CN111500638B - Method for constructing animal model of retinal neovascular disease by using gene manipulation technology, cultivation method and application - Google Patents
Method for constructing animal model of retinal neovascular disease by using gene manipulation technology, cultivation method and application Download PDFInfo
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- CN111500638B CN111500638B CN201911325954.9A CN201911325954A CN111500638B CN 111500638 B CN111500638 B CN 111500638B CN 201911325954 A CN201911325954 A CN 201911325954A CN 111500638 B CN111500638 B CN 111500638B
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knockout animals
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention discloses a method for constructing an animal model of retinal neovascular diseases by using a gene manipulation technology, a cultivation method and application, and relates to the technical field of gene editing. The construction method disclosed by the invention utilizes a gene manipulation technology to ensure that Ctnnd1 gene in vascular endothelial cells of a target animal is not expressed or the expression of the Ctnnd1 gene is inhibited. The method can obtain the animal model of the retinal neovascular disease, enriches the types of the animal model of the retinal neovascular disease, provides more model choices for the related research of the retinal neovascular disease, and is also beneficial to promoting the scientific research of the retinal neovascular disease, the development of related therapeutic drugs and the like.
Description
Technical Field
The invention relates to the technical field of gene editing, in particular to a method for constructing an animal model of retinal neovascular diseases by using a gene manipulation technology, a cultivation method and application.
Background
Retinal Neovascularization (RNV) -related eye diseases are diseases mainly characterized by pathological Retinal neovascularization, and mainly include Proliferative Diabetic Retinopathy (PDR), retinopathy of prematurity (ROP), and the like. The diseases have high incidence rate and poor treatment prognosis, so the blindness rate is high, and the vision health of human is seriously harmed. Currently, there is no more effective treatment method for RNV clinically, except for laser photocoagulation and intraocular injection of anti-Vascular Endothelial Growth Factor (VEGF) drugs in early stage of the disease. Also, studies have shown that anti-VEGF therapy is not effective in all patients; meanwhile, anti-VEGF treatment has the defects of high price, repeated injection, increased infection risk of patients and the like, so that the clinical application of the anti-VEGF treatment is limited. Therefore, it is a medical problem to be solved urgently that an effective animal model is constructed to search for a new effective treatment means or a new effective treatment target while the research on the pathogenesis of RNV is carried out.
Currently, there is a lack of animal models of retinal neovascular disease that can be used for RNV studies.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a novel method for constructing an animal model of retinal neovascular diseases by using a gene manipulation technology, a cultivation method and application. The invention provides a new method for constructing an animal model of the retinal neovascular disease, the animal model of the retinal neovascular disease can be obtained by the method, the variety of the animal model of the retinal neovascular disease is enriched, more model choices are provided for the relevant research of the retinal neovascular disease, and the method is favorable for promoting the scientific research of the retinal neovascular disease, the development of relevant treatment drugs and the like.
The invention is realized by the following steps:
in a first aspect, the embodiments of the present invention provide a method for constructing an animal model of retinal neovascular disease by using genetic manipulation technology, which is used to make the retinal neovascular disease in the vascular endothelial cells of a target animalCtnnd1The gene is not expressed or its expression is suppressed.
Ctnnd1The gene (NM _ 001085458) is located on chromosome 11, is a member of the catenin family, is located in the cell adhesion junction region, and interacts with cadherin family member Cadherinn. In addition, it is reported to participate in various signaling pathways, such as Wnt signaling pathway, Rho GTPase signaling pathway, TGF- β signaling pathway, and the like. The invention has found that the preparation method has the advantages of,Ctnnd1the gene is related to retinal neovascular diseases, so thatCtnnd1The non-expression or the inhibition of the expression of the gene in the vascular endothelial cells of the animal can make the animal have the phenotype of the retinal pigment degeneration disease, which is an ideal retinal pigment degeneration disease model.
In an alternative embodiment, the target animal is a non-human mammal.
