CN113957074B - Construction method and application of cerebellar ataxia disease model - Google Patents
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Abstract
The invention discloses a construction method and application of a cerebellar ataxia disease model, and relates to the technical field of biology. The construction method is that Wtap genes in target animal genome are not expressed or are inhibited, so that a cerebellar ataxia disease model is obtained. The model can show the characteristics of cerebellar ataxia disease, can be used for researching the cerebellar ataxia disease, can help to elucidate the pathogenesis and mechanism of HA, and provides a new target for treating or preventing the disease. In addition, the invention also provides application of the cerebellar ataxia disease model in research of pathogenesis, mechanism research or screening of medicines for preventing or treating the cerebellar ataxia disease.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for constructing a cerebellar ataxia disease model and application thereof.
Background
Ataxia (Ataxia), also known as hereditary Ataxia (Hereditary Ataxia, HA), is a pathological condition in which patients cannot maintain a fine gait in a certain form, perform fine movements, and any pathology involving the afferent or efferent pathways of the cerebellum may lead to Ataxia, mostly due to genetic factors. Three major features of HA are pathological changes, genetic background of generational phase transmission, ataxia manifestations, which are a group of genetically altered diseases characterized by chronic progressive cerebellar ataxia.
At present, cerebellar ataxia is not known to be particularly effective in treatment, and pathogenesis is not clear.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a method for constructing a cerebellar ataxia disease model and application thereof, so as to provide a cerebellar ataxia disease model, thereby providing a favorable foundation for deeply researching the treatment and etiology of Wtap on cerebellar ataxia.
The invention is realized in the following way:
the invention provides a method for constructing a cerebellar ataxia disease model, which comprises the following steps: so that the Wtap gene in the genome of the target animal is not expressed or is inhibited.
Wtap (Wilms tumours 1-associating protein, MGI: 1926395), this gene is located on mouse chromosome 17 at 13185686-13211430bp, full length 25.7kb, and its cDNA at full length 4967bp, contains 8 exons. Wtap, as part of the methyltransferase complex, is involved in the m6A methylation modification process of RNA, which is widely distributed in various types of tissue cells. The formation process of m6A methylation mainly comprises methyltransferase complex (METTL 3, METT14, WTAP and the like), demethylase (FTO and ALKBH 5) and reading protein (YTDDF 1/2/3, YTDYC 1/2) for dynamic regulation and control, and is closely related to gene expression regulation and control. Second, m6A may be involved in biological processes such as mRNA transcription, selective cleavage, nuclear transport, translation, and degradation, resulting in RNA dysfunction, which in turn affects a range of animal vital activities. At present, the research on WTAP protein functions is gradually increased, including influences on tumorigenesis, organism development and the like, but the detailed action mechanism and the biological functions thereof in retina are not clear, so that the development and application of the WTAP protein are limited.
The inventor finds that the Wtap gene in the genome of a target animal is not expressed or is inhibited, a cerebellar ataxia disease model can be constructed, the model can show characteristics of the cerebellar ataxia disease, such as degenerative disease and loss of Purkinje cells, mainly shown by progressive thinning of a cerebellar Molecular Layer (ML), granulocytopenia and cerebellar atrophy, and the model can be used for research of the cerebellar ataxia disease, can help elucidate the pathogenesis and mechanism of HA, and provides a new target for treatment or prevention of the disease.
The inventors found that WTAP proteins have important functions in the retina and are able to regulate gene expression by m6A methylation of mRNA, thereby affecting purkinje cell function, directly or indirectly affecting purkinje cell survival, resulting in cerebellar ataxia.
In a preferred embodiment of the present invention, the above construction method comprises modification by one or a combination of mutation, deletion and insertion such that the Wtap gene in the genome of the target animal is not expressed or is inhibited.
In one embodiment, the mutation modification means that the corresponding protein site of the Wtap gene is subjected to amino acid change by corresponding mutation, so that the protein expressed by the Wtap gene does not have normal bioactive function or translation is terminated prematurely.
When a deletion modification is used, it may be a deletion of one or more nucleotides, thereby rendering the Wtap gene sequence non-expressed or less active. For example, deletion modification of the target gene may be achieved by deleting a part or all of the exons.
When the insertion modification is adopted, at least one nucleotide can be inserted into the Wtap gene, for example, one or more nucleotides are inserted into an exon to cause frame shift mutation, so that the primary structure, the secondary structure or the tertiary structure of the expressed protein is changed, and the protein expressed by the Wtap gene does not have normal biological activity function.
