A method for constructing a model of retinal degenerative disease and application thereof

Abstract

This invention discloses a method for constructing a model of retinal degenerative diseases and its application, relating to the field of medical engineering technology. The method for constructing a model of retinal degenerative diseases includes: [the following steps are described in the original text, but the translation is incomplete and requires further context.] Fto Gene knockout, silencing, or decreased expression levels. This model involves conditionally knocking out gene expression in rod cells of a model animal. Fto The gene successfully induced typical pathological features such as thinning of the outer nuclear layer of the retina, shortening of the outer segments of photoreceptors, and glial activation. This invention provides a new tool model for studying the pathogenesis of retinal degenerative diseases and screening therapeutic drugs.

Description

Technical Field

[0001] This invention relates to the field of medical engineering technology, and more specifically, to a method and application for constructing a model of retinal degenerative diseases. Background Technology

[0002] Retinal degeneration (RD) is a group of inherited blinding eye diseases characterized by progressive damage to cone and rod cells. Its inheritance patterns are diverse, including autosomal dominant, recessive, and X-linked recessive inheritance. Initially, patients present with night blindness and narrowed visual field. As the disease progresses, progressive visual loss, abnormal electroretinograms, and characteristic retinitis pigmentosa may occur, often ultimately leading to blindness. Currently, there is a lack of ideal animal models for research on this disease.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a method and application for constructing a model of retinal degenerative diseases, thereby enriching the disease models of retinal degeneration (RD).

[0005] This invention is implemented as follows: In a first aspect, the present invention provides a method for constructing a model of retinal degenerative diseases, enabling the use of rod cells from non-human mammals... Fto Gene knockout, silencing, or decreased expression levels.

[0006] Secondly, the present invention also provides the application of the retinal degenerative disease model constructed by the above method in screening drugs for the prevention or treatment of retinal degenerative diseases.

[0007] Thirdly, the present invention also provides a method for breeding animal models of retinal degenerative diseases, wherein the retinal degenerative disease models constructed by the above method are crossbred.

[0008] The present invention has the following beneficial effects: This invention achieves its goal by specifically knocking out rod cells in non-human mammals. Fto This invention successfully constructed an animal model that stably mimics the main pathological phenotypes of human retinal degeneration (RD). By specifically knocking out or silencing this gene, or by reducing the protein expression level of the gene, damage to the animal's retina can be induced, manifested as abnormalities in the outer nuclear layer structure and photoreceptor outer segments, thus successfully constructing an animal model that mimics the human RD phenotype. This invention provides a stronger experimental model foundation for further exploration of the pathogenesis of this disease, the development of early molecular screening methods, and the screening of specific targeted therapeutic drugs.

[0009] Furthermore, the animal models obtained by the above methods have demonstrated significant application value in basic research on RD, early biomarker discovery, and the development and evaluation of therapeutic drugs targeting human retinal degenerative diseases, and have broad application prospects. Attached Figure Description

