Establishment method of mouse model with tubular structure of outer retina

Abstract

The invention discloses a method for establishing a mouse model with an outer-layer retina tubular structure. The mouse model is obtained by specifically knocking out an Lss gene from the retina of a mouse. According to the invention, by means of a CRISPR / Cas9 gene editing technology, an Lss gene is specifically knocked out from the retina, and the mouse model with the tubular structure of the outer retina is constructed. The outer-layer retina tubular structure mouse model constructed by the method is similar to the retinopathy phenotype clinically, and an ideal experimental animal model is provided for researching the development regulation and control of the retina of mammals and the pathogenesis of retinal degeneration.

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 outer retinal tubulation structure. Background Art

[0002] Outer retinal tubulation (ORT) is a pathological change occurring in the outer retina. The examination by spectral domain-optic coherence tomography (SD-OCT) shows round or oval hyperreflective tubular structures in the outer nuclear layer, which can be seen in various retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, crystalline retinopathy, Best disease, Stargardt disease, cone dystrophy, acute zonal occult outer retinopathy, etc.

[0003] At present, the formation mechanism of ORT is not fully understood. It is crucial to study the formation mechanism of ORT and explore possible treatment options with the aid of an ideal experimental animal model. However, the relevant experimental animal models are currently lacking. Summary of the Invention

[0004] The present invention provides a method for establishing a mouse model of this type of retinal disease for the basic research on the outer retinal tubulation structure. The mouse model of outer retinal tubulation structure constructed by this method is similar to the phenotype of this type of retinal disease clinically, providing an ideal experimental animal model for studying the developmental regulation of the mammalian retina and the pathogenesis of retinal degeneration.

[0005] With the aid of the CRISPR / Cas9 gene editing technology, the present invention constructs an ORT mouse model by specifically knocking out the Lss gene in the retina. The specific method includes the following steps:

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

[0007] S2. Hybridizing the Lss flox / flox mice with commercially available Six3-Cre mice to obtain Lss flox / + ; Six3-Cre mice;

[0008] S3. Self-crossing the Lss flox / + ; Six3-Cre mice to obtain the target gene mice Lss flox / flox ; Six3-Cre mice.

[0009] Preferably, step S1 is specifically as follows: 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 of C57BL / 6J wild-type mice by CRISPR / Cas9 gene editing technology to obtain Lss flox / + Mouse, Lss flox / + The mice were further selfed to obtain Lss flox / flox Mouse.

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

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

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

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

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

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

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

[0017] 1. The present invention constructs a mouse model of outer retinal tubular structure, which can better simulate the phenotype of such retinal diseases in clinical practice.

[0018] 2. This model can be used to explore the molecular mechanism of Lss regulation of retinal development and the occurrence mechanism of outer retinal tubular structure.

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

[0020] 4. This model can be used to study the regulatory mechanisms of mammalian retinal development and the pathogenesis of retinal degeneration, and provide possible therapeutic targets for such retinal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Figure 2 The results of mouse genotype identification.

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

[0024] Figure 4 For Lss flox / flox ; Six3-Cre mice have abnormal visual function. DETAILED DESCRIPTION

[0025] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

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

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

[0028] A method for establishing an outer retinal tubular structure mouse model comprises the following steps:

[0029] (1) CRISPR / Cas9 gene editing technology was used to insert a LoxP site in the upstream of the third exon region and the downstream of the sixth exon region of the Lss gene (GeneID: 16987) in wild-type (WT) mice with a C57BL / 6J background to obtain Lss flox / + Mouse, Lss flox / + The mice were further selfed to obtain Lss flox / flox Mouse.

[0030] (2) Lss flox / flox The mice were crossed with commercial Six3-Cre mice to obtain Lss flox / + ; Six3-Cre mice.

[0031] (3) Lss flox / + ; Six3-Cre mice were self-bred to obtain the target gene mouse Lss flox / flox ; Six3-Cre mice.

