A method for establishing a mouse model of congenital cryptophthalmia

Abstract

This invention discloses a method for establishing a mouse model for cryptophthalmia, which is obtained by overexpressing the IRX2 gene in mice. By overexpressing the IRX2 gene in mice, the invention successfully constructs a cryptophthalmia mouse model, providing an alternative animal model for basic research on such diseases. This model can be used to study the regulatory mechanisms of mammalian eye development and the pathogenesis of cryptophthalmia, providing a reference for drug development.

Description

Technical Field

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

[0002] Cryptophthalmos, also known as ablepharoptosis, is a congenital malformation of the eyeball and eyelid. The eyeball is covered by continuous skin, leaving only traces of the eyeball or no eyeball at all. This condition may be accompanied by optic nerve abnormalities. It is an autosomal recessive genetic disorder currently associated with mutations in genes such as FRAS1, FREM1, FREM2, and GRIP1. It is more common in females than in males. Cryptophthalmos can occur unilaterally or bilaterally, presenting as an isolated ocular deformity or as part of the clinical phenotype of Fraser Syndrome.

[0003] The pathogenesis of congenital cryptophthalmos is still unclear, and there is no effective treatment in clinical practice. Therefore, a lot of research is needed to study this disease. Using an ideal experimental animal model is crucial for studying the pathogenesis of this disease. Summary of the Invention

[0004] The present invention is aimed at basic research on congenital cryptophthalmia and provides a method for establishing a mouse model of this disease. The congenital cryptophthalmia mouse model constructed by this method is similar to the clinical manifestations of this type of disease, and the incidence rate in female mice is higher than that in male mice. It provides a relatively ideal experimental animal model for studying the regulatory mechanism of mammalian eye development and the pathogenesis of congenital cryptophthalmia.

[0005] The object of the present invention is achieved through the following technical solutions:

[0006] A method for establishing a mouse model of congenital cryptophthalmia is obtained by overexpressing the IRX2 gene (Gene ID: 153572) in mice.

[0007] Specifically, the method for establishing the congenital cryptophthalmia mouse model comprises the following steps:

[0008] S1. Using transgenic technology, the transgenic fragment containing CAG-IRX2-PolyA constructed in vitro was microinjected into fertilized eggs of C57BL / 6J mice to obtain F0 generation mice;

[0009] S2. Genotype identification of F0 generation mice was performed to obtain F0 generation IRX2-TG mice;

[0010] S3. F0 generation IRX2-TG mice were crossed with C57BL / 6J mice, and the genotypes were identified to obtain F1 generation IRX2-TG mice;

[0011] S4. Self-cross F1 IRX2-TG mice to obtain F2 IRX2-TG mice;

[0012] S5. Model mice with congenital cryptophthalmia were obtained from IRX2-TG mice of the F2 generation and their offspring.

[0013] The sequence of the CAG-IRX2-PolyA transgenic fragment is shown in SEQ ID NO: 1.

[0014] The present invention also provides application of the above-mentioned establishment method in the field of animal model construction.

[0015] The present invention also provides the use of the congenital cryptophthalmia mouse model obtained by the above-mentioned establishment method in the study of congenital cryptophthalmia diseases, wherein the research is for the purpose of non-disease diagnosis or treatment.

[0016] The present invention also provides the use of the congenital cryptophthalmia mouse model obtained by the above-mentioned establishment method in screening drugs for preventing or treating congenital cryptophthalmia diseases.

[0017] The present invention also provides a method for screening candidate drugs for preventing or treating cryptophthalmia, which comprises the step of administering the test candidate drug to the cryptophthalmia mouse model obtained according to the above-mentioned establishment method.

[0018] The present invention also provides an application of the IRX2 gene in constructing a congenital cryptophthalmia mouse model, and the congenital cryptophthalmia mouse model is obtained by overexpressing the IRX2 gene in mice.