In alternative embodiments, the mammal is selected from any one of cattle, sheep, horses, rabbits, mice, rats, sheep, and pigs.
The target animal of the present invention may be a mammal havingCtnnd1Any non-human mammal of the gene, provided that it is presentCtnnd1The gene can play a role in simulation.
In alternative embodiments, the gene manipulation technique is selected from one or a combination of gene knockout, gene editing, and RNA interference techniques.
Based on the present invention, those skilled in the art can select appropriate gene manipulation techniques such as gene knockout, gene editing, and RNA interference to achieve the goal of animal breedingCtnnd1The gene operation makes it not express in the vascular endothelial cell of the animal or the expression is inhibited, and the animal model of the retinal neovascular disease is obtained.
In an alternative embodiment, the gene knockout technique is a Cre-loxp gene knockout technique.
In an alternative embodiment, the method comprises: knock-out by Cre-loxp gene knockout technologyCtnnd1Exon sequence of gene so thatCtnnd1The gene is not expressed or its expression is suppressed in vascular endothelial cells.
In alternative embodiments, the exon sequences are selected fromCtnnd1At least one of exons 1 to 8 of the gene.
In alternative embodiments, the exon sequence is exon 7.
The embodiment of the invention knocks out by using Cre-loxp gene knockout technologyCtnnd1The 7 th exon of the gene, and thus the disease model with the retinal pigment degeneration disease manifestation, can be easily understood by those skilled in the art based on the disclosure of the present inventionCtnnd1The desired effect can also be achieved by knocking out other exons of the gene or suppressing its expression.
In a second aspect, embodiments of the present invention provide a method for breeding an animal model of retinal neovascular disease, wherein the animal model obtained by the method of any one of the preceding embodiments is selfed.
After obtaining the animal model of retinal neovascular disease by using the method described in the first aspect, those skilled in the art can easily think of selfing the animal model of retinal neovascular disease to obtain more offspring, and these also have retinal neovascular disease phenotype, in order to develop more animal models of retinal neovascular disease, and this method for developing animal models of retinal neovascular disease also belongs to the protection scope of the present invention.
In a third aspect, the embodiments of the present invention provide an application of the animal model obtained by the method for constructing the animal model of retinal neovascular disease by using the gene manipulation technology described in any one of the foregoing embodiments in research of retinal neovascular disease.
The animal model of the retinal neovascular disease obtained by the first aspect of the invention has a typical phenotype of the retinal neovascular disease, and can be applied to the research of the retinal neovascular disease, so that the full supply of research materials is ensured, the pathogenic process and mechanism of the retinal neovascular disease are clarified, a new target is provided for the treatment or prevention of the disease, and the model is favorable for promoting the continuous forward progress of the retinal neovascular disease. Therefore, the application of the above animal model of retinal neovascular disease in the research field of retinal neovascular disease also belongs to the protection scope of the present invention.
In an alternative embodiment, the above-described use is not for the purpose of diagnosis or treatment of a disease.
In a fourth aspect, the embodiments provide the use of the animal model obtained by the method for constructing the animal model of retinal neovascular disease by using gene manipulation technology according to any one of the preceding embodiments in screening drugs for preventing or treating retinal neovascular disease.
The animal model obtained according to the first aspect of the present invention may also be used in the field of drug screening, for example, by applying a candidate drug to the above-mentioned animal model of retinal neovascular disease, if the model's retinal neovascular disease phenotype is improved, indicating that the applied candidate drug has a potential as a prophylactic or therapeutic agent for retinal neovascular disease. Therefore, the application of the above animal model for retinal neovascular disease in screening drugs for preventing or treating retinal neovascular disease also belongs to the protection scope of the present invention.
