A method for establishing an animal model of early-onset inherited retinal degeneration

Abstract

This invention belongs to the field of biomedical technology, specifically disclosing a method for establishing an animal model of early-onset hereditary retinal degeneration. This animal model is achieved through the specific knockout of mouse retinal pigment epithelial cells. Pla2g5 Gene acquisition. The invention constructs... Pla2g5 flox / flox ;Best1‑Cre Mice exhibited stable STGD-like pathological and clinical manifestations at an early age, including yellowish-white lesions in the fundus, autofluorescence deposition, impaired retinopathy of prematurity (RPE), and progressive visual decline. This model effectively simulates the early onset and gradual worsening of STGD in clinical practice. This animal model provides an ideal and effective tool for studying early-onset, hereditary retinal degenerative diseases like STGD, and can be used for research on the pathogenesis of STGD, as well as drug screening and efficacy evaluation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for establishing an animal model of early-onset hereditary retinal degeneration. Background Technology

[0002] Hereditary retinal degeneration (IRD) is a group of retinal diseases characterized by high phenotypic heterogeneity and wide variation in genetic background, including macular dystrophies (MD), Stargardt disease (STGD), and Leber congenital amaurosis (LCA). STGD is the most common hereditary retinal degeneration, with a global incidence of 1 / 10,000. STGD-related blindness accounts for 12% of all blindness caused by hereditary retinal degeneration, and has become a pressing public health issue. Currently, the pathogenesis of STGD is not fully understood, and there are no effective preventive or therapeutic methods in clinical practice. Therefore, extensive research is still needed to address this disease.

[0003] Utilizing ideal experimental animal models is crucial for studying the pathogenesis of STGD and exploring potential treatment options. Currently, animal models used to study STGD include: Abca4 Knockout mouse models, STGD pig models induced by somatic cell frameshift mutation followed by nuclear transplantation, and canine models with homozygous loss-of-function ABCA4 mutations are among the models developed. However, these models have many limitations.

[0004] STGD is an early-onset, hereditary retinal degenerative disease characterized by progressive macular atrophy and lipofuscin deposition in the retinal pigment epithelium (RPE). STGD patients typically develop the disease in adolescence or young adulthood; generally, the earlier the onset, the more severe the retinal degeneration and the worse the prognosis. The main pathological phenotype of STGD is yellowish-white spots on the retina, usually surrounding the central area of ​​macular lesions or atrophy, with or without variable mid-to-peripheral distribution, which can be clearly seen through fundus autofluorescence imaging. Over time, the retinal structure and function of STGD patients slowly and gradually deteriorate.

[0005] The main clinical manifestation of STGD is bilateral central vision loss in childhood or early adulthood, with varying degrees of visual decline. Electroretinography (ERG) is commonly used clinically to assist in the examination of visual function to determine if there are any abnormalities. Almost all STGD patients show abnormalities in ERG, which are mostly manifested as lower than normal responses in cone and rod cells, combined with delayed dark adaptation. In severe cases, the ERG response may even disappear.

[0006] Combining the two evaluation criteria of pathological changes and clinical manifestations, the ideal animal model of STGD is one that can simulate the pathogenesis of clinical patients to the greatest extent, that is, early onset and gradual progression of the disease over time, while being able to monitor the main pathological phenotypes and clinical manifestations of the disease through existing examination methods (fundus endoscopy, OCT and ERG, etc.).

[0007] Based on the above evaluation criteria, existing models all have many limitations and are unable to fully reproduce STGD: the most commonly used Abca4 While knockout mouse models exhibit many clinical features associated with Stargardt disease, significant retinal degeneration does not appear until 8 months of age, and even with sensitive fundus autofluorescence detection, enhanced or diminished autofluorescence spots are not visible until 6 months of age. Furthermore, visual function impairment is not observed until 18 months of age. Currently, although there are treatments for... Abca4 Improvements to knockout mouse models, such as feeding them under strong light stimulation or double knockout with other genes, have not yielded significant improvements. Large animal models, such as the STGD pig model and the ABCA4 mutant canine model, have retinal sizes similar to humans, rod / cone cell ratios comparable to humans, and regions rich in cone cells similar to the human macula. They are structurally and functionally more similar to the human eye. However, the model construction process is very complex, the experimental cycle is long (at least 2 years), the breeding cost is high, and the number of available animals is limited, which restricts the application of the models.