[0019] The present invention has the following beneficial effects:

[0020] 1. The present invention constructs a mouse model of congenital cryptophthalmia, which can better simulate the phenotype of this type of disease in clinical practice.

[0021] 2. This model can be used to explore the molecular mechanism by which IRX2 regulates eye development, and the research results can be used in drug development.

[0022] 3. This model can be used to study the regulatory mechanisms of mammalian eye development and the pathogenesis of congenital cryptophthalmia, and provide possible therapeutic targets for such diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Strategy for the construction of IRX2-Tg mice.

[0024] Figure 2 The genotype identification results of cryptophthalmos mice and control mice are shown in Figure 1. TG refers to cryptophthalmos mice, and WT refers to C57BL / 6J background control mice.

[0025] Figure 3 Figure 1 shows cryptophthalmia in IRX2-TG mice. A: IRX2-TG mice and their littermate WT mice on postnatal day 14; B and C: Normal eyelids in both eyes of WT mice on postnatal day 14; D and E: Absent eyelids in both eyes of IRX2-TG mice on postnatal day 14; F and G: Opening the eye skin of an IRX2-TG mouse reveals the absence of the eyeball; H: Absent eyelid on the ipsilateral side of an adult cryptophthalmia mouse; I: Removal of the skin reveals the absence of the ipsilateral eyeball; J: Absent eyeball and optic nerve on the ipsilateral side.

[0026] Figure 4 The incidence of congenital cryptophthalmia in IRX2-TG mice is gender-specific. "normal" refers to normal mice; "abnormal" refers to mice with congenital cryptophthalmia; "MALE" indicates the incidence of congenital cryptophthalmia in male IRX2-TG mice; and "FEMALE" indicates the incidence of congenital cryptophthalmia in female IRX2-TG mice. DETAILED DESCRIPTION

[0027] In order to make the technical problems and technical solutions to be solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] Example 1 Construction of a congenital cryptophthalmia mouse model

[0029] (1) Using transgenic technology, the transgenic fragment containing CAG-IRX2-PolyA constructed in vitro was microinjected into the fertilized eggs of C57BL / 6J mice to obtain F0 generation mice.

[0030] (2) The tails of F0 generation mice were cut 5-7 days after birth, and genomic DNA was extracted for PCR identification to determine their genotypes, thus obtaining F0 generation IRX2-TG mice.

[0031] (3) F0 generation IRX2-TG mice were hybridized with C57BL / 6J mice, and the genotypes were identified to obtain F1 generation IRX2-TG mice.

[0032] (4) The F1 generation IRX2-TG mice were self-crossed to obtain the F2 generation IRX2-TG mice.

[0033] (5) We obtained congenital cryptophthalmia model mice from the F2 generation and its offspring IRX2-TG mice.

[0034] The construction strategy of congenital cryptophthalmia mice (IRX2-TG mice) is as follows Figure 1 shown.

[0035] The sequence of the transgenic fragment of CAG-IRX2-PolyA is shown in SEQ ID NO: 1, specifically:

[0036]

[0037] Example 2 Genotype identification of mice with congenital cryptophthalmia

[0038] 1. DNA extraction from mouse tail tissue:

[0039] 1) Remove and restrain the mouse to be identified. Number the mouse by clipping its toes. Cut a 0.5 cm section of the mouse's tail, which has been thoroughly wiped with alcohol, and place it in a 1.5 mL sterile EP tube. Label the mouse with the breeding cage number.

[0040] 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.

[0041] 3) Add 100 μL of 50 mM sodium hydroxide solution to each EP tube. After the lysed tissue is suspended, lyse in a 95°C metal bath for 10 minutes. After lysis is complete, centrifuge briefly at low speed to collect any liquid on the tube cap and walls at the bottom of the tube.

[0042] 4) Add 10 μL of 1 M Tris-HCl balanced solution (pH=8.0) and 100 μL of ddH 2 O to each tube, vortex to mix, and centrifuge (room temperature, 12,000×g, 5 min) to obtain the extracted mouse DNA.

