Construction method of retinal degenerative disease model, application of retinal degenerative disease model and medicine

Abstract

The invention provides a method for constructing a retinal degenerative disease model, which comprises the following steps of: performing targeted knockout on an Flvcr1 gene sequence in a non-human target mammal retinal tissue, and performing targeted knockout on the Flvcr1 gene sequence in the non-human target mammal retinal tissue. The non-human mammal can show the corresponding characteristics of the retinal degenerative disease by knocking out the long sequence of the Flvcr1 gene in the retinal tissue of the non-human target mammal or knocking out part of the sequence of the Flvcr1 gene, namely the construction of the retinal degenerative disease model is realized. The retinal degenerative disease model constructed by adopting the construction method of the retinal degenerative disease model provided by the invention has an important application value in screening medicines for preventing or treating retinal degenerative diseases; the medicine containing choline for treating the retinal degenerative disease is screened out on the basis of the constructed retinal degenerative disease model.

Description

Technical Field

[0001] The present invention relates to the technical field of disease model construction, and specifically relates to a method for constructing a retinal degeneration disease model and its application. Background Art

[0002] Inherited retinal diseases (IRDs) are a group of retinal degenerative diseases caused by gene mutations, with a global prevalence of about 0.07%. Among them, retinitis pigmentosa (RP), as the most common type, has an incidence of about 1 / 4000. This disease is mainly characterized by progressive visual acuity decline, night blindness, and visual field defects, and some patients may become blind in the late stage. Current research faces two major challenges: one is the high genetic heterogeneity and the complex genotype-phenotype association, resulting in a gene diagnosis rate of only 52.2% in the East Asian population, which is significantly lower than the global average level; the other is that existing therapies are only applicable to specific mutation types, and patients in the late stage are difficult to obtain curative effects due to irreversible damage to retinal cells. There is an urgent need to develop broad-spectrum treatment strategies and optimize the cost-effectiveness of gene detection. Clinically, about 40% of IRDs patients still cannot find the cause of the disease even after whole-genome sequencing. Therefore, it is particularly important to construct a pathogenic model of IRDs and conduct in-depth research on its molecular mechanism. Therefore, in the research on inherited retinal diseases, there is an urgent need to construct an effective retinal degeneration disease model.

[0003] Therefore, the present invention provides a method for constructing a retinal degeneration disease model to solve the above problems. Summary of the Invention

[0004] The present invention provides a method for constructing a retinal degeneration disease model, aiming to solve the problems raised in the background art.

[0005] To achieve the above object, the present invention provides the following technical solution: A method for constructing a retinal degeneration disease model: characterized in that the Flvcr1 gene sequence in the retinal tissue of a non-human target mammal is targeted and knocked out.

[0006] Furthermore, in the implementation manner of targeting and knocking out the Flvcr1 gene sequence in the retinal tissue of a non-human target mammal, it can be knocking out the long Flvcr1 gene sequence in the retinal tissue of a non-human target mammal or knocking out a partial sequence of the Flvcr1 gene. No matter which type of sequence (partial or full-length) is knocked out, as long as the expression of the Flvcr1 gene can be silenced in the retina, the purpose of targeting and knocking out the Flvcr1 gene sequence in the retinal tissue of a non-human target mammal can be achieved, thereby enabling the non-human mammal to exhibit the corresponding characteristics of retinal degeneration disease, that is, realizing the construction of a retinal degeneration disease model.

[0007] Furthermore, for the 10 exons of the Flvcr1 gene, the ATG start codon is located in exon 1, and the TGA stop codon is located in exon 10 (transcript: ENSMUST00000085635). Knocking out any one of the 10 exons of the Flvcr1 gene can lead to the loss of the function of the mouse Flvcr1 gene, thereby enabling non-human mammals to exhibit the corresponding characteristics of retinal degenerative diseases, that is, to construct a model of retinal degenerative diseases.

[0008] Furthermore, knocking out exon 2 among the 10 exons of the Flvcr1 gene can better enable non-human mammals to exhibit the corresponding characteristics of retinal degenerative diseases, that is, it can better construct a model of retinal degenerative diseases.

[0009] Furthermore, both the gene editing method and the animal hybridization method can be used to knock out the Flvcr1 gene sequence in the retinal genome of non-human target mammals.