[0008] In summary, existing models have many limitations. Therefore, there is a need to provide an animal model of early-onset hereditary retinal degeneration that can better simulate the clinical STGD phenotype. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention aims to provide a method for establishing an animal model of early-onset hereditary retinal degeneration (STGD). The mouse model constructed using this method exhibits scattered yellowish-white lesions in the fundus and decreased visual function at 6-8 weeks of age, with the lesions worsening further with age, similar to the clinical phenotype of this disease. This provides an ideal and effective animal model for STGD research.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] On the one hand, the present invention provides a method for establishing an animal model of early-onset hereditary retinal degeneration, by specifically knocking out retinal pigment epithelial cells. Pla2g5 Gene acquisition. This specifically includes the following steps:

[0012] S1. Using CRISPR / Cas9 gene editing technology in C57BL / 6J wild-type mice Pla2g5 Gene insertion at the Loxp site to construct Pla2g5 flox / + Mice; Pla2g5 flox / + Mice were self-crossed to obtain Pla2g5 flox / flox Mice;

[0013] S2, will Pla2g5 flox / flox Mice and commercial Best1-Cre mouse hybridization Pla2g5 flox / + Best1- Cre Mice;

[0014] S3, will Pla2g5 flox / + Best1-Cre Mice with the target gene obtained through self-crossing of mice Pla2g5 flox / flox Best1- Cre Mice.

[0015] Preferably, the Pla2g5 flox / + The mice were constructed using the following steps: CRISPR / Cas9 gene editing technology was used in C57BL / 6J wild-type mice. Pla2g5 Inserting a LoxP site in the same direction upstream of exon 3 and downstream of exon 6 of the gene yields... Pla2g5 flox / + Mice.

[0016] On the other hand, the present invention also provides the application of the above-described method in the field of animal model construction.

[0017] Preferably, the animal model is an early-onset hereditary retinal degeneration animal model.

[0018] On the other hand, the present invention also provides the use of the animal model of early-onset hereditary retinal degeneration obtained by the above-described method in screening or evaluating drugs for the prevention and / or treatment of early-onset hereditary retinal degeneration.

[0019] Preferably, the application includes the following steps: administering the candidate drug to the animal model of early-onset hereditary retinal degeneration, and evaluating the intervention effect of the candidate drug on early-onset hereditary retinal degeneration by one or more of electroretinography (ERG), fundus imaging observation, or retinal histopathological analysis. Specifically, the candidate drug can be administered to the animal model via intravitreal injection, subretinal injection, systemic administration, or local administration. At predetermined time points after administration, the preventive or therapeutic effects of the candidate drug on early-onset hereditary retinal degeneration are evaluated by detecting changes in retinal function (e.g., ERG), retinal structure (e.g., optical coherence tomography (OCT),) and retinal histopathological changes.

[0020] On the other hand, this invention also provides the application of the animal model of early-onset hereditary retinopathy obtained by the above-described method in studying the pathogenesis of early-onset hereditary retinopathy. Specifically, retinal tissue or intraocular fluid samples can be collected at different time points before and after the onset of the disease in the animal model. Molecular biology, cell biology, histopathology, and omics technologies can be used to detect changes in target gene expression, signaling pathway activation, apoptosis levels, oxidative stress levels, inflammatory factor release, metabolite accumulation, and retinal ultrastructural changes, thereby elucidating the molecular and cellular mechanisms of the development and progression of early-onset hereditary retinopathy. Compared with wild-type control animals, the pathological differences exhibited by the model animals can be used to identify key drivers and potential intervention targets of the disease.

[0021] On the other hand, the present invention also provides the application of the animal model of early-onset hereditary retinal degeneration obtained by the above-described method in evaluating gene replacement therapy for target gene loss of function.

[0022] On the other hand, the present invention also provides Pla2g5 Application of genes in constructing animal models of early-onset hereditary retinal degeneration. Preferably, this is achieved through specific knockout of retinal pigment epithelial cells. Pla2g5 Gene acquisition.

[0023] The present invention provides Pla2g5 flox / flox Best1-Cre The mice exhibit the following phenotypes:

[0024] 6-8 weeks old Pla2g5 flox / flox Best1-CreThe mouse fundus showed obvious mottled patterns and scattered yellowish-white lesions. Visual function showed varying degrees of decrease in the amplitude of dark-adapted a, b, and c waves, with the c wave amplitude showing the most significant decrease, approximately 50%. Light-adapted b wave also showed a decreasing trend, while retinal thickness did not show significant changes.