[0010] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0011] Picture 1 Knockout of retinal rod cells Fto Mouse construction strategy and genotyping of genes; A, in Fto LoxP sites were inserted flanking exon 3 of the gene, and exon 3 was removed by Cre enzyme. B. Using Rod-Cre tools, mice and... Fto floxed / floxed Mice were mated, and eventually... Fto floxed / floxed Rod-Cre (RKO) mice. C, results of F1 generation mouse identification by PCR; Cre - No stripes, Cre + : 600bp; in the figure, loxP / loxP indicates homozygote; + / loxP indicates heterozygote; + / + indicates wild type; Rod-Cre refers to Cre enzyme that specifically recognizes the loxP site in rod cells; Picture 2 : Fto Verification of gene knockout efficiency in the retina; A, Western blot analysis of the retina of RKO knockout mice. Fto Gene knockout efficiency, with GAPDH as an internal control; B, Statistical analysis results of grayscale values ​​in the Western blot experiment; Picture 3 Demonstrates specific knockout of retinal rod cells. Fto H&E staining results of paraffin sections of the retina of gene-infected mice; Figures A and C show the stained sections of knockout mice at different ages (5, 8, and 10 months); Figure DF shows the statistical analysis of the outer nuclear layer thickness of the retina of knockout mice and control mice at the corresponding age. Fto-RKO refer to Fto Gene knockout homozygous mice, Fto-Ctrl For those that do not carry Cre recombinase Fto-floxedControl mice; ONL represents the outer nuclear layer, and Distance ON indicates the distance from the optic nerve; Picture 4 : Specific knockout of retinal rod cells Fto Immunofluorescence staining results of gene-modified mice; Figures AF show the staining of the retinas of mice at 5 (A, B), 8 (C, D), and 10 (E, F) months of age, respectively. The markers used included nuclear dye DAPI, rhodopsin antibody, phosphodiesterase 6B (PDE6B) antibody, and sodium-potassium ATPase (NaK) antibody; the scale bar in the figure is 30 micrometers. Picture 5 : Western blot results of RKO mouse retina; A, Immunoblot results of different photosensitizing proteins in the retina, with GAPDH as an internal control; B, Statistical analysis of the grayscale values ​​of protein expression levels of different photosensitizing proteins. Picture 6 : Specific knockout of retinal rod cells Fto Immunohistochemical staining results of gene-modified mice (8 months old); scale bar in the figure is 30 micrometers. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0013] Definitions: Gene knockout refers to permanently destroying the coding sequence of a target gene, causing it to completely lose its function.

[0014] Gene silencing refers to the suppression of gene transcription or translation without altering the DNA sequence, and is often a reversible regulation; it includes methods such as RNA interference (RNAi), shRNA (short hairpin RNA), and antisense oligonucleotides (ASOs).

[0015] FTO protein is an important RNA m 6 A demethylase is involved in regulating gene expression and various physiological and pathological processes. As an m 6As a key member of the "erasers," FTO plays a central role in maintaining cellular metabolic homeostasis and responding to internal and external environmental signals by dynamically removing methylation modifications on RNA. Currently, research on FTO function has extended to multiple fields; it not only plays a role in metabolic diseases such as obesity and diabetes but is also considered an important regulatory factor in neurodegenerative diseases such as Alzheimer's and Parkinson's. Recent research progress further demonstrates that small molecule inhibitors targeting FTO (such as FB23 and Dac51) show translational potential in areas such as cancer treatment, proving that intervening in FTO activity is a feasible therapeutic strategy.

[0016] However, the expression patterns, physiological functions, and mechanisms of action of FTO in retinal tissues remain unclear, and relevant research models are also scarce. This gap limits in-depth exploration of its potential value in the visual system and related diseases.

[0017] In a first aspect, the present invention provides a method for constructing a model of retinal degenerative diseases, enabling the use of rod cells from non-human mammals... Fto Gene knockout, silencing, or decreased expression levels.

[0018] This invention reveals for the first time the knockout of rod cells in target animals Fto Genes can induce the RD phenotype. Based on this, the present invention provides a specific method for constructing a model, namely, using gene editing to specifically reduce or completely eliminate retinal rod cells in a model animal. Fto Gene expression.

[0019] mice Fto The gene (MGI: 1347093) is located on mouse chromosome 8, 92,040,052-92,395,161 bp, with a full length of 355.109 kb. Its cDNA is 3558 bp in length and contains 9 exons.

[0020] In a preferred embodiment of the present invention, Fto Gene knockout or silencing Fto This can be achieved by using partial exon sequences or complete coding regions of a gene.

[0021] For example, it can target any one or more of its nine exons. Regardless of the type of sequence knocked out (partial or full-length), as long as it can be achieved in rod cells. Fto The knockout of a gene, which causes an animal to exhibit characteristics of RD disease, falls within the scope of protection of this invention.

[0022] In a preferred embodiment of the present invention, some exon sequences are selected from... FtoThe third exon of the gene. This strategy has been shown to have high editing efficiency.

[0023] In a preferred embodiment of the present invention, gene knockout or decreased expression level is achieved through at least one of the following methods: CRISPR / Cas9 system, Cre / loxP recombinase system, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology.

[0024] In a preferred embodiment of the present invention, gene silencing or decreased expression levels are achieved through at least one of the following methods: siRNA, shRNA, and antisense oligonucleotides.