[0010] Furthermore, the animal hybridization method is as follows: mating Flvcr1 gene conditional knockout homozygous animals with animals carrying the Six3-Cre gene to obtain Flvcr1 gene knockout animals.

[0011] Furthermore, the gene editing method is selected from at least one of CRISPR / Cas9 technology, artificial nuclease-mediated zinc finger nuclease technology, transcription activator-like effector nuclease technology, and Cre-loxp gene knockout technology.

[0012] Furthermore, the CRISPR / Cas9 technology and Cre-loxp gene knockout technology are used to knock out the Flvcr1 gene sequence in the retinal genome of non-human target mammals.

[0013] Furthermore, the gRNA vector or guide RNA complex containing the Flvcr1 gene of non-human target mammals, Cas9 mRNA or Cas9 protein is transformed or chemically transfected into the fertilized eggs of non-human target mammals, and the embryonic cells are taken and transplanted into the uterus of pseudopregnant non-human target mammals to obtain the founder animals with conditional knockout of the Flvcr1 gene.

[0014] Transformation includes, but is not limited to, electroporation.

[0015] Furthermore, the founder animals are mated to screen for Flvcr1 gene conditional knockout homozygous animals; then the Flvcr1 gene conditional knockout homozygous animals are mated with animals carrying the Six3-Cre gene to obtain Flvcr1 gene knockout animals, which can be used as a model of retinal degenerative diseases.

[0016] Furthermore, the gRNA vector or guide RNA complex includes loxp sites.

[0017] Furthermore, the Cas9 protein is a Cas9 protein or a modified Cas9 protein. The modified Cas9 protein includes, but is not limited to, adding a functional domain such as EVE that inhibits activity to the C-terminus of the Cas9 protein, which can specifically inhibit the expression of the target gene.

[0018] Furthermore, the gRNA vector or guide RNA complex containing the non-human target mammalian Flvcr1 gene, dCas9 mRNA or dCas9 protein is transformed or chemically transfected into the fertilized eggs of non-human target mammals. Embryonic cells are taken and transplanted into the uterus of pseudopregnant non-human target mammals to obtain animals with the Flvcr1 gene knocked out; the 3' end of the dCas9 mRNA is linked to the mRNA sequence of the repressor complex or the C-terminus of the dCas9 protein is linked to the repressor complex, which can directly inhibit the transcription of the Flvcr1 gene, thereby achieving the effect of Flvcr1 gene silencing.

[0019] Furthermore, the repressor complex is selected from Kruppel, H-NS (nucleoid structuring protein), StpA, LRP (leucine-responsive regulatory protein) or CRP (cAMP receptor protein) repressors. The repressor is encoded by a prophage contained in the host cell, such as the AcrIIA protein, such as AcrIIA2 and / or AcrIIA4. It can also be encoded by the acr, aca1 and aca2 genes or their orthologs, homologs or paralogs, or encoded by the acrIIA2 and acrIIA4 genes or their orthologs, homologs or paralogs.

[0020] Furthermore, non-human target mammals include, but are not limited to: mice, rats, dogs, pigs, monkeys and apes. No matter which animal is selected, as long as it is an animal with the Flvcr1 gene, it can be used as the target (non-human mammalian) animal in the above construction method of the present invention. The Flvcr1 gene is knocked out in its retina, making it show the disease characteristics of retinal degeneration, and used as a retinal degeneration disease model in the field of retinal degenerative disease research, which all belong to the protection scope of the present invention.

[0021] The present invention also provides the use of a retinal degeneration disease model constructed by the construction method of the retinal degeneration disease model in screening drugs for preventing or treating retinal degeneration diseases.

[0022] The present invention also provides a drug for treating retinal degeneration diseases, characterized in that the drug contains choline.

