Method for establishing congenital hidden eye malformation mouse model

Abstract

The invention discloses a method for establishing a congenital hidden eye malformation mouse model. The mouse model is obtained by overexpressing an IRX2 gene in a mouse body. According to the present invention, the IRX2 gene is overexpressed in the mouse body, such that the congenital hidden eye malformation mouse model is successfully constructed, the alternative animal model is provided for the basic research of the diseases, and the model can be used for researching the regulation mechanism of the mammal eye development and the pathogenesis of the congenital hidden eye malformation so as to provide the reference for the drug research and development.

Description

Technical Field

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

[0002] Cryptophthalmos, also known as ablepharia, is a congenital malformation of the eyeball and eyelid. The patient's eyeball is covered by continuous skin, with only traces of the eyeball or no eyeball at all, and may be accompanied by abnormal development of the optic nerve. It is an autosomal recessive genetic disease, which is currently known to be related to gene mutations such as FRAS1, FREM1, FREM2, and GRIP1. It is more common in women than in men. Cryptophthalmos can occur unilaterally or bilaterally, and can be manifested as an isolated eye malformation, or as one of the clinical phenotypes of Fraser Syndrome.

[0003] At present, the pathogenesis of congenital cryptophthalmos is not fully understood, and there is no effective treatment in clinical practice, so a lot of research needs to be done on this type of disease. Using an ideal experimental animal model is crucial to studying the pathogenesis of this disease. Summary of the invention

[0004] The present invention aims at the basic research of congenital cryptophthalmos and provides a method for establishing a mouse model of such disease. The congenital cryptophthalmos mouse model constructed by this method is similar to the clinical manifestations of such diseases in clinic, 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 cryptophthalmos.

[0005] The objective 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 the mouse.

[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 the fertilized eggs of C57BL / 6J mice to obtain F0 generation mice;

[0009] S2, genotyping the F0 generation mice to obtain the 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-fertilizing the F1 generation IRX2-TG mice to obtain the F2 generation 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 cryptophthalmos mouse model obtained by the above-mentioned establishment method in the study of congenital cryptophthalmos disease, wherein the research is for the purpose of non-disease diagnosis or treatment.

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

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

[0018] The present invention also provides the use 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 the mouse.

[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 such diseases in clinical practice.

[0021] 2. This model can be used to explore the molecular mechanism of IRX2 regulating 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 cryptophthalmos, and provide possible therapeutic targets for such diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Strategy for constructing IRX2-Tg mice.

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

[0025] Figure 3 IRX2-TG mice with cryptophthalmia. A: IRX2-TG mice and WT mice on the 14th day after birth; B and C: WT mice with normal eyelids on the 14th day after birth; D and E: IRX2-TG mice with missing eyelids on the 14th day after birth; F and G: Cutting open the eye skin of IRX2-TG mice revealed missing eyeballs; H: Adult cryptophthalmia mice with missing eyelids on the affected side; I: Remove the skin and reveal missing eyeballs on the affected side; J: Missing eyeballs and optic nerves on the affected side.

[0026] Figure 4 The incidence of congenital cryptophthalmia in IRX2-TG mice has gender differences. Normal refers to normal mice; abnormal refers to mice with congenital cryptophthalmia; MALE represents the incidence of congenital cryptophthalmia in male IRX2-TG mice; FEMALE represents the incidence of congenital cryptophthalmia in female IRX2-TG mice. DETAILED DESCRIPTION

[0027] 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.

[0028] Example 1 Construction of 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 mice were cut 5-7 days after birth, and genomic DNA was extracted for PCR identification to determine their genotypes, thereby obtaining F0 IRX2-TG mice.

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

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

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

[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 congenital cryptophthalmia mice

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

[0039] 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 thoroughly wiped with alcohol, put it in a 1.5 mL sterile EP tube, and mark the mouse number and 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 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.

