Method and kit for constructing a model of retinal pigment degeneration

Abstract

The application provides a method and a kit for constructing a retinal pigment degeneration model, and relates to the technical field of biology. The method comprises administering a disulfiram-copper ion complex to a subject. The method alleviates at least one of the following defects in the prior art: a short model research window period, an unstable model, a complex construction method and high cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and in particular to a method for constructing a retinitis pigmentosa model and a kit. BACKGROUND

[0002] The following statements only provide background information related to the present application, and do not necessarily constitute the prior art.

[0003] Retinitis pigmentosa (RP) is a hereditary and degenerative disease characterized by progressive loss of photoreceptor and pigment epithelial function, and is a common hereditary eye disease. RP is a major blinding eye disease, and its genetic patterns include X-linked inheritance, autosomal recessive or dominant inheritance, and sporadic. The main feature of RP is rod cell degeneration, followed by loss of cone cells. The initial symptom is night vision loss, followed by concentric progressive loss of visual field. Ocular fundus abnormalities usually manifest as the triad of osteocyte-like pigmentation, retinal vascular narrowing, and optic disc waxy pallor. To date, various genetic mutations have been found to cause retinitis pigmentosa.

[0004] An effective model can simulate the disease, assist in studying the pathogenesis of retinitis pigmentosa, and perform drug screening and evaluation. Currently available retinitis pigmentosa models include the following:

[0005] 1. RD1 and RD10 model mice: These two model mice are the most commonly used mice for RP research. The variant genes of these two types of mice are phosphodiesterase subunit B (PDE6B), and mutations in this gene lead to the death of retinal photoreceptor cells, ultimately causing RP. The defects of this animal model include: the disease develops very quickly, RD1 often begins to develop at 1 week after birth, and enters the late stage at 2 weeks. RD10 begins to develop at 2 weeks after birth, and enters the late stage at 4 weeks. Therefore, the time left for researchers to conduct treatment research is very short. In particular, RD1 mice have not yet opened their eyes at 1 week after birth, and the eyeball and retina have not yet fully developed, so if a therapeutic drug is injected under the retina at this time, it is easy to cause the retina to fall off, and the drug efficacy cannot be observed well. If the experiment is started at 4 weeks of age when the mouse is basically mature, although the eyeball and retina have developed well, the disease is already in the late stage, with a photoreceptor cell death rate of more than 90%, and the effects of the drug on photoreceptor cell function and survival cannot be explored.

[0006] 2. Gene edited mice: Key genes such as RHO or RPGR in the visual cycle are knocked out using CRISPR / Cas9 technology to induce photoreceptor cell death, ultimately leading to RP. Knocking out specific genes in the mouse genome through CRISPR / Cas9 technology has a high technical threshold, a long cycle, and often takes more than 6 months, and requires high costs.

[0007] 3. Chemically induced animal model: wild-type mice are injected intraperitoneally with N-methyl-N-nitrosourea (MNU) to induce DNA damage in photoreceptor cells, which in turn causes RP. However, model mice constructed by intraperitoneal injection of MNU often exhibit systemic toxicity, leading to multiple organ damage, which in turn affects the study of specific organs.

[0008] 4. Physically induced animal model: wild-type mice are exposed to strong light for a long time to induce oxidative stress in photoreceptor cells, which ultimately leads to RP. However, strong light exposure can lead to uncontrolled damage, and individual differences and light-avoiding behavior in mice can easily lead to unstable models.

[0009] In summary, the existing art has the defects of short model research window period, unstable model, complex construction method, and high cost in constructing a retinal pigment degeneration model. How to improve the above defects is a technical problem to be solved.

[0010] Therefore, the present application is proposed. SUMMARY

[0011] The present application aims to provide a method for constructing a retinal pigment degeneration model to alleviate at least one of the defects of short model research window period, unstable model, complex construction method, and high cost in the prior art.

[0012] To solve the above technical problems, the present application adopts the following technical solutions:

[0013] In a first aspect, a method for constructing a retinal pigment degeneration model is provided, which comprises administering a disulfiram-copper ion complex to a subject, wherein the subject is a mouse or an organ, tissue or cell from a mouse.