[0023] By targeting and knocking out the Flvcr1 gene sequence in the retinal tissue of non-human target mammals, the present invention can induce the phenotypic characteristics of retinal degenerative diseases in the target mammals, thus realizing the construction of a retinal degenerative disease model. The Flvcr1 gene knockout animal model shows significant structural damage and progressive degeneration of photoreceptor cells, and its characteristic pathological changes include progressive reduction in the thickness of the outer nuclear layer of the retina, reduction in photoreceptor cells and photoreceptor proteins, and impairment of cellular mitochondrial homeostasis. This gene defect model successfully simulates the core pathological process of human retinal degenerative diseases, provides an ideal in vivo research platform for revealing the molecular mechanism of the FLVCR1 gene in retinal degenerative diseases, provides a good model basis for in-depth research on the treatment and etiology of retinal degenerative diseases, also provides a good application prospect for screening drugs for preventing or treating retinal degenerative diseases, and provides a drug for treating retinal degenerative diseases screened by using this model, which has been verified by experiments to have good effects. Brief Description of the Drawings

[0024] Figure 1 Schematic diagram of the construction strategy for the Flvcr1 conditional knockout mouse model. Among them, (a) the deletion of exon 2 leads to the loss of the function of the mouse Flvcr1 gene, and the PCR genotyping agarose gel electrophoresis results of SKO and RKO mice; (b) verification of the knockout efficiency of SKO and RKO mice;

[0025] Figure 2 H&E staining results and immunofluorescence staining results of the retina of SKO-30-day-old mice, showing that both the outer nuclear layer and the inner nuclear layer of SKO mice are thinned, and the number of photoreceptor cells and related structures are severely damaged;

[0026] Figure 3 ERG detection results of RKO mice after choline supplementation treatment, showing that the retinal function of RKO mice has been significantly restored after choline treatment;

[0027] Figure 4 H&E staining map of the retina of RKO mice after choline supplementation treatment, showing that the retinal thickness of RKO mice has been significantly restored after treatment;

[0028] Figure 5 Immunofluorescence staining map of the retina of RKO mice after choline supplementation treatment, showing that the photoreceptor cells and related structures of RKO mice have been significantly restored after treatment. Detailed Description of the Invention

[0029] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] Example 1

[0031] In this example, mice were used as the target animals to illustrate the construction method of the retinal degeneration disease model of the present invention. The roadmap for Flvcr1 gene knockout is as Figure 1 shown below. The specific operations are as follows:

[0032] 1. Use the CRISPR / Cas9 technology to construct loxP sites arranged in the same direction on both sides of the homologous arms of the conditional knockout Flvcr1 allele for replacing exon 2 of the Flvcr1 gene. The gRNA vector containing the mouse Flvcr1 gene and Cas9 mRNA were transformed into mouse fertilized eggs, and the embryonic cells were transplanted into the uterus of pseudopregnant mice to obtain the founder animals with conditional knockout of the Flvcr1 gene.

[0033] 2. The heterozygous mice with conditional knockout of the Flvcr1 gene obtained in step 1 were mated with each other to obtain homozygous mice with conditional knockout of the Flvcr1 gene.

[0034] 3. The homozygous mice with conditional knockout of the Flvcr1 gene obtained in step 2 were mated with mice carrying the Six3-Cre or Rhodopsin-Cre gene to obtain Flvcr1 knockout mice (SKO) or rod cell Flvcr1 knockout mice (RKO), which can be used as the retinitis pigmentosa disease model.

[0035] The obtained offspring mice were subjected to genotype identification. The identification method is as follows:

[0036] 1) Put 2 mm of the mouse tail into a 1.5 mL EP tube.

[0037] 2) Add 75 - 100 μL of lysis solution A (containing 0.2 mM EDTA2Na + 40 mM NaOH) to the EP tube; boil in a metal bath at 95 °C for 30 min, and take it out and cool for 10 min.

[0038] 3) Then add an equal volume of 75 - 100 μL of neutralization solution B (containing 40 mM Tri-HCl buffer) to the EP tube, centrifuge at 10000 g for 2 min, and take the supernatant for mouse genotype identification.

[0039] 4) PCR amplification:

[0040] The reaction system is as follows in the table:

[0041]

[0042] The primer sequences are as follows in the table:

[0043]

[0044] The amplification program is as follows in the table:

[0045]

[0046] 5) Gel electrophoresis includes the following steps:

[0047] A. Weigh 2 g of agarose and dissolve it in a conical flask containing 100 mL of 1×TAE, and heat it in a microwave oven at high power for 4 min;

[0048] B. Take out the conical flask with a wet towel, shake it gently, put it back into the microwave oven, and heat it at medium power for 2 min until the solution is completely transparent and has no obvious impurities;

[0049] C. Add 10 μL of nucleic acid dye to the conical flask and shake it well;