[0025] As time progresses, 3 months old Pla2g5 flox / flox Best1-Cre The mottled lesions in the mouse fundus were significantly aggravated, and the yellowish-white lesions were significantly aggravated, with an increase in the number of lesions, an increase in the size of the lesions, and an increase in the distribution range of the lesions.

[0026] 5 months old Pla2g5 flox / flox Best1-Cre In mice, a significant increase in yellowish-white lesions was observed in the fundus, with increased area and distribution. This was confirmed when a GFP filter (excitation wavelength: 469 / 35 (451.5-486.5nm), emission wavelength: 488- (above 488nm)) was used for detection. Pla2g5 flox / flox Best1-Cre In the fundus of mice, autofluorescence co-localized with the yellowish-white lesions can be observed, indicating that... Pla2g5 flox / flox Best1-Cre In mice, fundus deposits with autofluorescence may be observed. OCT scans of the yellowish-white lesions revealed highly reflective bulges in the RPE layer towards the outer nuclear layer (ONL), with some bulges penetrating the outer retinal membrane (NLM). Visual function was assessed, with further decreases in the amplitudes of dark-adapted a, b, and c waves on electroretinography, and a significant decrease in light-adapted b wave, indicating a worsening of visual impairment.

[0027] In summary, Pla2g5 flox / flox Best1-Cre The mouse model exhibits STGD-related clinical features at an early age (6-8 weeks), including yellow-white spots in the fundus and significant visual decline. These phenotypes gradually worsen over time, with numerous scattered yellow-white spots and further visual decline observed by 5 months of age. This mouse model has an early onset, similar to the clinical progression of STGD, and its pathological and clinical manifestations are similar to the clinical phenotype of STGD. Therefore, it can serve as a relatively ideal animal model for studying this early-onset hereditary retinal degenerative disease.

[0028] The invention constructs Pla2g5 flox / flox Best1-Cre Mice, compared with existing STGD animal models, have the following advantages:

[0029] 1. The onset time in this model is significantly earlier, with obvious yellowish-white lesions in the fundus and abnormal ERG appearing as early as 6-8 weeks of age, significantly earlier than in the traditional model. Abca4 The knockout mouse model shortens the experimental cycle and improves research efficiency. Compared with large animal models such as pigs and dogs, this model has a shorter construction cycle, higher reproductive efficiency, and lower feeding costs, making it more suitable for large-scale experimental research.

[0030] 2. This model can reproduce the key pathological and functional phenotypes of STGD, including yellowish-white lesions in the fundus, autofluorescence deposition, abnormal RPE structure, and abnormal ERG, etc. The model has a high similarity to the clinical disease.

[0031] 3. This model exhibits clear progressive disease characteristics, with fundus lesions, abnormal autofluorescence, and visual function impairment continuously worsening with age, which is more consistent with the natural course of clinical STGD.

[0032] 4. This model can be dynamically monitored using mature detection methods such as fundus photography, OCT, autofluorescence imaging, and ERG, and can be used for mechanism research, drug screening, and efficacy evaluation of STGD.

[0033] 5. This model is based on Pla2g5 The anomalous construction, independent of ABCA4 mutations, provides a new research platform for studying the pathogenesis of STGD, and has significant theoretical research value and application prospects. Attached Figure Description

[0034] Figure 1 : Pla2g5 flox / flox Best1-Cre Mouse construction strategy.

[0035] Figure 2 : Pla2g5 flox / flox Best1-Cre Mouse genotype identification results.

[0036] Figure 3 : Pla2g5 flox / flox Best1-Cre Results of fundus examination in mice.

[0037] Figure 4 : Pla2g5 flox / flox Best1-Cre Results of autofluorescence examination of mouse fundus.

[0038] Figure 5 : Pla2g5 flox / flox Best1-Cre Results of optical coherence tomography (OCT) examination of the mouse retina.

[0039] Figure 6 : Pla2g5 flox / flox Best1-Cre Results of visual function examination in mice (n=12). Detailed Implementation

[0040] To make the technical problem to be solved and the technical solution of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0041] This invention Pla2g5 flox / flox Best1-Cre Mouse construction strategies, such as Figure 1 As shown.

[0042] Example 1: Pla2g5 flox / flox Best1-Cre Mouse model construction

[0043] This embodiment employs the Cre-LoxP conditional gene knockout strategy. The specific principle is as follows: In Pla2g5 Constructing LoxP sites by inserting them in the same direction on both sides of the gene Pla2g5 flox / flox Mice, and compared with Best1-Cre Tool mouse hybridization; Best1 promoter-driven Cre recombinase is specifically expressed in retinal pigment epithelial (RPE) cells, mediating recombination between two co-directional LoxP sites, with partial deletion. Pla2g5 The gene sequence is incorrect, resulting in the inability to synthesize the PLA2G5 functional protein, thus achieving... Pla2g5 Conditional knockout in RPE cells.