In an alternative embodiment, the above-described use is not for the purpose of diagnosis or treatment of a disease.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1:Ctnnd1construction of vascular endothelial cell conditional knockout mice. A: in mouse vascular endothelial cells using Cre-loxp SystemCtnnd1Seventh exon knockout. B:Ctnnd1and (3) identifying the genotype of the knockout mouse. C: induction time for tamoxifen and mouse treatment time.
FIG. 2:Ctnnd1a breeding roadmap for vascular endothelial cell conditional knockout mice.
FIG. 3:Ctnnd1study of knockout efficiency in mouse blood vessels. A: wild type mice at postnatal day 25 andCtnnd1and (5) carrying out immunoblotting experiment on the lung lysate of the mice by conditional knockout.Ctnnd1For the experimental group, Gapdh is the internal control. B:Ctnnd1statistics of the western blotting result. C: wild type mice at postnatal day 25 andCtnnd1and (3) carrying out real-time fluorescence quantitative PCR (polymerase chain reaction) on lung mRNA of the mice subjected to conditional knockout.
FIG. 4:Ctnnd1and (3) detecting the retinal plaquettes of the knockout mice. Vascular endothelial cell specific knockoutCtnnd1Gene mouse retinaAnd (3) paving and staining results, incubating Isonectin B4 and Ter-119 antibody together. Where the arrows represent red blood cells leaking out of the blood vessel. Vascular endothelial cell specific knockoutCtnnd1Statistical analysis of superficial vascular length development in retinal slides of transgenic mice. C, vascular endothelial cell specific knockoutCtnnd1Statistical analysis of superficial blood vessel density in retinal slides of transgenic mice.
FIG. 5: vascular endothelial cell specific knockoutCtnnd1And (3) immunohistochemical staining results of eyeball sections and vitreous blood vessels of the gene mice. A: wild type at the ninth postnatal day andCtnnd1results of retinal sections of vascular endothelial cell-specific knockout mice. B: wild type at the ninth postnatal day andCtnnd1the result of the vitreous vascular degeneration of mice is specifically knocked out by vascular endothelial cells.
FIG. 6:Ctnnd1counting the number of cells at the top of the retinal plate of the knockout mouse.
FIG. 7:Ctnnd1proliferation experiment of vascular endothelial cells of knockout mice and statistical results thereof.
FIG. 8:Ctnnd1vascular endothelial cell-specific knockdown results in retinal vascular arteriovenous crossing.
In fig. 1-8: p6 Ctrl represents wild-type mice at the sixth postnatal day; p6Ctnnd1 iECKO/iECKORepresents the vascular endothelial cells of the sixth postnatal dayCtnnd1Knocking out a mouse; p9 Ctrl represents wild-type mice at the ninth day after birth; p9Ctnnd1 iECKO/iECKORepresenting vascular endothelial cells at the ninth postnatal dayCtnnd1A knockout mouse.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
In this embodiment, a mouse is used as a target animal, and a model of a retinal neovascular disease animal is constructed by using a Cre-loxp conditional knockout system, in the following way:
(1) mice carrying loxp sequence(s) (ii)Ctnnd1 loxp/loxpThe loxp sequence is positioned at two sides of the 7 th exon, and the target sequence for knockout isCtnnd1Exon 7 of the gene, designatedCtnna1cKO Mouse, purchased from Mutant Mouse Resource&Research Center at University of Californian at Davis, USA) and carryPdgfb-CreTool mouse (turn to)PDGFβ-creTransgenic mice, which specifically express Cre in vascular endothelial cells) to obtain mice carrying loxp sequence and Cre simultaneously, and is expressed asCtnnd1 loxp/+-Pdgfb-CreThen will beCtnnd1 loxp/loxpAndCtnnd1 loxp/+-Pdgfb-Cremating to obtainCtnnd1 Pdgfb-Creloxp/loxp-Mice (see fig. 1 a and fig. 2 for propagation).