Thus, whatever modification is chosen, e.g., combination modification, it is within the scope of the present invention to have the target animal's genome not express or to have expression inhibited, so that the animal exhibits the corresponding characteristics of the cerebellar ataxia disease.
In one embodiment, the modification by one or a combination of a mutation, deletion, and insertion results in the full-length or partial sequence of the Wtap gene in the genome of the target animal not being expressed or being inhibited; preferably, the partial sequence is selected from the group consisting of the exon sequences of the Wtap gene.
Preferably, the modification is performed by one or a combination of a mutation, a deletion and an insertion such that at least one of the 1 st to 8 th exons of the Wtap gene in the genome of the target animal is not expressed or is inhibited from being expressed.
In one embodiment, the modification by one or a combination of a mutation, deletion, and insertion results in the non-expression or the inhibition of expression of exon 3 of the Wtap gene in the genome of the target animal.
In a preferred embodiment of the present invention, the target animal is selected from any one of mice, rats, dogs, pigs, monkeys, and apes. One skilled in the art can adaptively select any non-human mammal as a target animal according to needs, and all the non-human mammals belong to the protection scope of the invention.
In a preferred embodiment of the present invention, the above construction method is such that Wtap gene in the genome of the purkinje cells of the target animal is not expressed or is inhibited.
In a preferred embodiment of the invention, the above construction method comprises the combined modification of mutation, deletion or insertion by means of a combination of one or more of the following techniques:
gene knockout technology and gene editing technology;
preferably, the gene knockout technology is Cre-loxP gene knockout technology; the gene editing technology is selected from any one or a combination of a plurality of CRISPR/Cas9 technology, ZFN technology and TALEN technology.
In a preferred embodiment of the invention, the construction method adopts a Cre-loxP gene knockout technology and a CRISPR/Cas9 technology to realize the deletion of Wtap genes, and comprises the following steps:
mating the Wtap gene conditional knockout animal with the Pcp2-cre transgenic animal to obtain the cerebellum purkinje cell knockout Wtap gene animal.
And mating the heterozygote Wtap gene conditional knockout animals to obtain the homozygous Wtap gene conditional knockout animals.
In a preferred embodiment of the invention for use, the cerebellar ataxia disease model described above exhibits at least one of the following characteristics:
(1) Gait disorder, shortened step length, tail hind limb curling, coordination and balancing capacity reduction;
(2) Progressive atrophy of the cerebellum;
(3) Progressive loss of cerebellar purkinje cells;
(4) Astrocytosis occurs in cerebellar purkinje cells.
The cerebellar ataxia disease model obtained by the method for constructing the cerebellar ataxia disease model is applied to the research of cerebellar ataxia diseases, and the research aims at the treatment of non-diseases.
The cerebellar ataxia disease model provided by the invention is beneficial to the research of pathogenesis and mechanism of cerebellar ataxia diseases or the research of screening medicines for preventing or treating cerebellar ataxia diseases.
The invention has the following beneficial effects:
the construction method provided by the invention can obtain a cerebellar ataxia disease model, the model can show the characteristics of the cerebellar ataxia disease, the model can be used for researching the cerebellar ataxia disease, can help to elucidate the pathogenesis and mechanism of HA, and provides a new target for treating or preventing the disease. In addition, the invention also provides application of the cerebellar ataxia disease model in research of pathogenesis, mechanism research or screening of medicines for preventing or treating the cerebellar ataxia disease.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of Wtap gene knockout mouse construction strategy;
FIG. 2 is a diagram of Wtap knockout mice genotyping;
FIG. 3 is a graph showing the result of WB demonstrating the reduced expression level of WTAP protein in the brains of knockout mice;
FIG. 4 is a graph showing the result of Q-PCR that the expression level of Wtap gene mRNA is reduced in the brain of a knockout mouse;
FIG. 5 is Wtap PKO A mouse gait analysis result diagram;
FIG. 6 is Wtap PKO Hind limb contractual figures when mice lift tail;
FIG. 7 is Wtap PKO Mouse coordinationA balancing capability test result graph;
FIG. 8 is Wtap PKO Mice developed a cerebellar contraction pattern;
FIG. 9 is Wtap PKO A graph of purkinje cell loss results from atrophy of the mouse cerebellum molecular layer;
FIG. 10 is Wtap PKO Results of progressive reduction of mouse cerebellum purkinje cells;
FIG. 11 is Wtap PKO Results of significant astrocyte increase in mouse cerebellum.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The embodiment provides a method for constructing a cerebellar ataxia disease model, and the embodiment uses a mouse as a target animal. FIG. 1 is a diagram of a mouse knockout strategy, wtap conditional gene knockout mouse (designated Wtap cko, loxp sequence flanking exon 3, target sequence for knockout is exon 3 of Wtap gene) purchased from Shanghai's model biotechnology Co., ltd (Shanghai Model Organisms Center, inc)
(https://www.modelorg.com/portal/article/index/id/3724/post_type/3.html)。
2) Mating and breeding the heterozygote Wtap gene knockout mice obtained in the step 1) to obtain Wtap gene knockout homozygote mice.