[0032] Example 2 Identification of mouse model genotype

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

[0034] 1) Take out and immobilize the mice to be identified, number the mice by cutting their toes, cut 0.5 cm of the tail of the mice that have been wiped thoroughly with alcohol, place it in a 1.5 mL sterilized EP tube, and label the mouse number and the breeding cage number.

[0035] 2) Put the EP tube containing the mouse tail into a centrifuge, centrifuge (at room temperature, 1000 g, 1 min), and centrifuge the mouse 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, lysate it in a metal bath at 95 °C for 10 minutes. After the lysis is completed, centrifuge at low speed instantaneously to gather the liquid on the tube cap and the tube wall to the bottom of the tube.

[0037] 4) Add 10 μL of 1 M Tris-HCl equilibration solution (pH = 8.0) to each tube, centrifuge (at room temperature, 1000 g, 1 min), and then add 100 μL of ddH 2 O and centrifuge at low speed instantaneously, which is the DNA extracted from this mouse.

[0038] 2. Mouse genotype identification:

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

[0040]

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

[0042]

[0043]

[0044] 3) Agarose gel electrophoresis:

[0045] After the PCR reaction is completed, weigh 2 g of agarose powder into a 250 mL conical flask, pour in 100 mL of 1×TAE, seal the conical flask mouth with aluminum foil, place it in a microwave oven and heat it to 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, paying attention not to have bubbles. Let it stand at room temperature for 30 minutes. After the agarose gel is 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, add it into the well of the agarose gel, and electrophorese for 30 minutes under the voltage condition of 150 V. Take out the agarose gel to detect whether there is a target band in the PCR product.

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

[0047]

[0048] 5) The genotype identification results are shown in Figure 2 .

[0049] As Figure 2 shown, in the PCR reaction system of the Lss gene, a single band of 270 bp was visible in WT (C57BL / 6J) mice, Lss flox / flox mice and Lss flox / flox ; a single band of 373 bp was visible in Six3-Cre mice, Lss flox / + ; a double band of 270 bp and 373 bp was visible in Six3-Cre mice; in the PCR reaction system of the Six3-Cre gene, no band was observed in WT (C57BL / 6J) mice and Lss flox / flox mice, Lss flox / + ; Six3-Cre mice and Lss flox / flox ; a single band of 480 bp was visible in Six3-Cre mice.

[0050] Example 3 Hematoxylin Staining of Mouse Retinal Tissue

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

[0052] 2. Hydration (rehydration): The deparaffinized sections were rehydrated through a gradient of alcohol, successively in 100% alcohol (Ⅰ) for 5 minutes, 100% alcohol (Ⅱ) for 5 minutes, 90% alcohol for 3 minutes, 70% alcohol for 3 minutes, 30% alcohol for 3 minutes, and finally rinsed in tap water for 1 minute.

[0053] 3. Hematoxylin staining: The rehydrated sections were placed in hematoxylin stain for 2 - 3 minutes, then soaked in pure water for 1 minute, and then differentiated with 1% hydrochloric acid ethanol for 3 s to make the staining of the nucleus and cytoplasm clearer. Immediately after that, it was slowly rinsed with running water for 10 minutes.

[0054] 4. Dehydration: The stained sections were dehydrated again, successively soaked in 95% alcohol (Ⅰ) for 30 s, 95% (Ⅱ) alcohol for 30 s, 100% alcohol (Ⅰ) for 1 minute, and 100% alcohol (Ⅱ) for 1 minute.

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

[0056] 6. Mounting the section: Place the cleared section flat, gently wipe away the remaining liquid with a cotton swab, add a drop of neutral balsam on the section, and then cover it with a cover slip for fixation (avoid generating air bubbles). Let it stand at room temperature for about half an hour. After the balsam is slightly dry, it can be stored or photographed.

[0057] As Figure 3 shown, Lss flox / flox ; Multiple round or oval-shaped tubular structures with different diameters were formed in the outer nuclear layer of the retina of Six3-Cre mice. Control group mice refer to Lss flox / flox mice.