[0043] 2. Mouse genotype identification:

[0044] 1) The PCR reaction system is as follows:

[0045]

[0046] 2) The PCR reaction procedure is as follows:

[0047]

[0048] 3) Agarose gel electrophoresis:

[0049] After the PCR reaction is complete, weigh 2g of agarose powder into a 250mL Erlenmeyer flask and pour in 100mL of 1X TAE. Seal the flask with aluminum foil and heat in a microwave until boiling. Gently shake and reheat until the colloid is homogeneous. After cooling slightly, add GelStain solution at a 1:10,000 ratio and gently shake to mix. Pour the mixture into a gel-casting tank with a gel comb inserted, taking care not to create bubbles. Let it stand at room temperature for 30 minutes. Once the agarose gel has completely solidified, remove the gel comb and place the gel in an electrophoresis tank filled with fresh TAE. Add 7μL of PCR product to the wells of the agarose gel comb and perform electrophoresis at 150V for 30 minutes. Remove the agarose gel and examine the PCR product for the desired band.

[0050] 4) The primer sequences for mouse genotyping are as follows:

[0051]

[0052] 5) Identification results of IRX2-TG mice are shown in Figure 2 .

[0053] Depend on Figure 2 It can be seen that compared with the C57BL / 6J background control mice (WT), the congenital cryptophthalmia mice (TG) showed a single band under the amplification conditions of CAG-IRX2 and IRX2-PolyA primers, indicating that the CAG-IRX2-PolyA transgenic fragment was successfully introduced into the congenital cryptophthalmia mice.

[0054] Example 3 Detection of related indicators in mice with congenital cryptophthalmia

[0055] 1. Collect images of mouse eye appearance

[0056] The appearance of mouse eyes was observed using a Zeiss SteREO Discovery.V8 stereo microscope with zoom, and images were acquired using a Zeiss AxioCam ERc5s microscope camera as follows:

[0057] (1) Anesthesia: Anesthesia was performed by intraperitoneal injection of 1% sodium pentobarbital (dose: 70 mg / kg).

[0058] (2) After the mouse is anesthetized for about 3-5 minutes, use a cotton swab moistened with saline to clean the mouse's ocular surface secretions and hair around the eye.

[0059] (3) Place the mouse on the microscope stage and use a cotton swab to elevate the head and expose the eyeballs.

[0060] (4) Use the focus knob to adjust the focal plane to the eye surface and the zoom knob to adjust the magnification to 1.0x-2.0x.

[0061] (5) Open the ZEISS ZEN software and start microscope image acquisition. During the image acquisition process, 0.9% saline solution is dripped onto the eye surface to keep the mouse eye surface moist. Then the mouse position is adjusted so that the center of the cornea is located at the center of the captured image.

[0062] (6) Adjust the white balance and exposure time to make the image clear and free of color difference, and then collect the image.

[0063] (7) Save the image data, use ZEISS ZEN software to adjust the brightness and contrast to optimize the image, and export the mouse eye appearance image.

[0064] 2. Collect images of the ventral side of the mouse brain

[0065] The ventral surface of the mouse brain was observed using a Zeiss SteREO Discovery.V8 stereo microscope with zoom, and images were acquired using a Zeiss AxioCam ERc5s microscope camera as follows:

[0066] (1) Mouse perfusion: Intraperitoneally inject 1% sodium pentobarbital into the mouse for anesthesia (dose of 70 mg / kg). After 5 minutes, fix the mouse's limbs on the mouse operating table and cut the mouse's abdominal and chest skin with scissors to expose the mouse's thorax. Cut the mouse's thorax from the midline to expose the mouse's heart. Insert the perfusion needle near the apex of the mouse's heart and cut a small incision in the mouse's right ventricle. Turn on the perfusion pump and irrigate the mouse's blood vessels with normal saline until the liver turns white. Then irrigate with 4% paraformaldehyde until the mouse's muscles contract and harden, the limbs twitch, and the tail hangs in the air.