[0014] In an optional embodiment, the retinal pigment degeneration model is an animal model, and the subject is a mouse at least 4 weeks old.

[0015] In an optional embodiment, the method for constructing a retinal pigment degeneration model comprises administering a disulfiram-copper ion complex to the subretina of a mouse.

[0016] In an optional embodiment, the method for constructing a retinal pigment degeneration model comprises administering a 5 mM disulfiram-copper ion complex to the subretina of a mouse.

[0017] In an optional embodiment, the retinal pigment degeneration model is an in vitro model, and the subject is an organ, tissue or cell from a mouse; the method for constructing a retinal pigment degeneration model comprises allowing the subject to be in sufficient contact with a disulfiram-copper ion complex.

[0018] In an alternative embodiment, the subject is a mouse retinal photoreceptor cell, and the constructing method comprises administering 5 μM disulfiram-copper ion complex to the mouse retinal photoreceptor cell.

[0019] In an alternative embodiment, the disulfiram-copper ion complex is prepared by mixing a disulfiram solution and a salt solution providing copper ions, and vortexing to form the disulfiram-copper ion complex.

[0020] In an alternative embodiment, the solvent of the disulfiram solution is PBS; and / or, the salt solution providing copper ions is a copper sulfate solution.

[0021] In a second aspect, a kit for constructing a retinal pigment degeneration in vitro model is provided, the kit comprising (i) and (ii); or the kit comprising (i) and (iii):

[0022] (i) an organ, a tissue or a cell;

[0023] (ii) a disulfiram-copper ion complex;

[0024] (iii) disulfiram and a salt providing copper ions.

[0025] In a third aspect, the retinal pigment degeneration model obtained by the constructing method of the first aspect, or the kit of the second aspect is used in any one of (X1) to (X6):

[0026] (X1) studying the pathogenesis of retinal pigment degeneration;

[0027] (X2) preparing a product for studying the pathogenesis of retinal pigment degeneration;

[0028] (X3) studying the molecular mechanism of retinal pigment degeneration;

[0029] (X4) preparing a product for studying the molecular mechanism of retinal pigment degeneration;

[0030] (X5) screening drugs for treating retinal pigment degeneration diseases;

[0031] (X6) preparing a product for screening drugs for treating retinal pigment degeneration diseases.

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] The application provides a method for constructing a retinal pigment degeneration model, which induces double death of copper death and necrotic apoptosis of photoreceptor cells by administering a disulfiram-copper ion complex to a subject to simulate a retinal pigment degeneration phenotype. The application proves by experiments that the photoreceptor cell layer of the animal model obtained by the construction method has a reduced thickness, but other layers of the retina have no obvious changes, and the pathological changes are consistent with the pathological characteristics of retinal pigment degeneration; at the same time, the expression of visual transduction proteins such as PDE6B and RHO in the retina of the constructed animal model is significantly down-regulated, and the most common RP pathogenic gene is PDE6B and RHO, which indicates that the model can well simulate retinal pigment degeneration. In a cell model, the disulfiram-copper ion complex down-regulates the expression of PDE6B and RHO in cells and inhibits cell growth.

[0034] The construction method provided by the application can selectively kill high-metabolic-activity photoreceptor cells and has little effect on other layers of the retina, and has good specificity and safety. The application also has cost and timeliness advantages: taking a mouse model as an example, the construction cost of a single model is less than 100 yuan, which significantly reduces the construction cost of an animal model; from the administration of the disulfiram-copper ion complex to the stabilization of the phenotype, only 2 weeks are needed, while a gene editing model needs more than 6 months, which shortens the model preparation period. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0036] Figure 1 OCT images for observing the retinal morphology of each experimental group in Example 2;

[0037] Figure 2 HE staining images of retinal sections of each experimental group in Example 2;

[0038] Figure 3 Western blotting results of immunoblotting for detecting the expression of visual transduction proteins PDE6B and RHO in Example 2;

[0039] Figure 4 OCT images for observing the retinal morphology of each experimental group in Example 3;

[0040] Figure 5 HE staining images of retinal sections of each experimental group in Example 3;

[0041] Figure 6Growth curves for cells in each experimental group in Example 4, P <0.05, P <0.01, P <0.001.

