CN111073909A - Mouse xerophthalmia animal model and application thereof - Google Patents
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Abstract
The invention provides a stable dry eye animal model, which is constructed by knocking out AQP5 gene in animals; the AQP5 gene knockout is a nucleotide fragment of 99589876-99594444 region on the 15 th chromosome of a mouse. The dry eye mouse model provided by the invention is applied to screening or evaluating the effect of dry eye treatment drugs. AQP5 of the present invention‑/‑The model mouse has high repeatability and stable model, is beneficial to the research of the dry eye pathological mechanism and the pharmaceutical intervention, and can avoid the influence of other influencing factors on the experimental result. Also in accordance with the present invention AQP5‑/‑Mouse model, with characteristic changes of reduced tear secretion immediately after birth, extraorbital lacrimal glands appear after birthPathological features of existing vacuolar degeneration; and the amount of lacrimal secretion has no obvious change with age and no sex difference.
Description
Technical Field
The invention belongs to the technical field of medical animal models, and particularly relates to a method for establishing a dry eye animal model.
Background
Dry eye refers to a group of diseases in which the tear film is unstable and/or the surface of the eye is abnormal due to abnormalities in the quality and quantity and kinetics of tear fluid, with the accompanying symptoms of ocular discomfort. Is one of the most common diseases in ophthalmology, can cause eye surface damage, blurred vision and the like, and influences the life quality of patients.
Dry eye can be classified into excessive evaporation type dry eye, aqueous fluid deficient type dry eye, mucin abnormal type dry eye, tear dynamics abnormal type dry eye, and mixed type dry eye. Clinical investigation shows that dry eye is a common ocular surface disease at present, and keratitis, corneal neovascularization, corneal ulcer and the like can be caused secondarily, so that the life quality and the work efficiency of people are seriously influenced. Common pathophysiological features of the onset of dry eye are ocular surface epithelial barrier breakdown, tear reduction, conjunctival goblet cell reduction, ocular surface epithelial squamous epithelialization, and the like. Due to the complexity and diversity of dry eye pathogenesis, a range of fundamental and clinical problems with dry eye pathogenesis, diagnosis and treatment remain to be studied in depth.
The establishment of animal models is an effective and practical means for researching the pathogenesis of xerophthalmia and exploring a treatment method. The establishment method of the dry eye animal model reported at present comprises the following steps: through nutrition factors, environmental factors, toxic substances, drug action, animal sex hormone secretion level change, lacrimal gland or ocular surface innervation removal, lacrimal gland induced autoimmune reaction, lacrimal gland surgical removal and transgenic animals and the like. The establishment of these animal models of dry eye often requires high technical conditions or long time and is subject to great variability.
Disclosure of Invention
The invention aims to overcome the defects of the existing model types and provide a stable xerophthalmia animal model, thereby making up the defects of the prior art.
The applicant designs sgRNA by using CRISPR/Cas9 technology, and obtains an AQP5 gene knockout mouse (AQP 5) by applying a high-flux electrotransformation fertilized egg mode-/-). And for the first time AQP5-/-Mice, which appeared dry eye after birth, were phenotypically stable and free of sex differences, thus contributing to the present invention.
The invention firstly provides a construction method of a dry eye animal model, which is characterized in that the dry eye animal model is constructed by knocking out AQP5 gene in an animal;
the AQP5 gene in the knockout animal is knocked out through a CRISPR/Cas9 system;
the invention also provides a dry eye mouse model which is constructed by knocking out the AQP5 gene by the method;
the AQP5 gene is knocked out of 99589876-99594444 region on the 15 th chromosome of a mouse, and the length of the nucleotide fragment is 4569 bp;
the invention also provides a method for detecting AQP5-/-Mouse products, wherein the products comprise primer pairs for detecting the deletion fragments;
wherein, the primer pair for detecting the deletion fragment has the following sequence information: AQP5-F1: CAAAGTGCTCAAACACTAACCGTAC (SEQ ID NO:1) AQP5-R1: GATTGGTGGTTTATTGGGAAACG (SEQ ID NO: 2). AQP5-R3: TGCAGGTCTTTGTTTCTGCCG (SEQ ID NO: 3).