[0044] The specific steps are as follows:

[0045] (1) Using C57BL / 6J mice as the genetic background, CRISPR / Cas9 gene editing technology was used to... Pla2g5 A LoxP site in the same orientation was inserted upstream of exon 3 and downstream of exon 6 of the gene (Gene ID: 18784) to obtain... Pla2g5 flox / + Mice. Pla2g5 flox / + Mice were self-crossed to obtain Pla2g5 flox / flox Mice.

[0046] (2) Pla2g5 flox / flox mice and Best1-Cre Mouse hybridization to obtain Pla2g5flox / + Best1-Cre Mice.

[0047] (3) Pla2g5 flox / + Best1-Cre Mouse self-crossing to obtain the target gene Pla2g5 flox / flox Best1-Cre Mice.

[0048] Example 2: Mouse Genotyping

[0049] 1. DNA extraction from rat tail tissue:

[0050] 1) Take out and restrain the mice to be identified, number the mice by clipping their toes, cut off 0.5 cm of the mouse tail that has been thoroughly wiped with alcohol, put it into a 1.5 mL sterile EP tube, and label the mouse number and the breeding cage number;

[0051] 2) Place the EP tube containing the rat tail into a centrifuge and centrifuge (room temperature, 1000 g, 1 min) to centrifuge the rat tail tissue to the bottom of the EP tube;

[0052] 3) Add 100 μL of 50 mM sodium hydroxide aqueous solution to each EP tube. After the lysed tissue is suspended, lyse it in a 95°C metal bath for 10 minutes. After lysis, centrifuge briefly at low speed to collect the liquid on the tube cap and wall at the bottom of the tube.

[0053] 4) Add 10 μL of 1 M Tris-HCl equilibration solution (pH=8.0) to each tube, centrifuge (room temperature, 1000 g, 1 min), then add 100 μL of ddH2O and centrifuge briefly at low speed. This is the extracted mouse DNA.

[0054] 2. Mouse genotyping:

[0055] 1) The PCR reaction system is as follows:

[0056]

[0057] 2) The PCR reaction procedure is as follows:

[0058]

[0059] 3) Agarose gel electrophoresis:

[0060] After the PCR reaction is complete, weigh 2 g of agarose powder into a 250 mL Erlenmeyer flask, pour in 100 mL of 1× TAE, seal the mouth of the flask with aluminum foil, and heat in a microwave oven until boiling. Gently shake and heat again until the gel solution is homogeneous. After it cools slightly, add GelStain solution at a ratio of 1:10000, gently shake to mix, and then pour into a gel casting tank with the gel comb inserted, taking care to avoid air bubbles. Let it stand at room temperature for 30 minutes until the agarose gel is completely solidified. Then remove the gel comb and insert the gel into an electrophoresis tank filled with fresh TAE. Take 5 μL of PCR product and add it to the well of the agarose gel. Electrophoresis is performed at 150 V for 30 minutes. The agarose gel is then removed to check for the presence of the target band in the PCR product.

[0061] 4) The primer sequences for mouse genotyping are as follows:

[0062]

[0063] 5) Genotype identification results are shown in Figure 2 .

[0064] like Figure 2 As shown, in Pla2g5 In the PCR reaction system for the gene, a single band of 385 bp was visible in WT (C57BL / 6J) mice. Pla2g5 flox / flox mice and Pla2g5 flox / flox Best1-Cre A single band of 489 bp was visible in mice. Pla2g5 flox / + mice and Pla2g5 flox / + Best1-Cre Double bands of 385 bp and 489 bp were visible in mice; Best1- Cre In the PCR reaction system of genes, WT (C57BL / 6J) mice and Pla2g5 flox / flox No band was observed in mice. Pla2g5 flox / + ； Best1-Cre mice and Pla2g5 flox / flox Best1-Cre A single band of 727 bp was visible in mice.

[0065] Example 3: Fundus Examination

[0066] The examination was performed using the Phoenix Small Animal Retinal Imaging System (MicronIV) fundus photography module. Pla2g5 flox / flox mice and Pla2g5 flox / flox Best1-Cre The mouse retina was examined using the following steps:

[0067] 1. Anesthesia: Anesthesia was administered via intraperitoneal injection of 1% sodium pentobarbital solution (dose: 10 μL / g).

