Method for establishing concurrent cataract mouse model

Abstract

The invention discloses a method for establishing a concurrent cataract mouse model. The mouse model is obtained by specifically knocking out a Phb2 gene from the retina. The method comprises the following steps: constructing Phb2flox / flox; six3-Cre mice have phenotypes of degenerative retinopathy at 6-8 weeks old, retinal atrophy and thinning, structural disorder of each layer and disorder distribution of retinal pigment can be seen in fundus examination, and visual function abnormality of the mice is shown in electrophysiological examination. The 8-10-month-old mouse has lens opacity and shows the phenotype of concurrent cataract. Therefore, the model mouse can better simulate the phenotype of the clinical concurrent cataract, and can be used as an animal model for related research of the concurrent cataract.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for establishing a mouse model of complicated cataract. Background Art

[0002] Complicated cataract is a cataract caused by ocular diseases. For example, corneal ulcer, glaucoma, uveitis, retinal detachment, retinitis pigmentosa, intraocular tumor, high myopia, etc. can all cause cataract. Its pathogenesis is not fully understood, and the etiology is complex. Inflammation, degenerative changes, metabolic disorders, etc. can lead to denaturation of lens proteins or nutritional disorders, thus causing vision loss and even blindness in severe cases. There is no effective preventive method for this disease clinically, and there is currently a lack of specific drugs for the treatment of complicated cataract. Therefore, it is particularly important to study its pathogenesis and develop corresponding treatment plans.

[0003] Establishing an ideal experimental animal model is crucial for studying the pathogenesis of such diseases and exploring possible treatment plans. Currently, researchers have tried to construct an animal model of complicated cataract through surgery. For example, an animal model of complicated cataract is established by performing vitrectomy on rabbit eyes combined with silicone oil or C3F8 gas filling in the vitreous cavity. This model has the following disadvantages: 1) Complex operation: Microsurgical techniques are required, and the success rate is greatly affected by the experience of the operator; 2) Pathological deviation: Mechanical trauma and the toxicity of the filler may mask the biological effects of the primary disease; 3) Narrow applicability: It is difficult to simulate chronic inflammatory processes such as uveitis or cataracts related to metabolic diseases.

[0004] Existing animal models of complicated cataract have deficiencies in etiology simulation, repeatability, and clinical relevance. Therefore, there is a need to provide an animal model that can better simulate the phenotype of complicated cataract clinically and can be used for related research on complicated cataract. Summary of the Invention

[0005] The object of the present invention is to provide a method for establishing a mouse model of complicated cataract, which can simulate the pathogenesis process and pathological characteristics of human complicated cataract to a certain extent, and provide an experimental basis for studying the pathogenesis and treatment methods of complicated cataract. With the help of this mouse model, researchers can better explore the etiology, pathophysiological process of complicated cataract and explore new treatment strategies, thus providing a theoretical basis and experimental support for clinical treatment.

[0006] The object of the present invention is achieved through the following technical solutions:

[0007] A method for establishing a mouse model of complicated cataract is obtained by specifically knocking out the Phb2 gene (GeneID: 11331) in the retina, and specifically includes the following steps:

[0008] S1. Using the CRISPR / Cas9 gene editing technology to insert Loxp sites into the Phb2 gene of C57BL / 6J wild-type mice to construct Phb2 flox / flox mice;

[0009] S2. Crossing the Phb2 flox / flox mice with commercially available Six3-Cre mice to obtain Phb2 flox / + ; Six3-Cre mice;

[0010] S3. Self-crossing the Phb2 flox / + ; Six3-Cre mice to obtain the target gene mice Phb2 flox / flox ; Six3-Cre mice.

[0011] Preferably, step S1 is specifically: inserting a LoxP site in the same direction at both the upstream and downstream ends of the fourth exon region of the Phb2 gene of C57BL / 6J wild-type mice through the CRISPR / Cas9 gene editing technology to obtain Phb2 flox / + mice, and further self-crossing the Phb2 flox / + mice to obtain Phb2 flox / flox mice.