3) Mating the Wtap gene conditional knockout homozygote animal obtained in the step 2) with a Pcp2-Cre transgenic animal (Pcp 2-Cre transgenic mouse (MGI: 2137515) purchased from Jackson laboratories (The Jackson Laboratory)) to obtain the cerebellum Purkinje cell Wtap conditional knockout homozygote animal, namely the model of ataxia disease.
Example 2
In this example, the offspring mice were genotyped using the PCR method.
The method comprises the following steps:
1) A small amount of tail tip samples of the mice are cut and placed in a 1.5ml clean centrifuge tube;
2) Adding 100ul of lysate (40mM NaOH,0.2mM EDTA solution) into the centrifuge tube, and heating the metal bath at 100 ℃ for 1h;
3) After cooling to room temperature, 100. Mu.l of a neutralization solution (40 mM Tris-HCl, pH 5.5) was added to the centrifuge tube, and 10000g was centrifuged for 2min to obtain a supernatant for genotyping of mice.
4) And (3) PCR amplification: the reaction system is as follows:
2×Taq Mix 10μL
tissue lysate 4. Mu.L
Upstream primer (Wtap-loxP-Forward (abbreviated as F2) or Pcp2-Cre-Forward (abbreviated as CreF)), 1. Mu.L (concentration: 10 mM)
Downstream primer (Wtap-loxP-Reverse (abbreviated as R2) or Pcp2-Cre-Reverse (abbreviated as CreR)), 1. Mu.L (concentration: 10 mM)
ddH 2 O 6μL。
The primer sequences were as follows:
F2：5’-GACGGCTCAAGGATGCAAAT-3’；
R2：5’-TCGGTTAACAGTGCTTGGAAC-3’；
CreF：GAACGCACTGATTTCGACCA；
CreR：GCTAACCAGCGTTTTCGTTC。
4) Amplification procedure:
after the system is prepared, the PCR instrument is preheated at 95 ℃ for 5min to fully denature DNA, and then the amplification circulation is carried out. The cycle conditions were 95℃for 20s to denature the template, 60℃for 20s to anneal the primer and template sufficiently, 72℃for 25s to extend the primer on the template, and the number of cycles was 35 to accumulate a large amount of amplified DNA fragments. Finally, the product was extended to completion at 72℃for 5min and stored at 4 ℃.
5) Gel electrophoresis
4.5g of agarose was weighed into 150ml of 1 XTAE buffer and melted in a microwave oven to prepare 3% agarose gel. 10ul of PCR product was taken in the wells and subjected to 160V constant pressure agarose electrophoresis for 30min. And then imaged with a gel imaging system.
The upper panel in FIG. 2 is the Wtap conditional knockdown assay, WT represents wild-type control, band size is 249bp; het represents a heterozygote with two bands 249bp and 283bp; KO represents homozygote and the band size is 283bp. The lower panel in FIG. 2 is the Pcp2-Cre assay result. The size of Pcp2-Cre is 232bp. Based on the results of FIG. 2, the genotype of the neonatal mice can be effectively identified for subsequent investigation. From preliminary determinations of FIG. 2, the homozygous Wtap conditional knockout model in example 1 (Wtap PKO Mice) were constructed successfully.
Example 3
Immunoblotting (Western blot) experiments analysis of gene knockout efficiency in the brains of Pcp2-Cre knockout mice.
The method comprises the following steps:
1) Brains of control and knockout mice were obtained after conventional sacrifice, and the tissues were thoroughly ground and added with 200ul of protein lysate RIPA.