[0025] In a preferred embodiment, a combination of CRISPR / Cas9 and homologous recombination is employed.

[0026] In a preferred embodiment of the present invention, the method includes the following steps: targeting... Fto The gRNA expression vector of the gene, the Cas9 protein, and a template sequence containing homologous arms and conditional elements were jointly introduced into the fertilized eggs of non-human mammals, and obtained via embryo transfer. Fto The first genetically modified animal; The first animals were bred and selected. Fto Homozygous individuals with conditional elements integrated into their genes were then crossbred with animal strains that specifically express Cre recombinase in rod cells to obtain individuals with rod cell-specific knockout. Fto Animal models of genes; Non-human mammals are selected from mammals including mice, rats, rabbits, dogs, pigs, monkeys, and apes. Any mammal possessing... Fto Mammals that exhibit retinal degeneration phenotypes by inactivating or reducing the expression of the gene in their retinal rod cells are all suitable for constructing the disease model described in this invention and can be used in related scientific research fields, and are all within the protection scope of this invention.

[0027] In addition, gene silencing technologies (such as RNAi or CRISPR / dCas9 transcriptional repression systems) can also be used to achieve this. Fto Downregulation of gene expression. For example, using dCas9 proteins linked to transcriptional repressor domains (such as KRAB) to bind to specific gRNAs, thereby repressing expression at the transcriptional level. Fto Gene expression.

[0028] A preferred option is to include a target Fto The gene's gRNA expression vector, Cas9 protein, and a homologous recombination template are jointly introduced into the fertilized egg of a model animal (such as a mouse). The homologous template contains... Fto The gene sequence was determined, and loxP sites were designed flanking exon 3. Implantation methods include electroporation. Subsequently, the treated embryos were transferred into the uterus of a pseudopregnant woman to obtain... Fto The first animal to have its genes conditionally knocked out.

[0029] The Cas9 protein used can be wild-type or a modified version, such as a variant with a transcriptional repressor domain (e.g., the EVE domain) fused to its C-terminus to precisely regulate gene activity.

[0030] Taking mice as an example, the specific construction process includes: Will get Fto Mice with loxP sites flanking the gene ( Fto flox / flox When 1,000 mice are mated with wild-type mice, heterozygotes are produced. Fto flox / + Descendants; Heterozygous mice were crossed to obtain homozygous conditional knockout mice. Fto flox / flox ); The homozygous mouse was mated with a mouse that expresses Cre recombinase in rod cells (such as the Rod-Cre mouse). The resulting offspring ( Fto flox / + Rod-Cre) again with Fto flox / flox Homozygous mating can produce retinal rod cell-specific knockout. Fto Mouse models of genes ( Fto flox / flox (; Rod-Cre), this model exhibits characteristics of retinal degeneration.

[0031] In one implementation, in addition to gene knockout, gene silencing technology can also be used. Fto Expression downregulation, such as through RNA interference (RNAi) or CRISPR / dCas9-based transcriptional repression systems. When using the latter approach, the target... Fto The gRNA component is delivered to the fertilized egg along with the dCas9 protein (or its mRNA). The C-terminus of the dCas9 protein or the 3' end of the corresponding mRNA must be linked to a transcriptional repression domain, thereby achieving repression at the gene transcription level. The repression domain can be selected from KRAB, H-NS, LRP, CRP, etc. In addition, some prophage-encoded repressor proteins (such as AcrIIA2, AcrIIA4) or their homologs can also be used for this purpose, and can be used in combination to enhance the repressive effect.

[0032] Secondly, the present invention also provides the application of the retinal degenerative disease model constructed by the above method in screening drugs for the prevention or treatment of retinal degenerative diseases.