[0042] 4) Add 10 μL 1M Tris-HCl (pH=8.0) and 100 μL ddHO to each tube. 2 O, 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 completed, weigh 2g of agarose powder into a 250mL conical flask, pour in 100mL 1X TAE, seal the conical flask with aluminum foil, place in a microwave and heat until boiling, shake gently and heat again until the colloidal liquid is uniform, wait for it to cool slightly, add GelStain solution at 1:10000, shake gently to mix, and then pour into the gel making tank with the gel comb inserted, be careful not to have bubbles. Let it stand at room temperature for 30 minutes, pull out the gel comb after the agarose gel is completely solidified, and insert the gel into the electrophoresis tank filled with fresh TAE. Take 7μL of PCR product, add it to the agarose gel comb hole, electrophoresed at 150V for 30 minutes, and take out the agarose gel to detect whether there is a target band in the PCR product.

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

[0051]

[0052] 5) IRX2-TG mouse identification results 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 cryptophthalmos mice (TG) showed a single band under the amplification conditions of CAG-IRX2 and IRX2-PolyA primers, indicating that the transgenic fragment of CAG-IRX2-PolyA was successfully introduced into the cryptophthalmos mice.

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

[0055] 1. Collect images of mouse eye appearance

[0056] The appearance of mouse eyes was observed using a Zeiss SteREO Discovery.V8 stereo microscope and zoom microscope, and images were collected using a Zeiss AxioCam ERc5s microscope camera. The steps were 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 soaked in saline to clean the mouse's ocular 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 use 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) Collect images by adjusting white balance and exposure time to make the image clear and free of color difference.

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

[0066] (1) Mouse perfusion: Inject 1% sodium pentobarbital into the mouse peritoneal cavity for anesthesia (dose 70 mg / kg). After 5 minutes, fix the mouse's limbs on the mouse operating table, 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 hole in the mouse's right ventricle. Turn on the perfusion pump, irrigate the mouse's blood vessels with saline until the liver turns white, and 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 head and 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 acquire microscope images.

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

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

[0073] The 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, and when the eye skin of the IRX2-TG mice was cut open, the eyeball was found to be absent; the eyelids on the affected side of adult cryptophthalmos mice were missing, and when the skin was removed, the eyeball and optic nerve on the affected side were found to be absent. These results indicate that the present invention successfully constructs a congenital cryptophthalmos mouse model, and the model can better simulate the phenotype of such diseases in clinical practice.

[0074] The incidence of congenital cryptophthalmia in IRX2-TG mice is shown in Figure 4 As shown. Figure 4It can be seen that the incidence of congenital cryptophthalmia in IRX2-TG mice is 20.35%, of which the incidence in male mice is 1.54% and the incidence in female mice is 45.83%. This result shows that the incidence of congenital cryptophthalmia in IRX2-TG mice is gender-differentiated, with a higher incidence in female mice than in male mice. This model can be used to study the regulatory mechanism of mammalian eye development and the pathogenesis of congenital cryptophthalmia, and provide possible therapeutic targets for such diseases.

[0075] The above detailed description is a specific description of the possible embodiments of the present invention. The embodiments are not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate 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: Obtained by overexpressing the IRX2 gene in mice.

2. The method for establishing a congenital cryptophthalmia mouse model according to claim 1, characterized in that: The following steps are involved: S1. 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; S2, genotyping the F0 generation mice to obtain the 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-fertilizing the F1 generation IRX2-TG mice to obtain the F2 generation IRX2-TG mice; S5. Model mice with congenital cryptophthalmia were obtained from IRX2-TG mice of the F2 generation and their offspring.

3. The method for establishing a mouse model of congenital cryptophthalmia according to claim 2, characterized in that: The sequence of the CAG-IRX2-PolyA transgenic fragment is shown in SEQ ID NO:

1.

4. Application of the establishment method according to any one of claims 1 to 3 in the field of animal model construction.

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

6. Use of the congenital cryptophthalmos mouse model obtained by the establishment method described in any one of claims 1 to 3 in screening drugs for preventing or treating congenital cryptophthalmos diseases.

7. A method for screening candidate drugs for preventing or treating congenital cryptophthalmos, characterized in that: The method comprises the step of administering a test candidate drug to the congenital cryptophthalmia mouse model obtained by the establishment method according to any one of claims 1 to 3.

8. Application of IRX2 gene in constructing a mouse model of congenital cryptophthalmia.

9. The use according to claim 8, characterized in that: The congenital cryptophthalmia mouse model is obtained by overexpressing the IRX2 gene in mice.

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