2) After cell disruption by sonication, the cells were lysed on ice for 20min.
3) Centrifuge at 4℃for 10min at 16000g, transfer the supernatant to another clean centrifuge tube, add 50. Mu.l protein loading solution, mix well and heat at 95℃for 5min.
4) After the sample was cooled, 20. Mu.l of each sample was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) at 160V to separate proteins.
5) After SDS-PAGE is finished, cutting a nitrocellulose membrane with proper size, paving filter paper, glue, the nitrocellulose membrane and the filter paper according to a certain sequence, removing bubbles, and then carrying out ice water bath and constant-current 0.28A membrane transfer for 2 hours.
6) After the film transfer is finished, the film is washed by pure water, dried and marked, and 8% of skimmed milk is sealed for 2 hours.
7) After blocking was completed, primary antibodies were added to blocking solution (8% skim milk) at a certain ratio (according to the instructions for antibody use) and incubated overnight at 4 ℃.
8) After recovery of primary antibodies, membranes were washed 3 times with 1 XTBE buffer for 10min each, appropriate secondary antibodies were selected depending on the source of primary antibody, horseradish peroxidase (HRP) -labeled secondary antibodies were diluted with 1 XTBE and incubated in a shaker at room temperature for 2h.
9) After the secondary antibody incubation, the membrane was washed 3 times with 10min each time using a 1 XTBST, and the protein was detected using a thermo ELC luminescence kit, the instrument being a Bio-Rad chemiluminescent gel imaging system.
Pcp2-Cre is expressed in the cerebellum Purkinje cells, and a three-number exon between two loxP loci is removed, so that Wtap protein is inactivated, and the Wtap gene is specifically knocked out in the cerebellum Purkinje cells, and the gene knocked-out homozygote mouse Wtap is obtained. Immunoblotting (western blot, WB) demonstrated the presence of a DNA sequence in Wtap PKO In mouse cerebellum purkinje cells, wtap protein was specifically knocked out (fig. 3).
Example 4
The gene knockout efficiency in the brains of Pcp2-Cre knockout mice was analyzed by real-time fluorescent quantitative PCR (Quantitative Real-time PCR, Q-PCR).
The method comprises the following steps:
1Trizol method for extracting RNA
1) Brains of control and knockout mice were obtained after conventional sacrifice, and tissues were sufficiently ground and added with Trizol 1ml as an RNA extraction reagent.
2) After sufficient lysis, 200ul of chloroform was added to dissolve RNA in the aqueous phase and left to stand for 10min.
3) Centrifuge 16000g for 10min at 4 ℃, transfer the upper aqueous phase to another clean centrifuge tube and add 0.5ml isopropyl alcohol. RNA was pelleted at 4℃and 16000g for 10min. 4) After the supernatant was sucked, 1ml of absolute ethanol was added, and the mixture was centrifuged at 16000g for 10min to wash; 1ml of 75% ethanol was then added and the mixture was washed by centrifugation at 16000g for 10min at 4 ℃.
Sucking the supernatant, airing at room temperature, and adding a proper amount of RNase-free water to redissolve RNA. 7) After determining the RNA concentration using Thermo NanoDrop, 1ug of RNA was reverse transcribed into cDNA (according to the proposed system of reagents).
2 real-time fluorescent quantitative PCR
The cDNA was diluted 20-fold with ddH2O for qPCR and its amplified primer sequences were as follows:
the system is configured as follows:
SYBR Green Premix TaqTm 5ul；
the reaction system is evenly mixed, instantly centrifuged and then is put into a qPCR instrument for detection, and the procedure is set as follows:
94℃、30s；94℃、5s；60℃、30s(go to 2for 40cycle)；4℃、Hold。
3) According to 2 -△(△Ct) The relative expression level of Wtap is calculated by a statistical method, and Gapdh is used as an internal reference gene.
Real-time fluorescent quantitative PCR (Quantitative Real-time PCR, Q-PCR) demonstrated a PCR reaction at Wtap PKO mRNA expression of Wtap was reduced in mouse cerebellum purkinje cells (fig. 4).
Example 5
The gait analysis experiment of the mice detects the action balance ability.
The method comprises the following steps: a white recording paper is paved on a runway with the length of 50cm and the width of 5cm, the front paws of the mice are dyed with red ink, and the rear paws are dyed with black ink, so that the mice run from one section to the other end of the runway rapidly. Multiple consecutive footprints were measured and the average of two groups of mice was recorded and the results are shown in figure 5.