（2）Ctnnd1 Pdgfb-Creloxp/loxp-Mice were induced by intraperitoneal injection of 50 μ g Tamoxifen (Tamoxifen) on postnatal days 1, 2, 3, and 5Ctnnd1Knockout (refer to C in FIG. 1), homozygous knockout mice are shownCtnnd1 iECKO/iECKO。
Tamoxifen induction method: preparing an absolute ethanol solution of tamoxifen, wherein the concentration is 10mg/mL, and storing the solution at the temperature of 4 ℃ in a dark place. Before use, a tamoxifen absolute ethanol solution was prepared: the tamoxifen corn oil solution with corn oil =1:9 was vortexed and mixed, and the tamoxifen solution was aspirated by a 1mL sterile syringe, and the mice were injected intraperitoneally.
(3) On the 5 th day after birth (P5), the mice were numbered by the toe-cutting method, and the DNA was extracted by dissolving the tissue by 50. mu.L of 40mM NaOH solution at 95 ℃ for 40min, after cooling, an equal volume of 40mM Tris-HCl pH 5.5 solution was added to adjust the pH, and thereafter, the genotyping PCR reaction was carried out, and the results of the identification gel electrophoresis pattern are shown in FIG. 1B.
For homozygous knockout mice (Ctnnd1 iECKO/iECKO) Amplification thereofCtnnd1The band of (A) is about 440bp, and simultaneously, the band of the amplified Pdgfb-Cre beta is present; amplification of heterozygous miceCtnnd1The two bands are about 400bp of a wild band, the other band is consistent with a homozygous band, and a Pdgfb-Cre beta band can be amplified; a wild mouse homozygous mouse capable of amplifying a 400bp band and incapable of amplifying a Pdgfb-Cre beta band in months (Ctnnd1 iECKO/iECKO) The model can be used as an animal model of the retinal neovascular disease; in other embodiments, the cells can be selfed at a later stage to breed more animal models of retinal neovascular diseases.
The reaction conditions for identifying PCR are as follows: 94 ℃ for 2min, (94 ℃ for 20s, 60 ℃ for 20s, 72 ℃ for 20 s), 34 cycles, 4 ℃ hold, and primers used for identifying the PCR reaction are as follows:Ctnnd1-Forward：5’- AGGGAGAGAGTTCAGTTGGTGAAATG-3’；Ctnnd1-Reverse：5’- CCTCTTCACCAATCATGTCTTCATAGCT-3’；Pdgfb-Cre-Forward：5’-GCCGCCGGGATCACTCTCG-3’；Pdgfb-Cre -Reverse：5’-CCAGCCGCCGTCGCAACTC-3’。
examples of the experiments
Knockout mouse of example 1Ctnnd1 iECKO/iECKOPhenotypic identification of
1 detection by Western blottingCtnnd1Protein expression in lung tissue at day 25 after birth in the target animal.
(1) Collecting lung tissues of a littermate wild type mouse and a knockout type mouse, putting the lung tissues into 1xPBS containing a protease inhibitor, and ultrasonically cracking the lung tissues on ice for 10 minutes; centrifuging at 16000g speed at 4 deg.C for 10 min, transferring the supernatant to another clean centrifuge tube, adding protein loading buffer solution, mixing, and heating at 95 deg.C for 5 min.
(2) After the samples are cooled, 20 mu l of protein samples are respectively taken, and electrophoresis is carried out for 25 minutes at constant pressure of 70v and 40 minutes at 160v by adopting 10% separation gel.
(3) The film transfer conditions are as follows: the transfer was done at constant flow 0.3A for 1 hour 30min, followed by rinsing the membrane once with deionized water and blocking with a 5% skim milk block at room temperature for 1 hour.
(4) The membrane was placed in primary antibody in 5% skim milk blocking solution and incubated overnight in shaker at 4 ℃. The next wash was performed 3 times for 5 minutes with 1 XTSST, and HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour.