[0042] Figure 7 Results of immunoblotting to detect the amount of PDE6B and RHO protein expression in cells in each experimental group in Example 4;

[0043] Figure 8 Growth curves for cells in each experimental group in Example 5, P <0.05, P <0.01. DETAILED DESCRIPTION

[0044] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0045] In the present application, all the embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions, and all the technical features and preferred features mentioned herein can be combined to form new technical solutions, and the components involved or the preferred components thereof can be combined to form new technical solutions, unless otherwise specified.

[0046] In the present application, unless otherwise specified, the numerical range "a~b" represents a shorthand notation for any real number combination between a and b, wherein a and b are both real numbers. For example, the numerical range "6~22" represents that all the real numbers between "6~22" have been listed herein, and "6~22" is only a shorthand notation for these numerical combinations. The "range" disclosed herein in the form of lower limit and upper limit can be one or more lower limits and one or more upper limits, respectively.

[0047] In the present application, unless otherwise specified, each reaction or operation step can be carried out in sequence or not in sequence. Preferably, the reaction method herein is carried out in sequence.

[0048] In the present application, "prepared from" is synonymous with "comprising". The terms "comprising", "including", "having" "with" or any other similar forms are intended to cover non-exclusive inclusion. For example, a composition, step, method, article or device comprising the listed elements does not necessarily limit to only those elements, but can include other elements not explicitly listed or inherent to such composition, step, method, article or device.

[0049] As used herein, the conjunctive term "consisting of" excludes any element, step, or component not specified. If used in a claim, this phrase will close the claim to the addition of any element not specified, but will not exclude conventional impurities that occur in the production of the specified materials.

[0050] As used herein, "and / or" is used to indicate one or both of the stated conditions can occur, for example A and / or B includes (A and B) and (A or B).

[0051] As used herein, unless otherwise indicated, any number is used to distinguish one entity or action from another, and does not necessarily imply any actual such relationship, order, or importance of the entities or actions, for example, the numbers i, ii,...; first, second,...; X1, X2,... etc.

[0052] As used herein, unless otherwise indicated, "optionally", "optional", "optional" or "may" means that the event or circumstance subsequently described can or can not occur, and the description includes instances where the event or circumstance occurs and instances where it does not.

[0053] In a first aspect, a method for constructing a retinitis pigmentosa model is provided, the method comprising administering a disulfiram-copper ion (DSF-Cu 2+ ) complex to a subject. The subject is a mouse or an organ, tissue or cell from a mouse.

[0054] DSF-Cu 2+ By binding to fatty acylated proteins in the mitochondria of photoreceptor cells (such as DLAT), protein toxic stress is induced, selectively killing high-metabolic-activity photoreceptor cells (such as rod cells), while other layers of cells in the retina (such as bipolar cells, ganglion cells) are minimally affected, and the resulting model conforms to the pathological changes of retinitis pigmentosa.

[0055] In an optional embodiment, the retinitis pigmentosa model is an animal model, and the subject is a mouse at least 4 weeks old. Using a mouse at least 4 weeks old, the eyeball retina has developed maturely, improving the success rate of injection of therapeutic drugs, and better enabling detection of retinal function and research on drug intervention.

[0056] In an optional embodiment, the method for constructing a retinitis pigmentosa model comprises administering a disulfiram-copper ion complex to the subretinal of a mouse. As used herein, the subretinal refers to the gap between the retinal pigment epithelial layer (RPE) and the neurosensory retina layer (NSR).

[0057] In an optional embodiment, the method for constructing a retinitis pigmentosa model comprises administering a 5 mM disulfiram-copper ion complex to the subretinal of a mouse.

[0058] In an alternative embodiment, the subretinal administration of the disulfiram-copper ion complex to the subject comprises subretinal injection of the disulfiram-copper ion complex. Subretinal injection is a local administration method that can maintain the drug concentration in the retina at an effective level while keeping the systemic circulation concentration below the safety threshold, avoiding liver and kidney damage in animal models, and can avoid systemic toxicity in animal models.