[0033] In a preferred embodiment of the present invention, the application includes at least one of the following application methods: (1) If the visual function of the animal model improves after treatment with the candidate drug, it indicates that the candidate drug can be used as a drug for the prevention or treatment of retinal degenerative diseases. (2) If the thickness of the outer nuclear layer and / or inner nuclear layer of the retina of the animal model increases or shows a thickening trend after treatment with the candidate drug, it indicates that the candidate drug can be used as a drug for the prevention or treatment of retinal degenerative diseases; (3) If the length of the extracellular segment of the retinal photoreceptor cells in the animal model increases after treatment with the candidate drug, it indicates that the candidate drug can be used as a drug for the prevention or treatment of retinal degenerative diseases. (4) If the activation of glial cells in the retina of the animal model is reduced or the inflammatory response is suppressed after treatment with the candidate drug, it indicates that the candidate drug can be used as a drug for the prevention or treatment of retinal degenerative diseases. (5) If the expression level of at least one of the following proteins in the animal model is downregulated after treatment with the candidate drug, it indicates that the candidate drug cannot be used as a drug for the prevention or treatment of retinal degenerative diseases.

[0034] The animal model obtained by the method of this invention exhibits typical pathological features of rheumatoid arthritis (RD) and has broad application prospects. For example, this model can be used to explore the occurrence and development of RD and its underlying mechanisms, providing an important foundation for a deeper understanding of the disease. Furthermore, this model is also suitable for screening candidate drugs with preventive or therapeutic potential, or for evaluating the efficacy and prognosis of drugs.

[0035] Thirdly, the present invention also provides a method for breeding animal models of retinal degenerative diseases, wherein the retinal degenerative disease models constructed by the above method are crossbred.

[0036] After obtaining the model animal for the first time, those skilled in the art can easily expand the population through this breeding method to obtain a sufficient number of experimental animals. Therefore, this method of breeding RD model animal offspring through the above-described mating method also falls within the scope of protection of this invention.

[0037] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0038] Example 1 This embodiment constructs a model of retinal degenerative disease. The specific construction method involves gene knockout; the gene knockout route is described in [link to gene knockout protocol]. Picture 1 As shown, the specific operation is as follows: 1) Obtained C57BL / 6NCya- from Cyagen Biosciences. Fto em1flox / Cya mice (abbreviated) Fto flox ).

[0039] 2) The above Fto flox / flox Homozygous mice were crossed with Rod-Cre transgenic mice (Rod-Cre mice were purchased from Jackson Laboratory in Maine, USA, B6.Cg-Pde6b+Tg(Rho-icre)1Ck / Boc). The resulting offspring showed specific knockout of retinal rod cells. Fto homozygous individuals of the gene Fto flox / flox Rod-Cre (RKO) mice can serve as a model of retinal degenerative diseases. In Rod-Cre transgenic mice, the Cre recombinase is expressed driven by a rod cell-specific opsin promoter, which can recognize and cleave LoxP sites on the genome to achieve conditional gene knockout.

[0040] Example 2 retinal rod cells constructed in Example 1 Fto Genotyping of gene knockout homozygous mice is performed using the following specific methods: 1) Take mouse tail tip tissue, add lysis buffer (40 mM NaOH, 0.2 mM EDTA), and heat at 100℃ for 45 min.

[0041] 2) After cooling, add neutralization solution (40 mM Tris-HCl, pH 5.5), centrifuge, and take the supernatant as a template.

[0042] 3) Perform PCR amplification. The reaction system includes: 10 μL of 2×Taq Mix, 4 μL of template DNA, 1 μL (10 mM) of each specific primer pair, and ddH2O to a final volume of 20 μL.

[0043] The primer sequences used are: Fto-loxP-Forward (SEQ ID NO:1): 5'-TCTTCCAGAGAGGTGAGTTTTCTT-3' Fto-loxP-Reverse (SEQ ID NO:2): 5'-TAAATGTTAGCCCAGGATTGAAGC-3' Rod-Cre-Forward (SEQ ID NO:3): 5'-TCAGTGCCTGGAGTTGCGCTGTGG-3' Rod-Cre-Reverse (SEQ ID NO:4): 5'-CTTAAAGGCCAGGGCCTGCTTGGC-3' 4) Amplification procedure: Primer pair 1: Preheat at 94℃ for 3 min; (denaturation at 94℃ for 30 s, annealing at 62℃ for 35 s, extension at 72℃ for 35 s) cycle 35 times; 72℃ for 5 min; store at 4℃.

