Method for establishing an outer retinal tubular structure mouse model

Abstract

This invention discloses a method for establishing a mouse model of outer retinal tubular structures, which is obtained by specifically knocking out the Lss gene in the mouse retina. This invention utilizes CRISPR / Cas9 gene editing technology to construct a mouse model of outer retinal tubular structures by specifically knocking out the Lss gene in the retina. The mouse model of outer retinal tubular structures constructed by this method is similar to the clinical phenotype of this type of retinal disease, providing a relatively ideal experimental animal model for studying the developmental regulation of the mammalian retina and the pathogenesis of retinal degeneration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for establishing a mouse model of the outer retinal tubular structure. Background Technology

[0002] Outer retinal tubulation (ORT) is a pathological change occurring in the outer retina. Spectrum domain-optic coherence topography (SD-OCT) reveals it as round or near-round, highly reflective tubular structures in the outer nuclear layer. It can be seen in various retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, crystalline retinal degeneration, Best's disease, Stargardt's disease, cone cell dystrophy, and acute regional occult outer retinal disease.

[0003] The formation mechanism of ORT is not yet fully understood. Using ideal experimental animal models is crucial for studying the formation mechanism of ORT and exploring possible treatment options. However, there is currently a lack of relevant experimental animal models. Summary of the Invention

[0004] This invention addresses the fundamental research on the tubular structure of the outer retina and provides a method for establishing a mouse model of this type of retinal disease. The mouse model of the tubular structure of the outer retina constructed by this method has a similar phenotype to that of this type of retinal disease in clinical practice, providing a relatively ideal experimental animal model for studying the developmental regulation of the mammalian retina and the pathogenesis of retinal degeneration.

[0005] This invention utilizes CRISPR / Cas9 gene editing technology to construct an ORT mouse model by specifically knocking out the Lss gene in the retina. The specific method includes the following steps:

[0006] S1. Using CRISPR / Cas9 gene editing technology, the LoxP site was inserted into the Lss gene of C57BL / 6J wild-type mice to obtain Lss. flox / flox Mice;

[0007] S2, Lss flox / flox Lss was obtained by crossing mice with commercial Six3-Cre mice. flox / + Six3-Cre mice;

[0008] S3, Lss flox / + Six3-Cre mice were self-crossed to obtain Lss mice, which contained the target gene. flox / flox Six3-Cre mice.

[0009] Preferably, step S1 specifically involves inserting a LoxP site in the same direction upstream of the third exon region and downstream of the sixth exon region of the Lss gene in C57BL / 6J wild-type mice using CRISPR / Cas9 gene editing technology, thereby obtaining Lss. flox / + Mice, Lss flox / + Lss was obtained by further self-crossing of mice. flox / flox Mice.

[0010] On the other hand, the present invention also provides the application of the above-described method in the field of animal model construction.

[0011] On the other hand, the present invention also provides the application of the mouse model of the outer retinal tubular structure obtained by the above-described method in the study of the regulatory mechanism of retinal development and the pathogenesis of retinal degeneration.

[0012] On the other hand, the present invention also provides the application of the mouse model of outer retinal tubular structure obtained by the above-described method in studying the molecular mechanism of Lss regulation of retinal development and the mechanism of occurrence of outer retinal tubular structure.

[0013] On the other hand, the present invention also provides the application of the mouse model of the outer retinal tubular structure obtained by the above-described method in drug screening and / or treatment efficacy evaluation.

[0014] On the other hand, the present invention also provides a method for drug screening and / or evaluation of therapeutic effects, which includes the step of administering a test candidate drug to a mouse model of the outer retinal tubular structure obtained according to the above-described establishment method.

[0015] On the other hand, the present invention also provides the application of the Lss gene in constructing a mouse model of the outer retinal tubular structure, which is obtained by knocking out the Lss gene in the mouse retina.

[0016] The present invention has the following beneficial effects:

[0017] 1. This invention constructs a mouse model of the outer retinal tubular structure, which can effectively simulate the phenotype of this type of retinal disease in clinical practice.

[0018] 2. This model can be used to explore the molecular mechanism by which Lss regulates retinal development and the mechanism of the formation of tubular structures in the outer retina.

[0019] 3. This model can be used to screen drugs and evaluate their efficacy.

[0020] 4. This model can be used to study the regulatory mechanisms of mammalian retinal development and the pathogenesis of retinal degeneration, and to provide potential therapeutic targets for such retinal diseases. Attached Figure Description

[0021] Figure 1 For Lss flox / flox Six3-Cre mouse construction protocol.

[0022] Figure 2 The results are for mouse genotype identification.

[0023] Figure 3 Lss staining for hematoxylin flox / flox Tubular structures in the outer retina of Six3-Cre mice.

[0024] Figure 4 For Lss flox / flox Six3-Cre mice exhibit visual dysfunction. Detailed Implementation

[0025] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0026] The construction scheme of the ORT mouse model of the present invention is as follows: Figure 1 As shown.