[0059] In an alternative embodiment, the retinal pigment degeneration model is an in vitro model, and the subject is an organ, tissue or cell from a mouse; and the method of construction comprises contacting the subject with the disulfiram-copper ion complex.

[0060] In an alternative embodiment, the subject is a mouse retinal photoreceptor cell, and the method of construction comprises administering 5 μM of the disulfiram-copper ion complex to the mouse retinal photoreceptor cell.

[0061] In an alternative embodiment, the method of construction comprises treating the retinal photoreceptor cell with 5 μM of the disulfiram-copper ion complex for 12-36 h, for example, but not limited to, 12, 15, 20, 24, 30 or 36 h, and further alternatively, 24 h.

[0062] In an alternative embodiment, the retinal photoreceptor cell is a mouse 661w retinal photoreceptor cell.

[0063] In an alternative embodiment, in the method of construction, the disulfiram-copper ion complex is prepared by mixing a disulfiram solution and a salt solution providing copper ions, and vortexing to form the disulfiram-copper ion complex.

[0064] In an alternative embodiment, the vortexing is performed for 5-15 min, for example, but not limited to, 5, 10 or 15 min.

[0065] In an alternative embodiment, the disulfiram solution and the salt solution providing copper ions are mixed in an equimolar ratio, based on the molar concentration of disulfiram in the disulfiram solution and the molar concentration of copper ions in the salt solution providing copper ions.

[0066] In an alternative embodiment, the solvent of the disulfiram solution is PBS.

[0067] In an alternative embodiment, the molar concentration of disulfiram in the disulfiram solution is 10 mM.

[0068] In an alternative embodiment, the molar concentration of copper ions in the salt solution providing copper ions is 10 mM.

[0069] In an alternative embodiment, the salt solution providing copper ions is a copper sulfate solution.

[0070] In a second aspect, there is provided a kit for constructing an in vitro model of retinitis pigmentosa, the kit comprising (i) and (ii);

[0071] (i) an organ, tissue or cell;

[0072] (ii) a disulfiram-copper ion complex.

[0073] In an optional embodiment, the disulfiram-copper ion complex is prepared by the method of preparing a disulfiram-copper ion complex as described in the first aspect.

[0074] Alternatively, the kit for constructing an in vitro model of retinitis pigmentosa comprises (i) and (iii):

[0075] (i) an organ, tissue or cell;

[0076] (iii) disulfiram and a salt that provides copper ions.

[0077] In an optional embodiment, the kit comprises a disulfiram solution, and further optionally, the solvent of the disulfiram solution is PBS.

[0078] In an optional embodiment, the salt that provides copper ions comprises copper sulfate.

[0079] In a third aspect, there is provided use of the retinitis pigmentosa model of the first aspect, or the kit of the second aspect, in any of (X1) to (X6):

[0080] (X1) studying the pathogenesis of retinitis pigmentosa;

[0081] (X2) preparing a product for studying the pathogenesis of retinitis pigmentosa;

[0082] (X3) studying the molecular mechanism of retinitis pigmentosa;

[0083] (X4) preparing a product for studying the molecular mechanism of retinitis pigmentosa;

[0084] (X5) screening for a drug for treating retinitis pigmentosa;

[0085] (X6) preparing a product for screening for a drug for treating retinitis pigmentosa.

[0086] It is noted that the uses of (X1) to (X6) are for non-diagnostic and non-therapeutic purposes.

[0087] The application is further illustrated by the following specific examples, but it should be understood that these examples are only used to illustrate in more detail, and should not be understood as limiting the application in any form.

[0088] Example 1

[0089] Establishment of a mouse model of retinal degeneration

[0090] I. Preparation of disulfiram-copper ion complex:

[0091] 1. Dissolve disulfiram (DSF) in PBS solution (final concentration 10 mM) and store in the dark.

[0092] 2. Prepare a copper sulfate (CuSO4) solution (10 mM, pH 7.4) and mix with DSF at a 1:1 molar ratio, vortex for 10 minutes to form a DSF-Cu2+ complex (final concentration 5 mM).