[0050] D. Pour the mixed gel solution into the gel-making plate with a comb inserted, and use a pipette tip to suck out the bubbles, and cool it to room temperature; after the gel solidifies, pull out the comb and place it in a horizontal electrophoresis tank containing 1×TAE;

[0051] E. Add 15 μL of sample to each well, and add 5 μL of DNA maker to the last well of each group of samples. Replace the pipette tip when changing the sample addition to avoid sample contamination;

[0052] F. After the sample addition is completed, turn on the power supply, keep the voltage at 120 V for 30 - 50 min. The moving direction of the sample should be from the negative pole to the positive pole, and stop the electrophoresis when the bromophenol blue indicator moves to 2 - 3 cm from the edge of the gel;

[0053] G. Put the run gel into an ultraviolet gel imager, expose it, and save the image.

[0054] Figure 1 As shown in the electrophoresis result in (a): Genotype identification result of Flvcr1-specific knockout mice, Flvcr1 + / + was used as a control mouse (labeled as Ctl, that is, wild-type mouse), and the band size was 215 bp; Flvcr1 + / flox was a heterozygous mouse with conditional knockout of the Flvcr1 gene; Flvcr1 flox / flox was a homozygous mouse with conditional knockout of the Flvcr1 gene, and the band size was 283 bp; Flvcr1 flox / +-Six3-Cre represents the heterozygote obtained after mating the heterozygous mouse with conditional knockout of the Flvcr1 gene and the mouse transfected with the Six3-Cre gene, having two bands at 215 bp and 283 bp;

[0055] Flvcr1 flox / flox -Six3-Cre represents the homozygous mouse with retinal knockout of the Flvcr1 gene (labeled as SKO); Flvcr1 flox / flox -Rod-Cre represents the homozygous mouse with rod cell knockout of the Flvcr1 gene (labeled as RKO). The symbols Cre- and Cre+ indicate whether the mouse carries six3-Cre or Rhodopsin-Cre.

[0056] According to Figure 1 the results shown in (a) herein, the identification method adopted in this example can effectively identify the genotypes of neonatal mice for subsequent research. Figure 1 (a) The preliminary identification results show that mice with knockout of the Flvcr1 gene are obtained. According to Figure 1 (b) the results of the Western blot experiment, it is found that the FLVCR1 protein in the retina is significantly reduced, proving the successful construction of SKO and RKO mice.

[0057] Example 2

[0058] In this example, retinal H&E staining and immunofluorescence staining are performed.

[0059] Paraffin sections of the mouse retina are stained by the hematoxylin-eosin staining method (H&E staining method), and the specific operations are as follows:

[0060] 1) Quickly take the mouse eyeball tissue and fix it in the fixative for 24 h; embed it in paraffin and section it with a thickness of 4 μm;

[0061] 2) The sections are routinely dewaxed with xylene and rinsed with water through multiple grades of ethanol: xylene (I) for 5 min → xylene (II) for 5 min → 100% ethanol for 2 min → 95% ethanol for 1 min → 80% ethanol for 1 min → 75% ethanol for 1 min → distilled water rinse for 2 min;

[0062] 3) Stain with hematoxylin for 5 minutes and rinse with tap water;

[0063] 4) Differentiate with hydrochloric acid ethanol for 30 seconds;

[0064] 5) Soak in tap water for 15 minutes;

[0065] 6) Place in eosin solution for 2 minutes;

[0066] 7) Conventional dehydration, clearing, and mounting: 95% ethanol (I) for 1 min → 95% ethanol (II) for 1 min → 100% ethanol (I) for 1 min → 100% ethanol (II) for 1 min → xylene - phenol (3:1) for 1 min → xylene (I) for 1 min → xylene (II) for 1 min → mounting with neutral resin.

[0067] 8) Photograph under a microscope.

[0068] Immunostaining of retinal frozen sections: After sacrificing the mice constructed in Example 1 by cervical dislocation, quickly remove the eyeballs and place them in 4% PFA. Fix on ice for 15 min, make an incision on the cornea, and then continue to fix on ice. After 2 h, rinse three times with PBS buffer, then dehydrate the eyeballs in 30% sucrose solution for 2 h. Then, under a dissecting microscope, cut off the cornea and lens, embed in OCT and quickly freeze in a -80 °C refrigerator. After about 10 min, take out the OCT - embedded eyeballs, place them in a cryostat at -25 °C and equilibrate for about 30 min before sectioning. The section thickness is 12 μm.