[0027] Example 1: Construction of a mouse model of outer retinal tubular structure

[0028] A method for establishing a mouse model of the outer retinal tubular structure includes the following steps:

[0029] (1) Using CRISPR / Cas9 gene editing technology, a LoxP site was inserted upstream of the third exon region and downstream of the sixth exon region of the Lss gene (GeneID: 16987) in C57BL / 6J wild-type (WT) mice to obtain Lss. flox / + Mice, Lss flox / + Lss was obtained by further self-crossing of mice. flox / flox Mice.

[0030] (2) Lss flox / flox Lss was obtained by crossing mice with commercial Six3-Cre mice. flox / + Six3-Cre mice.

[0031] (3) Lss flox / + Six3-Cre mice were self-crossed to obtain Lss mice, which contained the target gene. flox / flox Six3-Cre mice.

[0032] Example 2: Genotyping of a Mouse Model

[0033] 1. DNA extraction from rat tail tissue:

[0034] 1) Take out and restrain the mice to be identified, number the mice by clipping their toes, cut off 0.5cm of the mouse tail that has been thoroughly wiped with alcohol, put it into a 1.5mL sterile EP tube, and label the mouse number and the breeding cage number.

[0035] 2) Place the EP tube containing the rat tail into a centrifuge and centrifuge (room temperature, 1000g, 1min) to transfer the rat tail tissue to the bottom of the EP tube.

[0036] 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 is complete, centrifuge briefly at low speed to collect the liquid on the tube cap and tube wall at the bottom of the tube.

[0037] 4) Add 10 μL of 1M Tris-HCl equilibration solution (pH=8.0) to each tube, centrifuge (room temperature, 1000g, 1min), then add 100 μL of ddH2O and centrifuge briefly at low speed. This is the extracted mouse DNA.

[0038] 2. Mouse genotyping:

[0039] 1) The PCR reaction system is as follows:

[0040]

[0041] 2) The PCR reaction procedure is as follows:

[0042]

[0043]

[0044] 3) Agarose gel electrophoresis:

[0045] After the PCR reaction is complete, weigh 2g of agarose powder into a 250mL Erlenmeyer flask, add 100mL of 1×TAE, seal the flask opening with aluminum foil, and heat in a microwave oven until boiling. Gently shake and reheat until the gel solution is homogeneous. After slightly cooling, 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 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 5μL of PCR product and add it to the well of the agarose gel comb. Electrophoresis is performed at 150V for 30 minutes. The agarose gel is then removed to check for the presence of the target band in the PCR product.

[0046] 4) The primer sequences for mouse genotyping are as follows:

[0047]

[0048] 5) See the genotype identification results. Figure 2 .

[0049] like Figure 2 As shown, in the PCR reaction system of the Lss gene, a single band of 270 bp was observed in WT(C57BL / 6J) mice. flox / flox Mice and Lss flox / flox A single band of 373 bp was observed in Six3-Cre mice, Lss flox / + Double bands of 270bp and 373bp were observed in Six3-Cre mice; in the PCR reaction system of the Six3-Cre gene, WT(C57BL / 6J) mice and Lss mice showed... flox / flox No bands were observed in mice, Lss flox / + Six3-Cre mice and Lss flox / flox A single band of 480 bp was observed in Six3-Cre mice.

[0050] Example 3: Hematoxylin staining of mouse retinal tissue

[0051] 1. Dewaxing: Paraffin sections stored at room temperature were placed in a 60°C oven for 30 minutes, and then placed in xylene to remove the paraffin from the sections. The sections were immersed in xylene (I, II, III) three times, 10 minutes each time.

[0052] 2. Hydration (rehydration): The dewaxed sections were rehydrated in an alcohol gradient, in 100% alcohol (Ⅰ) for 5 min, 100% alcohol (Ⅱ) for 5 min, 90% alcohol for 3 min, 70% alcohol for 3 min, 30% alcohol for 3 min, and finally rinsed in tap water for 1 min.

[0053] 3. Hematoxylin staining: After rehydration, place the sections in hematoxylin staining solution for 2-3 minutes, then soak in pure water for 1 minute, and then differentiate with 1% hydrochloric acid ethanol for 3 seconds to make the staining of the cell nucleus and cytoplasm clearer. After that, rinse slowly with running water for 10 minutes.

[0054] 4. Dehydration: The stained sections are then dehydrated and then immersed in 95% alcohol (I) for 30 seconds, 95% alcohol (II) for 30 seconds, 100% alcohol (I) for 1 minute, and 100% alcohol (II) for 1 minute.

[0055] 5. Clearing: The dehydrated sections were cleared with xylene by immersing them in xylene three times (I, II, III), each time for 10 minutes.

[0056] 6. Mounting: Lay the transparent section flat, gently wipe away any remaining liquid with a cotton swab, add a drop of neutral resin to the section, and then cover with a coverslip to seal (avoid air bubbles). Let it sit at room temperature for about half an hour until the resin dries slightly before storing or photographing.