[0093] II. Preparation method of mouse model:

[0094] 1. Subretinal injection procedure:

[0095] 1) Animal selection: Wild-type C57BL / 6J mice (4 weeks old) were bred in SPF conditions and purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0096] 2) Anesthetize the mice with isoflurane inhalation (5% induction, 1.5% maintenance) and perform local anesthesia by applying 0.5% propamocaine hydrochloride eye drops.

[0097] 3) Microinjection: Under a surgical microscope, use a 30G needle to puncture the corneal limbus into the subretinal space. Slowly inject the DSF-Cu2+ solution using a Hamilton microsyringe (1 μL capacity, 0.1 μL precision) at a speed of about 0.2 μL / s. The control group was injected with an equal volume of PBS.

[0098] Example 2

[0099] I. Postoperative detection of the retinal degeneration mouse model prepared in Example 1, in vivo layer evaluation of the effect of disulfiram-copper ion complex on inducing mouse photoreceptor degeneration:

[0100] 1. Short-term observation: Monitor for vitreous hemorrhage and retinal detachment within 24 hours after surgery. No vitreous hemorrhage was found by fundus photography at 24 hours after surgery, and no retinal detachment was found by OCT examination.

[0101] 2. Long-term evaluation: Measure the outer nuclear layer (ONL) thickness of the retina at 14 days after surgery to assess photoreceptor survival. Calculate the photoreceptor death rate by retinal sectioning and HE staining. Detect the expression level of visual cycle proteins by Western blotting.

[0102] 1) Disulfiram-copper ion complex causes photoreceptor layer atrophy:

[0103] Four-week-old C57 wild-type mice were injected subretinally with a disulfiram-copper ion complex. OCT examination of the mice 14 days later revealed a significant thinning of the photoreceptor layer in the experimental group. Figure 1 Subsequently, mouse eyeballs were taken for HE staining to further confirm that the thickness of the photoreceptor cell layer in the experimental group mice was significantly thinner. Figure 2 The thickness of the retina decreased from 65 μM ± 2.3 μM to 23 μM ± 1.8 μM. No significant changes were observed in other layers of the retina.

[0104] 2) The disulfiram-copper ion complex leads to a decrease in the level of visual signaling proteins.

[0105] Retinal tissue from mice 14 days after injection was collected, and the expression levels of visual conduction proteins were detected by Western blotting. The results showed that the levels of PDE6B and RHO proteins were significantly downregulated. Figure 3 These two proteins are specifically expressed in photoreceptor cells, and mutations in these two proteins are key factors leading to retinitis pigmentosa.

[0106] Example 3

[0107] Mice were administered N-acetylcysteine ​​(120 mg / kg body weight), a commonly used clinical treatment for retinitis pigmentosa, via intraperitoneal injection. The first injection of N-acetylcysteine ​​was administered in conjunction with DSF-Cu. 2+ Simultaneously, the complex was administered with injections every other day after the initial injection, for a total of 7 injections. Receptor cell viability was assessed by measuring the outer nuclear layer (ONL) thickness using OCT. Receptor cell mortality was statistically analyzed using retinal sections and HE staining.

[0108] N-acetylcysteine ​​is a commonly used drug in clinical treatment of retinitis pigmentosa. In this mouse model, intraperitoneal injection of N-acetylcysteine ​​showed that the atrophy of the photoreceptor layer was significantly alleviated in the treatment group. Figure 4 HE results also yielded the same conclusion. Figure 5 The thickness of the photoreceptor layer increased from 23 μM ± 1.8 μM to 45 μM ± 2.2 μM. The experimental results indicate that N-acetylcysteine, a commonly used treatment for retinitis pigmentosa, can alleviate photoreceptor layer atrophy in the model mice, and the model mice prepared in Example 1 were able to produce typical symptoms of retinitis pigmentosa.

[0109] Example 4

[0110] I. Construction of an in vitro model of the disulfiram-copper ion complex:

[0111] 1. Cell line 661w (mouse 661w retinal photoreceptor cells) was cultured with medium containing disulfiram-copper ion complex (5 μM, prepared according to the method of Example 1) for 24 h, and PBS was used as negative control.

[0112] 2. Cell viability was detected by CCK8 assay. Cell viability was detected by CCK8 assay at time points of 12 h, 24 h and 48 h after the addition of DSF-Cu2+ complex (0 h). 2+

[0113] 3. Expression levels of visual cycle proteins were detected by immunoblotting.