[0068] 2. Mydriasis and topical anesthesia: After the mice are anesthetized for about 3-5 minutes, compound tropicamide eye drops (mydriatic) and acecaine eye drops (topical anesthetic) are instilled into both eyes to dilate the pupils and perform topical anesthesia.

[0069] 3. After the pupils have fully dilated (about 5 minutes), wipe off the eye drops with a cotton swab and apply hydroxypropyl methylcellulose to keep the eye moist. Place the mouse on the small animal platform.

[0070] 4. Open the fundus photography software Discover and perform white balance before the retinal imaging experiment;

[0071] 5. By adjusting the knob on the stage, position the mouse's eyeball in the center of the software image. Rotate the knob at the bottom of the Micron to make the objective lens fit completely against the mouse's eyeball. Adjust the focus and brightness to acquire a retinal image.

[0072] 6. Save the image and export it.

[0073] 7. Fundus examination results are shown in [the image / document]. Figure 3 , Figure 4 :

[0074] like Figure 3 As shown, 6-8 weeks old Pla2g5 flox / flox Best1-Cre The mice exhibited mottled fundus findings with a few scattered yellowish-white lesions, primarily located in the mid-peripheral region of the fundus. These lesions progressed over time (at 3 and 5 months of age). Pla2g5 flox / flox Best1-Cre The mottled lesions in the mouse fundus were significantly aggravated, and the yellowish-white lesions were significantly aggravated, with an increase in the number of lesions, an increase in the size of the lesions, and an increase in the distribution range of the lesions.

[0075] like Figure 4 As shown, GFP filters (excitation filter wavelength: 469 / 35 (451.5-486.5nm), emission filter wavelength: 488- (above 488nm)) were used to detect 5-month-old infants. Pla2g5 flox / flox Best1-Cre In the fundus of mice, autofluorescence was observed at the yellowish-white lesions, indicating that... Pla2g5 flox / flox Best1-Cre Deposits with autofluorescence may appear in the fundus of mice.

[0076] Example 4: Retinal Optical Coherence Tomography (OCT) Examination

[0077] The examination used the Phoenix Small Animal Retinal Imaging System (MicronIV) OCT module to examine the mouse retina. The specific steps are as follows:

[0078] 1. Anesthesia: Anesthesia was administered via intraperitoneal injection of 1% sodium pentobarbital solution (dose: 10 μL / g).

[0079] 2. Mydriasis and topical anesthesia: After the mice are anesthetized for about 3-5 minutes, compound tropicamide eye drops (mydriatic) and acecaine eye drops (topical anesthetic) are instilled into both eyes to dilate the pupils and perform topical anesthesia.

[0080] 3. After the pupils have fully dilated (about 5 minutes), wipe off the eye drops with a cotton swab and apply hydroxypropyl methylcellulose to keep the eye moist. Place the mouse on the small animal platform.

[0081] 4. Open the OCT scanning software and adjust the reference arm and OCT focus;

[0082] 5. Locate the yellowish-white macules based on the retinal fundus images in the software, move the scan line to the lesion, and scan the OCT image of the lesion;

[0083] 6. Save the scanned image data;

[0084] 7. OCT examination results are shown below. Figure 5 :

[0085] like Figure 5 As shown, at 5 months of age Pla2g5 flox / flox Best1-Cre OCT horizontal scanning of the yellowish-white lesions in the fundus of mice revealed a highly reflective bulge in the RPE layer towards the outer nuclear layer (ONL) of the retina.

[0086] Example 5: Visual Function Test

[0087] The electroretinograms (ERGs) of mice were recorded using the Diagnosys Celeris rodent ERG device. The specific steps are as follows:

[0088] 1. Dark adaptation: Mice were placed in a dark environment for at least 8 hours in advance for dark adaptation;

[0089] 2. Anesthesia: Anesthesia was administered via intraperitoneal injection of 1% sodium pentobarbital solution (dose: 10 μL / g).