[0057] like Figure 3 As shown, Lss flox / flox In Six3-Cre mice, multiple round or near-round tubular structures of varying diameters are formed in the outer nuclear layer of the retina. In the Control group mice, Lss... flox / flox Mice.

[0058] Example 4: Visual function examination in mice

[0059] Electroretinography (ERG) of mice was recorded using the Diagnosys Celeris rodent ERG device. All mice to be subjected to electrophysiological testing underwent overnight dark acclimatization, as follows:

[0060] 1. Anesthesia: Anesthesia was administered via intraperitoneal injection of 1% sodium pentobarbital (dose: 70 mg / kg).

[0061] 2. Mydriasis and topical anesthesia: After the mice are anesthetized for about 3-5 minutes, compound tropicamide eye drops (mydriatic) and alcaine eye drops (topical anesthetic) are instilled into both eyes to dilate the pupils and perform topical anesthesia.

[0062] 3. After complete pupil dilation (approximately 5 minutes), place the mouse on the Diagnosys Celeris instrument's warming platform (maintaining the mouse's body temperature at 37°C). Instill a small amount of physiological saline onto the ocular surface to keep it moist.

[0063] 4. During the ERG experiment, the entire process was conducted in the dark. First, a scotopic dark adaptation test was performed on the mice. In the dark adaptation program, the stimulus was white light, with an intensity of 0.01 cd·s / m². 2 0.03 cd·s / m 2 0.1 cd·s / m 2 0.3 cd·s / m 2 1 cd·s / m 2 3cd·s / m 2 10 cd·s / m 2 The electroretinogram (e.g., a wave and b wave) of mice was recorded.

[0064] 5. Mice that had completed dark adaptation ERG underwent light adaptation for five minutes. Five minutes later, the light adaptation ERG results were assessed using light stimulation at an intensity of 0.3 cd·s / m². 2 1 cd·s / m 2 3cd·s / m2 10 cd·s / m 2 30 cd·s / m 2 The electroretinogram (e.g., a wave and b wave) of mice was recorded.

[0065] like Figure 4 As shown, Lss flox / flox The electrophysiological results of Six3-Cre mice were abnormal. Compared with control mice (Lss... flox / flox Compared to mice, Lss flox / flox In Six3-Cre mice, the amplitudes of both a-wave and b-wave decreased during dark adaptation. Figure 4 (A and B in the text). Compared with control mice (Lss) flox / flox Compared to mice, Lss flox / flox Six3-Cre mice showed a decrease in both the amplitude of the a and b waves due to adaptive adaptation. Figure 4 (C and D in the text).

[0066] 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 a mouse model of the outer retinal tubular structure, characterized in that, By knocking out the mouse retina Lss Gene acquisition includes the following steps: S1. Gene editing in C57BL / 6J wild-type mice using CRISPR / Cas9 gene editing technology. Lss One LoxP site in the same direction was inserted upstream of the third exon region and downstream of the sixth exon region of the gene, resulting in Lss. flox / + Mice, Lss flox / + Lss was obtained by further self-crossing of mice. flox / flox Mice; S2, Lss flox / flox Lss was obtained by crossing mice with commercial Six3-Cre mice. flox / + Six3-Cre mice; S3, Lss flox / + Six3-Cre mice were self-crossed to obtain Lss mice, which contained the target gene. flox / flox Six3-Cre mice.

2. The application of the method described in claim 1 in the field of animal model construction.

3. The application of the mouse model of the outer retinal tubular structure obtained by the method described in claim 1 in the study of the regulatory mechanism of retinal development and the pathogenesis of retinal degeneration.

4. The mouse model of the outer retinal tubular structure obtained by the method described in claim 1 is used in research... Lss Applications of molecular mechanisms regulating retinal development and the formation mechanism of tubular structures in the outer retina.

5. The application of the mouse model of outer retinal tubular structure obtained by the method described in claim 1 in drug screening.

6. A method for drug screening, characterized in that, The method includes the step of administering a test candidate drug to a mouse model of the outer retinal tubular structure obtained by the method described in claim 1.

7. Lss The application of genes in constructing a mouse model of the outer retinal tubular structure is characterized by, By knocking out the mouse retina Lss A mouse model of the outer retinal tubular structure obtained through gene therapy includes the following steps: S1. Gene editing in C57BL / 6J wild-type mice using CRISPR / Cas9 gene editing technology. Lss One LoxP site in the same direction was inserted upstream of the third exon region and downstream of the sixth exon region of the gene, resulting in Lss. flox / + Mice, Lss flox / + Lss was obtained by further self-crossing of mice. flox / flox Mice; S2, Lss flox / flox Lss was obtained by crossing mice with commercial Six3-Cre mice. flox / + Six3-Cre mice; S3, Lss flox / + Six3-Cre mice were self-crossed to obtain Lss mice, which contained the target gene. flox / flox Six3-Cre mice.

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