The dry eye mouse model provided by the invention is applied to screening or evaluating the effect of dry eye treatment drugs.
AQP5 of the present invention-/-The model mouse has high repeatability and stable model, is beneficial to the research of the dry eye pathological mechanism and the pharmaceutical intervention, and can avoid the influence of other influencing factors on the experimental result. Also in accordance with the present invention AQP5-/-A mouse model, wherein the characteristic change of tear secretion reduction occurs immediately after birth, and the pathological characteristic of vacuolar degeneration occurs in extraorbital lacrimal glands after birth; and the amount of lacrimal secretion has no obvious change with age and no sex difference.
Drawings
FIG. 1: A. b is AQP5 of example 1 of the present invention-/-Mouse information and KO region, C is AQP5-/-A sequencing identification result graph of the mouse;
FIG. 2: AQP5 in example 1 of the present invention-/-Electrophoresis chart of mouse gene identification result;
FIG. 3: AQP5 of different ages in example 1 of the present invention+/+And AQP5-/-A fluorescein sodium staining profile of the mouse ocular surface;
FIG. 4: book (I)AQP5 in inventive example 1+/+And AQP5-/-Tear secretion analysis result chart of the mouse;
FIG. 5: AQP5 in example 1 of the present invention+/+And AQP5-/-Photographs of extraorbital lacrimal HE staining of mice;
FIG. 6: AQP5 in example 1 of the present invention+/+And AQP5-/-Transmission electron microscopy scans of the extraorbital lacrimal gland of the mice.
Detailed Description
Applicants have found AQP5 that knocks out the Aquaporin5 gene (Aquaporin5, AQP5)-/-The mice appeared dry eye after birth, and their lacrimal secretion function did not change significantly with age and there was no sex difference.
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
Example 1: construction of AQP5-/-Mouse xerophthalmia animal model
1、AQP5-/-Construction and identification of mice
AQP5(NCBI ID:11830) is located on mouse chromosome 15, AQP5-/-The information and the knockout region of the mouse are shown in figure 1A, B, the sgRNA is designed by adopting CRISPR/Cas9 technology, the gene fragment with the length of 4569bp in the chr15:99589876-99594444 region is knocked out by applying a high-throughput electrotransformation zygote mode, and the AQP5 is obtained-/-A mouse. FIG. 1C is AQP5-/-The sequencing identification result of the mouse is seen by comparing with a wild type sequence, and a 4569bp sequence is deleted altogether, which indicates that the model mouse is successfully established. This mouse has been successfully bred in my unit and a stable population of homozygous mice has been obtained.
In subsequent routine feeding, the PCR amplification method is used for detecting AQP5-/-Knockout effect in mice.
PCR amplification of the fragment of interest: reaction conditions and reaction system:
(1) and (3) PCR reaction conditions: 3min at 94 ℃; 94 ℃ 30sec, 60 ℃ 30se, 65 ℃ 50sec, 33 cycles; 10min at 72 ℃.
(2) Reaction system: (TAKARA LA Taq polymerase)
Wherein the primer pairs are respectively
AQP5-F1:CAAAGTGCTCAAACACTAACCGTAC、
AQP5-R1:GATTGGTGGTTTATTGGGAAACG、
AQP5-F1:CAAAGTGCTCAAACACTAACCGTAC、
AQP5-R3:TGCAGGTCTTTGTTTCTGCCG。
The identification result of the breeding mouse is shown in figure 2, and the result of PCR amplification of the mouse tail genome DNA shows that the 1, 2, 6 and 7 columns only have 640bp fragments and are homozygote mice; 3. the two fragments of 640bp and 348bp are shown in columns 4 and 5, which are heterozygotes.
