease in muscle fluorescence area and a decrease in average muscle fluorescence intensity (…). Picture 6 Furthermore, qPCR analysis using the primers shown in Table 5 revealed that muscle-related genes such as... Myh7 , myod1 , foxo3a and Asog The expression of [a specific substance] was significantly reduced in the mutant, further confirming the altered muscle phenotype.

[0068] Table 5 qPCR primer sequences

[0069] As can be seen from the above, the present invention provides... adora2b Compared to wild-type zebrafish, mutant zebrafish exhibited reduced muscle fluorescence area and average intensity, increased cellular senescence, and significantly decreased expression of muscle function-related genes. This invention provides... adora2b Mutant zebrafish may serve as an animal model of sarcopenia. adora2b This research has laid a solid foundation for studying the relationship between gene mutations and the pathogenesis of sarcopenia, as well as for screening drugs for the prevention and treatment of sarcopenia.

[0070] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing an animal model of sarcopenia, characterized in that, Including knocking out the target animal's genome adora2b Gene.

2. The construction method according to claim 1, characterized in that, The target animals include zebrafish.

3. The construction method according to claim 2, characterized in that, The knockout includes the absence of zebrafish. adora2b The bases in exon 2 of the gene.

4. A combination of sgRNAs for constructing a zebrafish model of sarcopenia, characterized in that, include adora2b -sgRNA1, adora2b -sgRNA4, adora2b -sgRNA6 and adora2b -sgRNA8, wherein adora2b The nucleotide sequence of -sgRNA1 is shown in SEQ ID No.

1. adora2b The nucleotide sequence of -sgRNA4 is shown in SEQ ID No.

4. adora2b The nucleotide sequence of -sgRNA6 is shown in SEQ ID No.

6. adora2b The nucleotide sequence of -sgRNA8 is shown in SEQ ID No.

8.

5. A method for constructing a zebrafish model of sarcopenia, characterized in that, The process includes the following steps: injecting the mixture of the sgRNA combination and Cas9 protein as described in claim 4 into zebrafish fertilized eggs, culturing them to adulthood, and obtaining F0 generation mutant zebrafish; The F0 generation mutant zebrafish were crossed with wild-type zebrafish to obtain F1 generation embryos, and the F1 generation embryos were screened for the presence of [specific mutations]. adora2b The genetically mutated embryos were cultured into adult fish to obtain the sarcopenia zebrafish model.

6. The method according to claim 5, characterized in that, The final concentration of the sgRNA combination in the mixture is 1500 ng / μL, and the final concentration of the Cas9 protein is 400 ng / μL.

7. The method according to claim 5, characterized in that, The concentration ratio of each sgRNA in the sgRNA combination is 1:1:1:

1.

8. The method according to claim 5, characterized in that, The screening method includes extracting genomic DNA from fertilized eggs that have developed to 24 hpf; using the genomic DNA as a template, performing PCR amplification using upstream and downstream amplification primers to obtain PCR amplification products; and comparing the PCR amplification products with wild-type... adora2b Gene comparison; if the amplification results are different, then the fertilized egg is a valid knockout. adora2b The embryo with genes; The nucleotide sequences of the upstream amplification primers are shown in SEQ ID No. 9 and SEQ ID No. 11; the nucleotide sequences of the downstream amplification primers are shown in SEQ ID No. 10 and SEQ ID No.

12.

9. The method according to claim 8, characterized in that, The PCR amplification program includes: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 34 cycles; and 72℃ extension for 5 min.

10. The use of the sarcopenic zebrafish model constructed using the method described in any one of claims 5 to 9 in screening drugs for the prevention and / or treatment of sarcopenic diseases.

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