[0058] Example 4 Mouse Visual Function Examination

[0059] The electroretinogram (ERG) of mice was recorded using a Diagnosys Celeris rodent ERG device. All mice to be subjected to electrophysiological detection were dark adapted overnight in advance. The specific steps are as follows:

[0060] 1. Anesthesia: Anesthetize the mice by 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, instill compound tropicamide eye drops (mydriatic) and proparacaine eye drops (topical anesthetic) into both eyes for mydriasis and topical anesthesia.

[0062] 3. After complete mydriasis (about 5 minutes), place the mice on the warming platform of the Diagnosys Celeris instrument (maintain the body temperature of the mice at 37 °C). Drop a small amount of normal saline on the ocular surface to keep the ocular surface moist.

[0063] 4. During the ERG experiment, keep the light off throughout. First, perform scotopic detection on the mice. 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 , 3 cd·s / m 2 , 10 cd·s / m 2 , and record the electroretinogram responses (a-wave and b-wave) of the mice.

[0064] 5. The mice that have completed the scotopic ERG are light adapted for five minutes. After five minutes, detect the light-adapted ERG results by light stimulation. The stimulating light intensities are 0.3 cd·s / m 2 , 1 cd·s / m 2 , 3 cd·s / m2 、 10 cd·s / m 2 、 30 cd·s / m 2 , record the electroretinogram responses (a-wave and b-wave) of the mice.

[0065] As Figure 4 shown, Lss flox / flox ; the electrophysiological results of Six3-Cre mice were abnormal. Compared with the control mice (Lss flox / flox mice), Lss flox / flox ; Six3-Cre mice showed decreased amplitudes of the scotopic a-wave and b-wave ( Figure 4 A and B in flox / flox ). Compared with the control mice (Lss flox / flox mice), Lss Figure 4 ; Six3-Cre mice showed decreased amplitudes of the photopic a-wave and b-wave (

[0066] The above detailed description is a specific description of the embodiments of the present invention. The embodiments are not intended to limit the patent scope of the present invention. Any equivalent implementation or modification without departing from the present invention shall be included in the patent scope of this case.

Claims

1. A method for establishing a mouse model of outer retinal tubular structure, characterized in that: It is obtained by specifically knocking out the Lss gene in the mouse retina.

2. The method for establishing the outer retinal tubular structure mouse model according to claim 1, characterized in that: The following steps are involved: S1. CRISPR / Cas9 gene editing technology was used to insert LoxP sites into the Lss gene of C57BL / 6J wild-type mice to obtain Lss flox / flox Mice; S2, Lss flox / flox The mice were crossed with commercial Six3-Cre mice to obtain Lss flox / + ;Six3-Cre mice; S3, Lss flox / + ; Six3-Cre mice were self-bred to obtain the target gene mouse Lss flox / flox ; Six3-Cre mice.

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

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 outer retinal tubular structure mouse model obtained by the establishment method according to any one of claims 1 to 3 in studying the regulatory mechanism of retinal development and the pathogenesis of retinal degeneration.

6. Application of the outer retinal tubular structure mouse model obtained by the establishment method according to any one of claims 1 to 3 in studying the molecular mechanism of Lss regulating retinal development and the occurrence mechanism of outer retinal tubular structure.

7. Use of the outer retinal tubular structure mouse model obtained by the establishment method according to any one of claims 1 to 3 in drug screening and / or therapeutic effect evaluation.

8. A method for drug screening and / or therapeutic effect evaluation, characterized in that: The method comprises the step of administering a test candidate drug to the outer retinal tubular structure mouse model obtained according to the establishment method according to any one of claims 1 to 3.

9. Application of Lss gene in constructing a mouse model of outer retinal tubular structure.

10. The use according to claim 9, characterized in that: It is obtained by specifically knocking out the Lss gene in the mouse retina.

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