(5) An appropriate amount of chemiluminescent substrate was added dropwise to the membrane and the protein band was observed in a chemiluminescent detector (see FIG. 3, panel A). The protein bands were gray-scaled using ImageJ and analyzed for statistical differences using the t-test (see B in figure 3 for results).
2 detection by fluorescent quantitative PCRCtnnd1In the lungs of wild-type and targeted knockout mice at postnatal day 25.
Total lung RNA from wild-type and knockout mice at day 25 after birth was extracted and cDNA was synthesized using a cDNA synthesis kit (Invitrogen, Waltham, MA, USA). According toCtnnd1Designing a primer based on the cDNA sequence of (1):
Q-ms-Ctnnd1-F1：5’- CATGTCTCGGCGCAACTG-3'；
Q-ms-Ctnnd1-R1：5’- GGGTCCGTTGAGTTTCAGAT-3'。
the results of real-time fluorescent quantitative PCR experiments using the ABI 7500 machine using the extracted cDNA as a template and Gapdh as an internal reference are shown in FIG. 3C, which shows that the lung of knockout mice at day 25 after birthCtnnd1The mRNA level decreased significantly by about 85%.
3, detecting the retinal vessel development condition by using a retinal creeping (wall) technology.
The retina slide method is referred to as Chinese patent application with the application number of CN201910397466.2, which is named as a method for constructing a disease model based on a gene operation strategy and an application.
The results are shown in figure 4 at A, B and C,Ctnnd1 iECKO/iECKOthe Ter-119 signal of the mice is shown extravascularly, which indicates thatCtnnd1 iECKO/iECKOLocal leakage of red blood cells occurs in the blood vessels of mice; in addition, as can be seen from the result of B in FIG. 4,Ctnnd1 iECKO/iECKOthe radius of vascular radiation in the mice was about 62%, which was significantly reduced compared to about 85% in the wild-type mice, and in addition, the results of C in fig. 4 show that,Ctnnd1 iECKO/iECKOthe vascular density of the mice is also significantly high at about 72%There was an increase in wild type mice of about 57%, as indicated above,Ctnnd1 iECKO/iECKOthe retinal vascular development of the mice is disturbed.
4 eyeball cryosection staining and vitreous vessel (hyaloid) separation staining.
Eyeball frozen section staining and vitreous blood vessel separation staining method reference application number is CN201910397466.2, is named as method for constructing disease model based on gene operation strategy and Chinese patent application of application, and wild mouse of 9 th day of life are taken outCtnnd1 iECKO/iECKOMouse (i.e., P9 of example 1)Ctnnd1 iECKO/iECKOMouse) was performed.
As a result, as shown in FIG. 5, staining of eyeball sections referring to A in FIG. 5, retinal blood vessels of wild type mice had begun to extend deep into the retina at postnatal day 9 of the mice, andCtnnd1 iECKO/iECKOthe development of the blood vessels of the mice is only limited on the surface of the blood vessels, the development obviously shows a retardation phenomenon, and the density of the blood vessels can be obviously increased from a section. The vitreous vascular staining results are shown in fig. 5, panel B, postnatal day 9,Ctnnd1 iECKO/iECKOthe vitreous vessels of the mice were significantly more abundant than those of the wild type mice, indicating thatCtnnd1 iECKO/iECKOThe vitreous vessel degeneration of the mouse is slowed down.
5 mouse retinal plating apical cell count.
After retinal plating staining, the results are shown in FIG. 6, and the results are shown by detailed analysis of vascular apical cellsCtnnd1 iECKO/iECKOThe number of the retinal vascular apical cells of the mice is obviously increased compared with that of the wild mice, and further shows thatCtnnd1 iECKO/iECKOVascular development disorder in mice.
6, detecting the proliferation condition of the retinal vascular endothelial cells of the mice by using an Edu cell proliferation experiment.
The method reference application number is CN201910397466.2, which is named as a method for constructing a disease model based on a gene operation strategy and an applied Chinese patent application.