[0114] II. Experimental results:

[0115] Disulfiram-copper ion complex significantly inhibited the growth of mouse photoreceptor-like cell line: the mouse photoreceptor-like cell line 661w was treated with disulfiram-copper ion complex, and the results showed that disulfiram-copper ion complex significantly inhibited the growth of 661w cells (Fig. 2). Figure 6 At the same time, the results of immunoblotting also showed that disulfiram-copper ion complex down-regulated the expression of PDE6B and RHO in cells (Fig. 3). Figure 7

[0116] Example 5

[0117] 661w cell line was treated with disulfiram-copper ion complex (5 μM, prepared according to the method of Example 1) for 24 h; at the same time, 661w cell line was treated with disulfiram-copper ion complex (5 μM, prepared according to the method of Example 1) and N-acetylcysteine (10 μM) for 24 h, and cell viability was detected by CCK8 assay. The results showed that N-acetylcysteine significantly reversed the cell growth inhibition caused by disulfiram-copper ion, and promoted cell survival (Fig. 4). Figure 8 The experimental results showed that the constructed cell model could produce typical symptoms of retinitis pigmentosa.

[0118] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.​​

Claims

1. A method for constructing a model of retinal pigment degeneration, characterized by, The retinal pigment degeneration model comprises an animal model or an in vitro model. The construction method of the animal model comprises subretinally administering a disulfiram-copper ion complex to a mouse. The construction method of the in vitro model comprises administering a disulfiram-copper ion complex to a mouse retinal photoreceptor cell.

2. The construction method of claim 1, wherein, The mouse is at least 4 weeks old.

3. The construction method according to claim 2, characterized in that, The subretinal administration comprises administering a 5 mM disulfiram-copper ion complex to the mouse.

4. The construction method of claim 1, wherein, The administration to the mouse retinal photoreceptor cell comprises administering a 5 μM disulfiram-copper ion complex to the mouse retinal photoreceptor cell.

5. The construction method according to claim 4, characterized in that, The retinal photoreceptor cell is a mouse 661w retinal photoreceptor cell.

6. The construction method of claim 5, wherein, The treatment time is 12-36 h.

7. The construction method of claim 6, wherein, The treatment time is 24 h.

8. The construction method according to any one of claims 1 to 7, characterized in that, The disulfiram-copper ion complex is prepared by the following method: The disulfiram solution and the salt solution providing copper ions are mixed, and a disulfiram-copper ion complex is formed after vortex shaking.

9. The construction method of claim 8, wherein, The vortex shaking is performed for 5-15 min.

10. The construction method of claim 8, wherein, The solvent of the disulfiram solution is PBS; and / or, the salt solution providing copper ions is a copper sulfate solution.

11. The construction method of claim 10, wherein, The disulfiram solution and the salt solution providing copper ions are mixed in an equimolar ratio, in terms of the molar concentration of disulfiram in the disulfiram solution and the molar concentration of copper ions in the salt solution providing copper ions.

12. The construction method of claim 11, wherein, The molar concentration of disulfiram in the disulfiram solution is 10 mM, and / or the molar concentration of copper ions in the salt solution providing copper ions is 10 mM.

13. A kit for constructing an in vitro model of retinal pigmentosa, characterized in that, The kit comprises (i) and (ii); or the kit comprises (i) and (iii): (i) a mouse 661w retinal photoreceptor cell; (ii) a disulfiram-copper ion complex; (iii) disulfiram and a salt providing copper ions.

14. Use of the retinal pigment degeneration model obtained by the construction method of any one of claims 1-12 or the kit of claim 13 in any one of (X1)-(X6): (X1) studying the pathogenesis of retinal pigment degeneration; (X2) preparing a product for studying the pathogenesis of retinal pigment degeneration; (X3) studying the molecular mechanism of retinal pigment degeneration; (X4) preparing a product for studying the molecular mechanism of retinal pigment degeneration; (X5) screening a drug for treating a retinal pigment degeneration disease; (X6) preparing a product for screening a drug for treating a retinal pigment degeneration disease.

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