[0069] After sectioning, select the slides with better quality and place them in an oven at 37 °C for 30 min. Then, use an immunohistochemistry pen to draw a circle around the area with retinal tissue, wash three times with PBS to remove OCT, and then block and permeabilize with 5% NGS (containing 0.25% Triton) for 2 h. Incubate with the primary antibody overnight at 4 °C. The next day, after washing three times with PBS, incubate with the corresponding fluorescent secondary antibody, then wash three times with PBS again, mount the slides, and observe.

[0070] The results are as Figure 2 , the H&E staining results and immunofluorescence staining results of the retinas of SKO - 30 - day - old mice show that both the outer nuclear layer and the inner nuclear layer of SKO mice are thinned, and the number of photoreceptor cells and related structures are severely damaged.

[0071] Example 3

[0072] In this example, ERG visual acuity detection was performed on Flvcr1 gene - knockout mice:

[0073] 1) Weigh the mice to be subjected to the ERG experiment in advance and calculate the required anesthesia dose. Place them in a dark room overnight for light - avoidance feeding the night before the measurement.

[0074] 2) Turn on the ERG measuring instrument and the computer host, and adjust the screen brightness mode to the dark red light mode to minimize the influence of white light on the experimental results during the dark - response measurement as much as possible.

[0075] 3) First, prepare the required ketamine anesthetic (1 mL of ketamine + 4 mL of 0.9% saline + 50 μL of luminal), and anesthetize the mice according to their corresponding body weight (10 - 12 μL / g). When the activity of the mice weakens, drop an appropriate amount of mydriatic solution into the eyes of the two mice. After 2 - 3 minutes, place them on the lifting plate of the ERG measuring instrument.

[0076] 4) Connect the three electrodes of the ERG instrument to the cerebral cortex, corneal limbus, and tail root of the mice respectively according to the specification requirements. After the connection is completed, use a syringe to drop two drops of saline at the place where the corneal limbus of the mice contacts the metal ring of the electrode to promote its conductivity.

[0077] 5) After the ERG electrodes are connected, in order to ensure whether the installation position is appropriate, the measurement of the electrophysiological baseline can be started. If the baseline is stable, the subsequent measurement can be continued. If the baseline is unstable, the position of the cerebral cortex electrode or the electrode at the corneal limbus needs to be readjusted.

[0078] 6) According to the specification requirements of ERG measurement, perform the dark reaction under different light stimuli respectively, and record the relevant experimental results.

[0079] The ERG experiment is as Figure 3 shown. The ERG test results of RKO mice after choline supplementation therapy were found that the retinal function of RKO mice was significantly restored after choline treatment.

[0080] Example 4

[0081] This example is the ERG detection of RKO mice after choline supplementation therapy, and the detection method uses the method of Example 3:

[0082] As Figure 4 shown, it was found that the retinal thickness of RKO mice was significantly restored after treatment.

[0083] Example 5

[0084] This example is the immunofluorescence staining of the retina of RKO mice after choline supplementation therapy, and the staining method uses the method of Example 2:

[0085] It was found that the photoreceptor cells and related structures of RKO mice were significantly restored after treatment.

[0086] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution of the present invention and its inventive concept, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. A method for constructing a retinal degenerative disease model, characterized in that: By targeted knockout of the Flvcr1 gene sequence in the retinal tissue of non-human target mammals.

2. The method for constructing a retinal degenerative disease model according to claim 1, characterized in that: In the implementation method of targeted knockout of the Flvcr1 gene sequence in the retinal tissue of non-human target mammals, the long sequence of the Flvcr1 gene in the retinal tissue of the non-human target mammals can be knocked out, or a partial sequence of the Flvcr1 gene can be knocked out. No matter which type of sequence (partial or full-length) is knocked out, as long as the expression of the Flvcr1 gene in the retina can be silenced, the purpose of targeted knockout of the Flvcr1 gene sequence in the retinal tissue of the non-human target mammals can be achieved, thereby achieving the corresponding characteristics of retinal degenerative diseases in non-human mammals, that is, realizing the construction of a retinal degenerative disease model.

3. The method for constructing a retinal degenerative disease model according to claim 2, characterized in that: A partial sequence of the Flvcr1 gene is knocked out. For the 10 exons of the Flvcr1 gene, any one of the 10 exons of the Flvcr1 gene is knocked out.