2、AQP5-/-Dry eye symptoms in mice
AQP5+/+ and AQP 5-/-mice were selected for routine feeding, and AQP 5-/-mice exhibited characteristic changes in reduced tear secretion immediately after birth: the results of experiments involving corneal epithelial cell depletion and sodium fluorescein staining are shown in FIG. 3. The tear secretion was significantly reduced, as measured by the phenol red cotton line method, as shown in FIG. 4.
3、AQP5-/-Pathological changes in extraorbital lacrimal glands in mice
AQP5 of the present invention-/-After birth, the mouse picks up the extraorbital lacrimal gland, conventionally makes paraffin section, and carries out HE staining, finds that the extraorbital lacrimal gland has pathological feature change of vacuole degeneration, as shown in figure 5, the pathological feature change shows that the number of acini in a unit area is reduced, the number of acinar epithelial cells is reduced, and the area of a single acinus is increased; the number of vacuoles in the acinar epithelium in a unit area is obviously increased, and the proportion of the area occupied by the vacuoles is obviously increased.
4、AQP5-/-Transmission electron microscope scanning structure change of extraorbital lacrimal gland of mouse
Taking AQP5+/+Mouse and AQP5 of the present invention-/-The study of mouse lacrimal gland, conventionally making transmission electron microscope section, observing under transmission electron microscope, collecting image analysis, the result is shown in figure 6, AQP5+/+Group lacrimal gland pictureThe shape of the skin cells is slightly edematous, the mitochondria is slightly swollen, the size and the shape of secretory granules are uniform, and the intercellular spaces are clear. AQP5-/-The group of pictures show that epithelial cell damage is relatively serious, mitochondrial ridges disappear, membranes are damaged, stroma overflows, secretory granules are different in size, partial granules are fused, and intercellular spaces are obviously widened.
The animal model constructed by the invention can be used for researching the pathogenesis of tear generation deficiency type dry eye and screening or evaluating the action mechanism and effect evaluation of various dry eye treatment drugs; the method can also be used for researching the damage and mechanism of the dry eye to the ocular microenvironment and screening or evaluating the protective effect and the effect of various dry eye treatment drugs on the ocular microenvironment.
Sequence listing
<110> Qingdao university
<120> mouse xerophthalmia animal model and application thereof
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Claims (8)
1. A method for constructing a dry eye animal model, which is characterized in that the dry eye animal model is constructed by knocking out AQP5 gene in an animal.
2. The method of claim 1, wherein the AQP5 gene is knocked out in said knockout animal by the CRISPR/Cas9 system.
3. The method of claim 1 or 2, wherein the AQP5 gene in said knockout animal is a knockout mouse AQP5 gene.
4. The method as claimed in claim 3, wherein the AQP5 gene knock-out is a nucleotide fragment of 99589876-99594444 on chromosome 15 of a mouse.
5. A dry eye mouse model constructed by the method of any one of claims 1 to 4.
6. Use of the mouse model of dry eye of claim 5 to screen for or evaluate the effect of a dry eye treatment drug.
7. A preparation for detecting the dry eye mouse model of claim 5, wherein the preparation comprises a primer pair for detecting the deleted fragment.
8. The article of claim 8, wherein the primer pair has the sequence of SEQ ID NOS: 1-3.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112251467A (en) * | 2020-10-18 | 2021-01-22 | 青岛大学 | Animal model of neurotrophic keratitis of mice and application thereof |
| CN113519461A (en) * | 2021-07-06 | 2021-10-22 | 江西中洪博元生物技术有限公司 | Construction method and application of concanavalin A-induced mouse xerophthalmia model |
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| US5858702A (en) * | 1991-12-13 | 1999-01-12 | The Johns Hopkins University | Isolation, cloning and expression of transmembrane water channel Aquaporin 5 (AQP5) |
| CN102743577A (en) * | 2012-07-09 | 2012-10-24 | 南京中医药大学 | Traditional Chinese medicine composite for promoting expression of aquaporin-5 (AQP5) and preparation method and application of traditional Chinese medicine composite |
| CN107312859A (en) * | 2017-07-26 | 2017-11-03 | 青岛大学 | Application of the AQP5 genes in detection congenital cataract product is prepared |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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