[0012] On the other hand, the present invention also provides the application of the above-mentioned establishment method in the field of constructing animal models. Preferably, the animal model is a mouse model of complicated cataract.

[0013] On the other hand, the present invention also provides the application of the mouse model of complicated cataract obtained by the above-mentioned establishment method in studying the pathogenesis of complicated cataract in mammals.

[0014] On the other hand, the present invention also provides the application of the mouse model of complicated cataract obtained by the above-mentioned establishment method in screening drugs for preventing or treating complicated cataract.

[0015] On the other hand, the present invention also provides a method for screening candidate drugs for preventing or treating complicated cataract, including the step of administering a test candidate drug to a mouse model of complicated cataract obtained by the above-mentioned establishment method.

[0016] On the other hand, the present invention also provides the application of the Phb2 gene in constructing a mouse model of complicated cataract, which is obtained by specifically knocking out the Phb2 gene in the mouse retina.

[0017] By means of the CRISPR / Cas9 gene editing technology, through specifically knocking out the Phb2 gene in the retina, our team invented a new model of complicated cataract mice (Phb2 flox / flox ; Six3-Cre mice).

[0018] The complicated cataract mice model shows the phenotype of degenerative retinopathy at 6 - 8 weeks of age. Fundus examination reveals that the retina is atrophied and thinned, the structures of each layer are disordered, and the distribution of retinal pigment is disordered. Electrophysiological examination shows abnormal visual function in these mice. At 8 - 10 months of age, these mice develop lens opacity, showing the phenotype of complicated cataract.

[0019] In summary, Phb2 flox / flox ; Six3-Cre mice can better simulate the phenotype of complicated cataract clinically and can be used as an animal model for related research on complicated cataract.

[0020] The present invention has the following beneficial effects:

[0021] For the basic research on complicated cataract, the present invention provides a method for establishing a mice model of this kind of disease. The complicated cataract mice model constructed by this method is similar to the clinical phenotype of this kind of disease, providing an ideal experimental animal model for exploring the pathogenic mechanism of Phb2 in complicated cataract, studying the pathogenesis of mammalian complicated cataract, and screening drugs for preventing or treating complicated cataract. Brief Description of the Drawings

[0022] Figure 1 It is the construction scheme of Phb2 flox / flox ; Six3-Cre mice.

[0023] Figure 2 It is the result of genotype identification of mice.

[0024] Figure 3 It is that the Phb2 flox / flox ; Six3-Cre mice at 6 - 8 weeks of age develop degenerative retinopathy.

[0025] Figure 4 It is that the Phb2 flox / flox ; Six3-Cre mice at 6 - 8 weeks of age have abnormal visual function.

[0026] Figure 5 It is that the Phb2 flox / flox ; Six3-Cre mice at 8 - 10 months of age develop complicated cataract.

[0027] Figure 6 It is that the Phb2 flox / flox ; Six3-Cre mice at 8 - 10 months of age have lens opacity. DETAILED DESCRIPTION

[0028] In order to make the technical problem and technical solution to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] Phb2 of the present invention flox / flox ; The construction scheme of Six3-Cre mice is as follows Figure 1 shown.

[0030] Example 1 Phb2 flox / flox ; Construction of Six3-Cre mouse model

[0031] (1) CRISPR / Cas9 gene editing technology was used to insert a LoxP site in the same direction at both ends of the upstream and downstream of the fourth exon region of the Phb2 gene in wild-type (WT) mice with a C57BL / 6J background to obtain Phb2 flox / + Mouse, Phb2 flox / + The mice were further selfed to obtain Phb2 flox / flox Mice;

[0032] (2) Phb2 flox / flox Phb2 was obtained by crossing mice with commercial Six3-Cre mice. flox / + ;Six3-Cre mice;

[0033] (3) Phb2 flox / + ; Six3-Cre mice were self-bred to obtain the target gene mouse Phb2 flox / flox ; Six3-Cre mice.