[0067] (2) Remove the mouse's head and remove the skin.

[0068] (3) Remove the skull, muscles, conjunctiva and other tissues to expose the mouse brain, eyeballs and optic nerves.

[0069] (4) Place the mouse brain on the microscope stage with the eyeball and optic nerve facing up.

[0070] (5) Open the ZEISS ZEN software to capture microscope images.

[0071] (6) Adjust the focal plane to the chiasm using the focus knob and adjust the magnification to 1.0x using the zoom knob.

[0072] (7) Use ZEISS ZEN software to adjust the brightness and contrast to optimize the image, save and export the image.

[0073] Images of the eye appearance and ventral side of the brain of IRX2-TG mice are shown in Figure 3 As shown. Figure 3 It can be seen that on the 14th day after birth, the eyelids of both eyes of IRX2-TG mice were missing. When the skin around the eyes of IRX2-TG mice was cut open, the eyeballs were found to be absent. The eyelids on the affected side of adult cryptophthalmos mice were missing. When the skin was removed, the eyeballs and optic nerves on the affected side were found to be absent. These results indicate that the present invention successfully constructed a congenital cryptophthalmos mouse model, which can well simulate the phenotype of this type of disease in clinical practice.

[0074] The incidence of congenital cryptophthalmia in IRX2-TG mice is shown in Figure 4 As shown. Figure 4The incidence of congenital cryptophthalmia in IRX2-TG mice was 20.35%, with an incidence of 1.54% in males and 45.83% in females. These results suggest that there is a sex difference in the incidence of congenital cryptophthalmia in IRX2-TG mice, with a higher incidence in females than in males. This model can be used to study the regulatory mechanisms of mammalian eye development and the pathogenesis of congenital cryptophthalmia, and to provide potential therapeutic targets for such diseases.

[0075] The above detailed description is a specific description of the possible embodiments of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the present invention should be included in the patent scope of this case.

Claims

1. A method for establishing a congenital cryptophthalmia mouse model, characterized in that: By overexpression in mice IRX2 Gene acquisition includes the following steps: S1. Using transgenic technology, the transgenic fragment containing CAG-IRX2-PolyA constructed in vitro was microinjected into fertilized eggs of C57BL / 6J mice to obtain F0 generation mice; S2. Genotype identification of F0 generation mice was performed to obtain F0 generation IRX2-TG mice; S3. F0 generation IRX2-TG mice were crossed with C57BL / 6J mice, and the genotypes were identified to obtain F1 generation IRX2-TG mice; S4. Self-cross F1 IRX2-TG mice to obtain F2 IRX2-TG mice; S5. Congenital cryptophthalmia model mice were obtained from IRX2-TG mice of the F2 generation and their offspring; The sequence of the CAG-IRX2-PolyA transgenic fragment is shown in SEQ ID NO:

1.

2. Application of the establishment method according to claim 1 in the field of animal model construction.

3. Use of the congenital cryptophthalmia mouse model obtained by the establishment method according to claim 1 in the study of congenital cryptophthalmia diseases, wherein the research is for the purpose of non-disease diagnosis or treatment.

4. Use of the congenital cryptophthalmia mouse model obtained by the establishment method according to claim 1 in screening drugs for preventing or treating congenital cryptophthalmia diseases.

5. A method for screening candidate drugs for preventing or treating congenital cryptophthalmia, characterized in that: The method comprises the step of administering a test candidate drug to the congenital cryptophthalmia mouse model obtained according to the establishment method of claim 1.

6. IRX2 The application of the gene in constructing a mouse model of congenital cryptophthalmia is characterized in that: By overexpression in mice IRX2 The gene is obtained by the congenital cryptophthalmia mouse model established by the method described in claim 1.

Images