[0044] Primer pair 2: Preheat at 94℃ for 3 min; (denaturation at 94℃ for 30 s, annealing at 60℃ for 35 s, extension at 72℃ for 35 s) cycle 35 times; 72℃ for 5 min; store at 4℃.

[0045] 5) The PCR products were analyzed by agarose gel electrophoresis.

[0046] Fto Genotype determination: Wild-type (+ / +) band is 155 bp, heterozygote (+ / loxp) band is 155 bp and 209 bp double band, homozygote (loxp / loxp) band is 209 bp single band.

[0047] The Rod-Cre transgenic band was approximately 600 bp in size. Mice with the genotype loxp / loxp and carrying Rod-Cre were selected as the disease model.

[0048] PCR identification results refer to Picture 1 As shown in Figure C, the results indicate that retinal rod cells were successfully constructed. Fto Gene knockout homozygous mice.

[0049] Example 3 Detection of knockout efficiency at the protein level.

[0050] Western blot was used to analyze the expression of the Fto gene in the retina of knockout mice.

[0051] Western blot experimental method: 1) Place mouse retinal tissue in a 1.5 ml centrifuge tube, add 150 μL of protein RIPA lysis buffer, and incubate on ice for 20 min; 2) The ultrasonic disruptor is used to sonicate the sample (power 15%, pulse: 3 s operation, 3 s interval, repeated 10 times). 3) Place the ultrasonicated sample in a 4-degree centrifuge and centrifuge at 10000g for 10 minutes; 4) Carefully aspirate 120 μL of the supernatant and transfer it to a new 1.5 ml centrifuge tube; 5) Add 40 μL of 4× protein loading and heat in a 95°C metal bath for 5 min to denature; 6) Perform electrophoresis on the samples using SDS-PAGE gel (80V, 15min; 120V, 1h); 7) Electrospin the SDS-PAGE gel to transfer the protein bands onto a nitrocellulose membrane (NC membrane); 8) Block the NC membrane with 8% skim milk at room temperature for 1 hour, then add primary antibody and incubate overnight at 4°C on a shaker. 9) Wash 4 times with 1×TBST, add secondary antibody, and shake at room temperature for 2 hours; 10) Image the NC membrane using a chemiluminescence imager.

[0052] The results are as follows Picture 2 As shown, compared with the control (Fto-Ctrl), the expression level of FTO protein in the retina of retinal rod cell Fto gene knockout homozygous mice (Fto-RKO) was significantly reduced, confirming that... Fto Effective gene silencing.

[0053] Example 4 This embodiment performs histopathological analysis (H&E staining).

[0054] Paraffin sections and H&E staining were performed on the retinas of 5-, 8-, and 10-month-old model mice. The steps are as follows: 1) The ocular tissue was fixed, dehydrated, embedded in paraffin, and then sectioned.

[0055] 2) After dewaxing and rehydration, the sections were stained with hematoxylin and eosin.

[0056] 3) After routine dehydration, clearing, and mounting, observe under a microscope.

[0057] The results show that ( Picture 3 Compared with the control group, the outer nuclear layer (ONL) of the retina of 5-month-old RKO mice began to thin, and this phenotype progressively worsened with increasing age, indicating the loss and degenerative changes of photoreceptor cells.

[0058] Example 5 Morphological evaluation of the outer retinal segments (immunofluorescence).

[0059] Frozen sections of the retina were prepared from the eyeballs of mice at different ages (5, 8, and 10 months) and subjected to immunofluorescence staining.

[0060] 1) After fixation and dehydration, the eyeballs were embedded in OCT and frozen sections were prepared.

[0061] 2) After blocking, the sections were incubated overnight at 4°C with rhodopsin (Cell Signaling Technology, #27182) or PDE6B (Proteintech, #22063-1-AP) antibodies.

[0062] 3) After washing with PBS, incubate with fluorescently labeled secondary antibody, mount the slide, and observe under a fluorescence microscope.

[0063] The results are as follows Picture 4 As shown, in RKO mice, the signal marking the outer retinal segment significantly decreased with increasing age ( ). Picture 4 In the figure, A and B are 5-month-old mice, C and D are 8-month-old mice, and E and F are 10-month-old mice, indicating that the outer segment structure of the photoreceptor has degenerated.