[0034] Example 2 Mouse genotype identification

[0035] 1. DNA extraction from mouse tail tissue:

[0036] 1) Take out and restrain the mice to be identified, number them by clipping their toes, cut 0.5 cm of the mouse tail that has been wiped thoroughly with alcohol, put it in a 1.5 mL sterilized EP tube, and mark the mouse number and breeding cage number;

[0037] 2) Place the EP tube containing the mouse tail into a centrifuge and centrifuge (room temperature, 1000 g, 1 min) to centrifuge the mouse tail tissue to the bottom of the EP tube;

[0038] 3) Add 100 μL of 50 mM sodium hydroxide aqueous solution to each EP tube, and after the lysed tissue is suspended, lyse in a 95°C metal bath for 10 minutes. After lysis, centrifuge at low speed to collect the liquid on the tube cap and tube wall at the bottom of the tube;

[0039] 4) Add 10 μL of 1 M Tris-HCl equilibration solution (pH = 8.0) to each tube, centrifuge (at room temperature, 1000 g, for 1 min), and then add 100 μL of ddH2O and centrifuge briefly at low speed. The DNA extracted from the mouse is obtained.

[0040] 2. Genotyping of mice:

[0041] 1) The PCR reaction system is as follows:

[0042]

[0043] 2) The PCR reaction program is as follows:

[0044]

[0045] 3) Agarose gel electrophoresis:

[0046] After the PCR reaction is completed, weigh 2 g of agarose powder into a 250 mL conical flask, pour in 100 mL of 1X TAE, seal the conical flask mouth with aluminum foil, heat it in a microwave oven until boiling, gently shake it and then heat it again until the colloidal solution is uniform. After it cools slightly, add GelStain solution at a ratio of 1:10000, gently shake and mix well, and then pour it into the gel casting tank with the comb inserted, taking care not to have bubbles. Let it stand at room temperature for 30 minutes. After the agarose gel has completely solidified, pull out the comb, and place the gel into the electrophoresis tank filled with fresh TAE. Take 5 μL of the PCR product and add it into the well of the agarose gel. Electrophorese for 30 minutes at a voltage of 150 V, and take out the agarose gel to detect whether there is a target band in the PCR product.

[0047] 4) The primer sequences for mouse genotyping are as follows:

[0048]

[0049] 5) The genotyping results are shown in Figure 2 .

[0050] As Figure 2 shown, in the PCR reaction system of the Phb2 gene, a single band of 163 bp can be seen in WT (C57BL / 6J) mice, Phb2 flox / flox mice and Phb2 flox / flox ; a single band of 252 bp can be seen in Six3-Cre mice, Phb2 flox / + mice and Phb2 flox / + ; a double band of 163 bp and 252 bp can be seen in Six3-Cre mice; in the PCR reaction system of the Six3-Cre gene, WT (C57BL / 6J) mice, Phb2 flox / +Mouse and Phb2 flox / flox No band in the mouse, Phb2 flox / + ; Six3-Cre mouse and Phb2 flox / flox ; A single band of 480 bp was visible in the Six3-Cre mouse.

[0051] Detection of relevant indicators of mice in Example 3

[0052] Respectively for Phb2 flox / flox mice, Phb2 flox / flox ; The Six3-Cre mice were examined for visual function and other indicators at 6-8 weeks of age and 8-10 months of age. The specific methods are as follows:

[0053] 1. Slit lamp examination

[0054] The examination was performed using a slit lamp digital imaging system (Topcon) to examine the anterior segment of the mouse eye. The steps are as follows:

[0055] (1) Anesthesia: Intraperitoneal injection of 1% sodium pentobarbital for anesthesia (dose: 70 mg / kg);

[0056] (2) Mydriasis and surface anesthesia: After the mouse was anesthetized for about 3-5 minutes, compound tropicamide eye drops (mydriatic) and Alcaine eye drops (surface anesthetic) were instilled into both eyes for mydriasis and surface anesthesia;

[0057] (3) After complete mydriasis (about 5 minutes), place the mouse on the mouse stand and adjust the body position;

[0058] (4) Open the software, adjust the angle of the slit lamp, observe and collect images of the anterior segment of the eye under a 40x microscope. During the collection process, instill 0.9% normal saline on the eye surface to keep the eye surface of the mouse moist;

[0059] (5) Save the image and export it.