FIG. 5 shows that Wtap knockout mice have symptoms of cerebellar ataxia, manifested as gait disturbances, a shortened step size.
Example 6
The mouse tail suspension experiment detects the characterization of neurodegenerative diseases.
The method comprises the following steps: the mice were lifted and observed for hind limb extension, and when hind limb contracture exceeded 3 seconds, the recordings were taken, and the results are shown in figure 6.
FIG. 6 shows that Wtap knockout mice have symptoms of cerebellar ataxia, manifested by hind limb contracture when lifted.
Example 7
The mouse stick-turning experiment detects the action balance ability.
The method comprises the following steps: the mice were placed on a stationary rotarod apparatus, starting at 1r/min, reaching 30r/min within 3min, and the residence time of the mice on the rotarod apparatus was recorded. Each mouse was tested three times, 30min apart, and the longest residence time was recorded, with the results shown in figure 7.
FIG. 7 shows that Wtap knockout mice have symptoms of cerebellar ataxia, which are manifested as a decrease in coordination and balance ability.
Example 8
The mice brains were stained by paraffin section, hematoxylin-eosin staining (H & E staining method) as follows:
1) Rapidly taking mouse brain tissue, and fixing the mouse brain tissue in a fixing solution for 24 hours;
2) Embedding paraffin, slicing with thickness of 4 μm;
3) Slices were conventionally dewaxed with xylene, washed with multi-stage ethanol to water: xylene (I) 5 min- & gt xylene (II) 5 min- & gt 100% ethanol 2 min- & gt 95% ethanol 1 min- & gt 80% ethanol 1 min- & gt 75% ethanol 1 min- & gt distilled water washing 2min;
4) Hematoxylin staining for 5 minutes, washing with tap water;
5) Ethanol hydrochloride differentiation for 30 seconds;
6) Soaking in tap water for 15 minutes;
7) And (5) placing eosin solution for 2 minutes.
8) Conventional dehydration, transparency and sealing sheet: 95% ethanol (I) 1min, 95% ethanol (II) 1min, 100% ethanol (I) 1min, 100% ethanol (II) 1min, xylenol carbonic acid (3:1) 1min, xylene (I) 1min, xylene (II) 1min and neutral resin sealing.
9) And photographing under a microscope.
As a result, as shown in fig. 9, it was found that the Molecular Layer (ML) where purkinje cell dendrites are located in the brains of 2 month old knockout mice was significantly reduced in thickness (fig. 9a, b), and it was revealed that the number of purkinje cells (yellow arrows) was significantly reduced (fig. 9a, c). Indicating that the molecular layer in which purkinje cells reside is severely atrophic and the number of purkinje cells is decreased.
Example 9
The brain pathological changes of the Pcp2-Cre knockout mice are analyzed by a cerebellum frozen section immunostaining experiment.
The method comprises the following steps:
1) The cerebellum specific knockout Wtap gene mice constructed in the example 1 suitable for the month of age were taken, and after neck-broken and sacrificed, the cerebellum was quickly taken and placed in 4% PFA for overnight fixation at 4 ℃.
2) Washing with PBS buffer solution for 3 times, and dehydrating cerebellum in 30% sucrose solution overnight;
3) Embedding and slicing: OCT is embedded and quickly frozen in a refrigerator at-80 ℃. After about 10min, the OCT embedded cerebellum was removed and placed in a frozen microtome at-25℃for about 30min to obtain sections with a thickness of 12. Mu.m.
4) After slicing, higher quality plates were selected and placed in an oven at 37 ℃ for 30min, the immunohistochemical pen was circled around the area with cerebellum tissue, washed 3 times with PBS buffer to remove OCT,5% NDS (containing 0.25% triton) blocked through 2h, primary antibodies incubated, and overnight at 4 ℃. Washing 3 times by PBS buffer solution, incubating the corresponding fluorescent secondary antibodies, then washing three times by PBS, sealing, and observing.
Brains of 2 month old knockout mice were dissected and the knockout mice were found to undergo progressive atrophy of brains with age (fig. 8). Frozen section, immunohistochemical staining analysis of brains of knockout mice of different ages, staining for purkinje cell marker protein Calbindin-D28K, found that purkinje cells gradually decreased in brains with age in the knockout mice, and almost all purkinje cells died in the knockout mice at 12 weeks after birth (fig. 10).