Through the EdU staining experiment, it can be seen thatCtnnd1 iECKO/iECKOThe obvious reduction of the number of the vascular endothelial cells in the proliferation stage in the retinal blood vessels of the mice compared with the wild type mice strongly indicates thatCtnnd1 iECKO/iECKOThe proliferation of retinal vascular endothelial cells in the mice was slowed (fig. 7).
7 knockdown in vascular endothelial cellsCtnnd1Causing the retinal vessel arteriovenous crossing of the mouse
Detection ofCtnnd1Vascular endothelial cells knock out arteriovenous crossing in mice. Generally, the distribution of arteries and veins stained by retinal plating blood vessels is judged by sparsely populated periarterial capillaries and densely populated perivenous capillaries. As can be seen in FIG. 8, the arteries and veins of P6 Ctrl mice alternate, extend outward, and do not intersect, whereas P6Ctnnd1 iECKO/iECKO The mouse shows the phenomena of disordered arteriovenous arrangement, crossed arteriovenous vessels and disordered extending direction.
From the results of the above tests, the knockout mouse obtained in example 1 was usedCtnnd1 iECKO/iECKOHas obvious phenotype of retina neovascular diseases, such as retina vascular hypoevolutism, obvious reduction of vascular radiation radius, obvious increase of vascular density, vascular development disorder, local leakage of red blood cells appearing in blood vessels, limitation of retinal vascular development on the surface of blood vessels, multiple vitreous blood vessels, slow degeneration, obvious increase of the number of retinal vascular apical cells, obvious reduction of the number of vascular endothelial cells, disordered arteriovenous arrangement, arteriovenous crossing, disordered extending direction and the like, and indicates that the knockout mouse has the advantages of obvious phenotype of retina neovascular diseases, obvious reduction of the retinal vascular radiation radius, obvious increase of vascular density, blood vessel development disorder, local leakage of red blood cells appearing in blood vessels, obvious limitation of retinal vascular development on the surface of blood vessels, obvious reduction of the number of vascular endothelial cells, disordered arteriovenous arrangement, crossed arteriovenous crossing, disordered extending direction and the likeCtnnd1 iECKO/iECKOIs a relatively ideal animal model of the retinal neovascular disease, and can be used in the fields of research of the retinal neovascular disease, screening of medicaments for preventing or treating the retinal neovascular disease and the like in the later period.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A method for constructing an animal model of retinal neovascular disease by using gene manipulation technology, which is characterized in that the gene manipulation technology is used to make the cells in the vascular endothelial cells of a target animalCtnnd1The gene is not expressed or its expression is inhibited; the target animal is selected from any one of cattle, sheep, horses, rabbits, mice, rats, sheep and pigs,Ctnnd1GenBank accession number of the gene is NM-001085458.
2. The method of claim 1, wherein the genetic manipulation technique is selected from the group consisting of gene knockout techniques.
3. The method of claim 2, wherein the gene knockout technique is a Cre-loxp gene knockout technique.
4. The method of claim 3, wherein the method comprises: knock-out by Cre-loxp gene knockout technologyCtnnd1Exon sequence of gene so thatCtnnd1The gene is not expressed or its expression is suppressed in vascular endothelial cells.
5. The method of claim 4, wherein the exonic sequence is selected from the group consisting ofCtnnd1At least one of exons 1 to 8 of the gene.
6. The method of claim 5, wherein the exon sequences are exon 7.
7. A method for breeding an animal model of retinal neovascular disease, wherein the animal model obtained by the method of any one of claims 1 to 6 is selfed.
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| Endothelial β-Catenin Signaling Supports Postnatal Brain and Retinal Angiogenesis by Promoting Sprouting, Tip Cell Formation, and VEGFR (Vascular Endothelial Growth Factor Receptor) 2 Expression;Agnieszka Martowicz等;《Arterioscler Thromb Vasc Biol》;20191130;第39卷(第11期);2273-2288 * |
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