[0060] 2. Fundus examination

[0061] The examination was performed using the fundus photography module of a small animal retinal imaging system (Phoenix) to detect the retina of the mouse. The steps are as follows:

[0062] (1) Anesthesia: Intraperitoneal injection of 1% sodium pentobarbital for anesthesia (dose: 70 mg / kg);

[0063] (2) Mydriasis and surface anesthesia: After the mouse was anesthetized for about 3-5 minutes, compound tropicamide eye drops (mydriatic) and Alcaine eye drops (surface anesthetic) were instilled into both eyes for mydriasis and surface anesthesia;

[0064] (3) After complete mydriasis (about 5 minutes), place the mouse on the mouse stand of the small animal retinal imaging system and expose the eyeball;

[0065] (4) Open the fundus photography software, collect retinal images, and instill 0.9% normal saline on the ocular surface to keep the ocular surface of the mouse moist.

[0066] (5) Save the images and export them.

[0067] 3. Retinal optical coherence tomography (OCT) examination

[0068] The examination is performed using the OCT module of the small animal retinal imaging system (Phoenix) to detect the retina of the mouse. The steps are as follows:

[0069] (1) Anesthesia: Intraperitoneal injection of 1% sodium pentobarbital for anesthesia (dose: 70 mg / kg);

[0070] (2) Mydriasis and topical anesthesia: After the mouse is anesthetized for about 3 - 5 minutes, instill compound tropicamide eye drops (mydriatic) and proparacaine eye drops (topical anesthetic) into both eyes for mydriasis and topical anesthesia;

[0071] (3) After complete mydriasis (about 5 minutes), place the mouse on the mouse holder of the small animal retinal imaging system to expose the eyeball;

[0072] (4) Open the OCT scanning software and perform an overall scan of the retina. During the OCT scan, instill 0.9% normal saline on the ocular surface to keep the ocular surface of the mouse moist;

[0073] (5) Adjust the position of the mouse so that the center of the optic disc is located at the center of the scanning area;

[0074] (6) Centering on the optic disc, horizontally scan the retina of the mouse to collect images;

[0075] (7) Save the scanned images and export them.

[0076] 4. Visual function examination

[0077] The mouse electroretinogram (ERG) is recorded using the Diagnosys Celeris rodent ERG device. All mice prepared for electrophysiological detection are dark adapted overnight in advance. The specific steps are as follows:

[0078] (1) Anesthesia: Intraperitoneal injection of 1% sodium pentobarbital for anesthesia (dose: 70 mg / Kg);

[0079] (2) Mydriasis and topical anesthesia: After the mouse is anesthetized for about 3 - 5 minutes, instill compound tropicamide eye drops (mydriatic) and proparacaine eye drops (topical anesthetic) into both eyes for mydriasis and topical anesthesia;

[0080] (3) After complete mydriasis (about 5 minutes), place the mouse on the warming stage of the Diagnosys Celeris instrument (maintain the mouse body temperature at 37 °C). Drop a small amount of normal saline on the ocular surface to keep the ocular surface moist;

[0081] (4) During the ERG experiment, keep the light off throughout the process. First, perform scotopic testing on the mouse. In the scotopic program, the stimulating light is white light, and the stimulating light intensities are 0.01 cd·s / m 2 , 0.03 cd·s / m 2 , 0.1 cd·s / m 2 , 0.3 cd·s / m 2 , 1 cd·s / m 2 , and record the electroretinogram responses (a-wave and b-wave) of the mouse;

[0082] (5) The mouse that has completed the scotopic ERG is given 5 minutes of photopic adaptation. After 5 minutes, detect the photopic ERG results by light stimulation and record the electroretinogram responses of the mouse.