[0090] 3. Mydriasis and local anesthesia: After the mice are anesthetized for about 3-5 minutes, compound tropicamide eye drops (mydriatic) and acecaine eye drops (topical anesthetic) are instilled into both eyes to dilate the pupils and perform surface anesthesia;

[0091] 4. Moisturize: Wipe away the eye drops with a cotton swab and apply hydroxypropyl methylcellulose to keep the eye area moist;

[0092] 5. ERG Detection: Place the mouse on the operating table and attach the electrode pads to the mouse cornea. Confirm electrode fixation and normal resistance. During the ERG experiment, avoid light exposure throughout. First, perform a scotopic dark adaptation test on the mice. The stimulus in the dark adaptation program is flickering white light. Perform ERG tests at seven intensity gradients: 0.001, 0.01, 0.05, 0.1, 0.5, 1, and 10 cd·s / m². 2 The dark adaptation ERG response waveforms (wave a and wave b) of mice were recorded. After completing the dark adaptation ERG, mice underwent light adaptation for 5 minutes. The light adaptation ERG structure was then detected by light stimulation. ERG detection was performed continuously at three intensity gradients, with stimulation light intensities of 3, 10, and 30 cd·s / m². 2 Record the adaptive ERG response waveform (b wave) of mice at 150.0 cd·s / m 2 The ERG c-wave waveform of mice was recorded under the stimulation light intensity (the c-wave cannot be performed simultaneously with other ERG tests and must be tested separately). After the test, tobramycin eye ointment was applied, and the mice were placed on a heating pad for resuscitation.

[0093] 6. Results of visual function in mice are shown below. Figure 6 :

[0094] like Figure 6 As shown, compared to age-matched control mice, 6-8 week old mice... Pla2g5 flox / flox Best1-Cre In mice under dark adaptation, the amplitudes of waves a, b, and c all showed significant decreases to varying degrees, with the c wave showing the most significant decrease (approximately 50%). In light adaptation, wave b also showed a decreasing trend. At 5 months of age... Pla2g5 flox / flox Best1-Cre The amplitudes of dark-adapted a, b, and c waves in mice all decreased significantly, and the amplitude of light-adapted b wave also decreased significantly, suggesting that the degree of visual impairment has further worsened.

[0095] The above detailed description is a specific description of the embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. A method for establishing an animal model of early-onset hereditary retinal degeneration, characterized in that, By specifically knocking out retinal pigment epithelial cells Pla2g5 Gene acquisition.

2. The method for establishing an animal model of early-onset hereditary retinal degeneration according to claim 1, characterized in that, Includes the following steps: S1. Using CRISPR / Cas9 gene editing technology in C57BL / 6J wild-type mice Pla2g5 Gene insertion at the Loxp site to construct Pla2g5 flox / + Mice; Pla2g5 flox / + Mice were self-crossed to obtain Pla2g5 flox / flox Mice; S2, will Pla2g5 flox / flox Mice and commercial Best1-Cre mouse hybridization Pla2g5 flox / + Best1-Cre Mice; S3, will Pla2g5 flox / + Best1-Cre Mice with the target gene obtained through self-crossing of mice Pla2g5 flox / flox Best1-Cre Mice.

3. The method for establishing an animal model of early-onset hereditary retinal degeneration according to claim 2, characterized in that, The Pla2g5 flox / + The mice were constructed using the following steps: CRISPR / Cas9 gene editing technology was used in C57BL / 6J wild-type mice. Pla2g5 Inserting a LoxP site in the same direction upstream of exon 3 and downstream of exon 6 of the gene yields... Pla2g5 flox / + Mice.

4. The application of the method described in any one of claims 1-3 in the field of animal model construction.

5. The application according to claim 4, characterized in that, The animal model described is an early-onset hereditary retinal degenerative disease animal model.

6. The use of the animal model of early-onset hereditary retinal degeneration obtained by the method according to any one of claims 1-3 in screening or evaluating drugs for the prevention and / or treatment of early-onset hereditary retinal degeneration.

7. The application according to claim 6, characterized in that, Includes the following steps: The test candidate drug was administered to the animal model of early-onset hereditary retinal degeneration, and the intervention effect of the candidate drug on early-onset hereditary retinal degeneration was evaluated by one or more of the following methods: electroretinography, fundus imaging, or retinal histopathological analysis.

8. The application of the animal model of early-onset hereditary retinal degeneration obtained by the method described in any one of claims 1-3 in the study of the pathogenesis of early-onset hereditary retinal degeneration.

9. The use of an animal model of early-onset hereditary retinal degeneration obtained by the method described in any one of claims 1-3 in evaluating gene replacement therapy regimens targeting gene loss of function.

10. Pla2g5 The application of genes in constructing animal models of early-onset hereditary retinal degeneration is characterized by, By specifically knocking out retinal pigment epithelial cells Pla2g5 Gene acquisition.

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