To further investigate the effect of knockout Wtap on the cerebellum, we performed immunohistochemical staining analysis on frozen sections of the cerebellum of knockout mice. Astrocytes are the most abundant cell type in the central nervous system and have important functions in providing metabolic and nutritional support of the central nervous system, maintaining normal neuronal function, etc. Glial acidic protein (glial fibrillary acidic protein, GFAP) is the primary intermediate fibrin of astrocytesThe expression level of GFAP is up-regulated when the central nervous system is damaged. Therefore, we performed GFAP staining on the frozen sections of cerebellum, found that knockout of Wtap resulted in a significant increase in GFAP expression, damage to the central nervous system, wtap PKO Astrocytes were significantly increased in the mouse cerebellum (fig. 11).
Taken together, it can be seen that the embodiment of the invention takes a mouse as an example, and specifically knocks out the Wtap gene in the cerebellum of the mouse by the Cre-loxP knocking-out technology, so that the mouse shows typical characteristics of cerebellar ataxia: gait instability, cerebellar atrophy, purkinje cell progressive apoptosis and the like. Thus, it is fully demonstrated that conditional knockout of the Wtap gene in the mouse cerebellum can cause the target animal to exhibit a cerebellar ataxia disease phenotype. The animal model can be used as cerebellar ataxia disease model. The disease model can be used in the cerebellar ataxia research field, and provides a new model for the research of the disease, such as the pathogenesis, mechanism and screening of related drugs.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (12)
1. A method of constructing a model of cerebellar ataxia disease, comprising: such that in the genome of a mouse purkinje cellWtapThe gene is not expressed.
2. The method of claim 1, wherein the method of constructing comprises modifying the genome of the mouse by one or a combination of mutation, deletion and insertionWtapThe gene is not expressed.
3. The method of constructing according to claim 1, wherein the mouse group is modified by one or a combination of mutation, deletion and insertionIn the genomeWtapThe full-length sequence of the gene is not expressed.
4. The method of constructing according to claim 1, wherein the modification is performed by one or a combination of mutation, deletion and insertion so as to be in the genome of the mouseWtapAt least one of the exon 1 to exon 8 sequences of the gene is not expressed.
5. The method according to claim 4, wherein the modification is performed by one or more of mutation, deletion and insertionWtapExon 3 of the gene is not expressed.
6. The method of construction according to claim 1, characterized in that it comprises a combined modification of mutation, deletion or insertion by gene knockout technology.
7. The construction method according to claim 6, wherein the gene knockout technology is any one or a combination of several of Cre-loxP gene knockout technology, CRISPR/Cas9 technology, ZFN technology and TALEN technology.
8. The construction method according to claim 7, wherein the construction method is implemented by using Cre-loxP gene knockout technology and CRISPR/Cas9 technologyWtapA deletion of a gene comprising:
Wtapmating the conditional knockout mouse with the Pcp2-cre transgenic mouse to obtain the cerebellum purkinje cell knockoutWtapGene mice.
9. The construction method according to claim 8, wherein,Wtapthe conditional knockout mice of the gene were obtained by:
mice are put intoWtapCo-injecting donor vector of gRNA of gene and Cas9 mRNA into fertilized ovum to obtainWtapFirst-established mice with conditional gene knockouts, howeverMating the first-established mice with wild mice to obtain heterozygotesWtapConditional knockout of the gene in mice and heterozygoteWtapThe conditional knockout mice are mutually mated to obtain homozygoteWtapConditional knockout mice of the gene.
10. The method of construction according to any one of claims 1-9, wherein the cerebellar ataxia disease model exhibits at least one of the following characteristics:
(1) Gait disorder, shortened step length, tail hind limb curling, coordination and balancing capacity reduction;
(2) Progressive atrophy of the cerebellum;
(3) Progressive loss of cerebellar purkinje cells;
(4) Astrocytosis occurs in cerebellar purkinje cells.
11. Use of a cerebellar ataxia disease model obtained by the method of construction of a cerebellar ataxia disease model according to any of claims 1-9, in a cerebellar ataxia disease study, wherein the study is aimed at the treatment of non-diseases.
12. The use according to claim 11, wherein the study is a cerebellar ataxia disease pathogenesis, a mechanism study or a study to screen for a medicament for the prevention or treatment of a cerebellar ataxia disease.
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