[0083] 5. Collect ex vivo lens images

[0084] Observe using a Zeiss SteREO Discovery.V8 stereoscopic and zoom microscope, and collect images using a Zeiss AxioCam ERc5s microscope camera. The steps are as follows:

[0085] (1) Euthanasia: Euthanize the mouse by cervical dislocation;

[0086] (2) Specimen collection: Take out the mouse lens and place it in PBS solution, and carefully separate the surrounding tissues under the stereomicroscope;

[0087] (3) Adjust the focal plane through the focusing knob and adjust the magnification to 1.0x - 2.0x through the zoom knob;

[0088] (4) Open the ZEISS ZEN software, and by adjusting the white balance and exposure time, make the image clear and free of chromatic aberration, and collect the image;

[0089] (5) Save and export the image.

[0090] As Figures 3 - 4 shown, Phb2 flox / flox ; Six3-Cre mice show a phenotype of degenerative retinopathy at 6 - 8 weeks of age. Fundus examination shows retinal atrophy and thinning, disordered structures of each layer, and disordered distribution of retinal pigment. Electrophysiological examination shows that Phb2 flox / flox ; The visual function of Six3-Cre mice is abnormal.

[0091] AsFigures 5 - 6 As shown, Phb2 flox / flox ; The Six3-Cre mice showed lens opacity at 8-10 months of age, presenting the phenotype of complicated cataract.

[0092] Therefore, this model mouse can better simulate the phenotype of complicated cataract clinically and can be used as an animal model for related research on complicated cataract.

[0093] 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. Any equivalent implementation or change without departing from the present invention should be included in the patent scope of this case.

Claims

1. A method for establishing a concurrent cataract mouse model, characterized in that, Obtained by specifically knocking out the Phb2 gene in the retina.

2. The method for establishing a concurrent cataract mouse model according to claim 1, characterized in that, Including the following steps: S1. Use the CRISPR / Cas9 gene editing technology to insert Loxp sites into the Phb2 gene of C57BL / 6J wild-type mice to construct Phb2 flox / flox mice; S2. Cross Phb2 flox / flox mice with commercially available Six3-Cre mice to obtain Phb2 flox / + ; Six3-Cre mice; S3. Transfer Phb2 flox / + ; Self-cross the Six3-Cre mice to obtain the target gene mice Phb2 flox / flox ; Six3-Cre mice.

3. The method for establishing a concurrent cataract mouse model according to claim 2, characterized in that Step S1 is as follows: using CRISPR / Cas9 gene editing technology, a LoxP site in the same direction is inserted into the upstream and downstream ends of the fourth exon region of the Phb2 gene of C57BL / 6J wild-type mice to obtain Phb2 flox / + Mouse, Phb2 flox / + The mice were further selfed to obtain Phb2 flox / flox Mouse.

2. Use of the establishment method according to any one of claims 1-3 in the field of constructing animal models.

5. The application according to claim 4, wherein The animal model is a concurrent cataract mouse model.

6. Use of the concurrent cataract mouse model obtained by the establishment method according to any one of claims 1-3 in studying the pathogenesis of mammalian concurrent cataracts.

7. Use of the concurrent cataract mouse model obtained by the establishment method according to any one of claims 1-3 in screening drugs for preventing or treating concurrent cataracts.

8. A method for screening candidate drugs for preventing or treating complicated cataract, characterized in that Including the step of administering a test candidate drug to a concurrent cataract mouse model obtained by the establishment method according to any one of claims 1-3.

9. Use of the Phb2 gene in constructing a concurrent cataract mouse model.

10. The application according to claim 9, wherein Obtained by specifically knocking out the Phb2 gene in the mouse retina.

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