[0064] Example 6 This example analyzes the expression of phototransduction-related proteins in RKO mice.

[0065] The expression levels of various phototransduction-related proteins in the retina of 6-month-old RKO mice were detected by Western blot, and the experimental procedure was the same as in Example 3.

[0066] The results are as follows Picture 5 As shown, compared with the control group, the expression levels of key proteins such as rhodopsin, PDE6A, PDE6B, PDE6G, GRK1, PRPH2 and GNAT1 in the retina of RKO mice were downregulated to varying degrees, suggesting that FTO knockout leads to abnormal function of the retinal light signal transduction system.

[0067] Example 7 This embodiment detects glial activation and inflammatory response in RKO mice.

[0068] Frozen sections of the retina of 8-month-old mice were immunostained with glial fibrillary acidic protein (GFAP) (Cell Signaling Technology, #80788), following the same procedure as in Example 5.

[0069] The results are as follows Picture 6 As shown, compared to wild-type mice, FtoThe significantly enhanced GFAP positive signal in the retina of gene knockout mice indicates the activation and proliferation of astrocytes, suggesting damage and secondary inflammatory response in the retinal tissue.

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing a model of retinal degenerative diseases, characterized in that, This leads to the rod cells of non-human mammals Fto Gene knockout, gene silencing, or decreased expression levels.

2. The method according to claim 1, characterized in that, The Fto Gene knockout or silencing Fto This can be achieved by using partial exon sequences or complete coding regions of a gene.

3. The method according to claim 2, characterized in that, The partial exon sequence is selected from Fto The third exon of the gene.

4. The method according to claim 1, characterized in that, The gene knockout or decreased expression level is achieved through at least one of the following methods: CRISPR / Cas9 system, Cre / loxP recombinase system, TALENs and ZFNs.

5. The method according to claim 1, characterized in that, The gene silencing or decreased expression level is achieved through at least one of the following methods: siRNA, shRNA, and antisense oligonucleotides.

6. The method according to claim 1, characterized in that, The method includes the following steps: targeting Fto The gRNA expression vector of the gene, the Cas9 protein, and a template sequence containing homologous arms and conditional elements were jointly introduced into the fertilized eggs of non-human mammals, and obtained via embryo transfer. Fto The first genetically modified animal; Breeding the first animals described above, and screening to obtain... Fto Homozygous individuals with the conditional element integrated into their genes were then crossbred with animal strains that specifically express Cre recombinase in rod cells to obtain individuals with rod cell-specific knockout. Fto Animal models of genes; The non-human mammals are selected from mammals including mice, rats, rabbits, dogs, pigs, monkeys, and apes.

7. The use of the retinal degenerative disease model constructed by the method of any one of claims 1-6 in screening drugs for the prevention or treatment of retinal degenerative diseases.

8. The application according to claim 7, characterized in that, The application includes at least one of the following application methods: (1) If the visual function of the animal model improves after treatment with the candidate drug, it indicates that the candidate drug can be used as a drug for the prevention or treatment of retinal degenerative diseases. (2) If the thickness of the outer nuclear layer and / or inner nuclear layer of the retina of the animal model increases or shows a thickening trend after treatment with the candidate drug, it indicates that the candidate drug can be used as a drug for the prevention or treatment of retinal degenerative diseases; (3) If the length of the extracellular segment of the retinal photoreceptor cells in the animal model increases after treatment with the candidate drug, it indicates that the candidate drug can be used as a drug for the prevention or treatment of retinal degenerative diseases. (4) If the activation of glial cells in the retina of the animal model is reduced or the inflammatory response is suppressed after treatment with the candidate drug, it indicates that the candidate drug can be used as a drug for the prevention or treatment of retinal degenerative diseases. (5) If the expression level of at least one of the following proteins in the animal model is downregulated after treatment with the candidate drug, it indicates that the candidate drug cannot be used as a drug for the prevention or treatment of retinal degenerative diseases.

9. A method for breeding an animal model of retinal degenerative disease, characterized in that, The retinal degenerative disease model constructed by the method described in any one of claims 1-6 is crossbred.

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