4. The method for constructing a retinal degenerative disease model according to claim 3, characterized in that: Knocking out a portion of the sequence of the Flvcr1 gene, knocking out exon 2 of the 10 exons of the Flvcr1 gene, can better enable non-human mammals to exhibit the corresponding characteristics of retinal degenerative diseases, that is, can better achieve the construction of a retinal degenerative disease model.

5. The method for constructing a retinal degenerative disease model according to claim 1, characterized in that: The Flvcr1 gene sequence in the retinal genome of non-human target mammals can be knocked out by using a gene editing method or an animal hybridization method; the animal hybridization method is: mating a homozygous Flvcr1 gene conditional knockout animal with an animal carrying the Six3-Cre or Rhodopsin-Cre gene to obtain an animal with a conditional knockout of the Flvcr1 gene; the gene editing method is selected from at least one of CRISPR / Cas9 technology, artificial nuclease-mediated zinc finger nuclease technology, transcription activator-like effector nuclease technology and Cre-loxp gene knockout technology; CRISPR / Cas9 technology and Cre-loxp gene knockout technology are used to knock out the Flvcr1 gene sequence in the retinal genome of non-human target mammals; a gRNA vector or guide RNA complex containing the Flvcr1 gene of a non-human target mammal, Cas9 mRNA or Cas9 protein is transformed or chemically transfected into a fertilized egg of a non-human target mammal, and embryonic cells are transplanted into the uterus of a pseudo-pregnant non-human target mammal to obtain a founder animal with a conditional knockout of the Flvcr1 gene.

6. The method for constructing a retinal degenerative disease model according to claim 5, characterized in that: The first-established animals are mated to screen for homozygous animals with conditional knockout of the Flvcr1 gene; the homozygous animals with conditional knockout of the Flvcr1 gene are then mated with animals carrying the Six3-Cre or Rhodopsin-Cre gene to obtain animals with conditional knockout of the Flvcr1 gene. It can be used as a retinal degeneration model; the gRNA vector or guide RNA complex includes a loxp site; the Cas9 protein is a Cas9 protein or a modified Cas9 protein, and the modified Cas9 protein includes but is not limited to adding an inhibitory functional domain such as EVE to the C-terminus of the Cas 9 protein, which can specifically inhibit the expression of the target gene; the gRNA vector or guide RNA complex containing the Flvcr1 gene of the non-human target mammal, dCas9 mRNA or dCas9 protein is transformed or chemically transfected into the fertilized egg of the non-human target mammal, and the embryonic cells are transplanted into the uterus of the pseudo-pregnant non-human target mammal to obtain an animal with Flvcr1 gene knockout; the 3' end of the dCas9 mRNA is connected to the mRNA sequence of the repressor complex or the C-terminus of the dCas9 protein is connected to the repressor complex, which can directly inhibit the transcription of the Flvcr1 gene, thereby achieving the effect of Flvcr1 gene silencing; the repressor complex is selected from Kruppel, H-NS (nucleoid structured protein), StpA, LRP (leucine responsive regulatory protein) or CRP (cAMP receptor protein) repressor. The repressor is encoded by a prophage contained in the host cell, such as AcrIIA protein, such as AcrIIA2 and / or AcrIIA4. It can also be encoded by acr, aca1 and aca2 genes or their orthologs, homologs or paralogs, or acrIIA2 and acrIIA4 genes or their orthologs, homologs or paralogs.

7. The method for constructing a retinal degenerative disease model according to claims 1-6, characterized in that: Non-human target mammals include but are not limited to: mice, rats, dogs, pigs, monkeys and apes. No matter which animal is selected, as long as it is an animal with the Flvcr1 gene, it can be used as the target (non-human mammal) animal in the above-mentioned construction method of the present invention. The Flvcr1 gene is knocked out in its retina to make it show the disease characteristics of retinal degeneration, which can be used as a retinal degeneration disease model.

8. A method for constructing a retinal degenerative disease model according to claims 1-7, characterized in that: The use of the retinal degenerative disease model constructed by the method for constructing a retinal degenerative disease model in screening drugs for preventing or treating retinal degenerative diseases.

9. The drug screened out from the retinal degenerative disease model constructed by the method for constructing a retinal degenerative disease model according to claim 8, characterized in that: The drug contains choline.

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