AU2018357499A2

AU2018357499A2 - Method for constructing ataxia animal model and application thereof

Info

Publication number: AU2018357499A2
Application number: AU2018357499A
Authority: AU
Inventors: Shujin LI; Fang Lu; Mu Yang; Yeming YANG; Lin Zhang; Shanshan Zhang; Xianjun ZHU
Original assignee: Sichuan Provincial Peoples Hospital
Current assignee: Sichuan Provincial Peoples Hospital
Priority date: 2017-10-26
Filing date: 2018-09-18
Publication date: 2019-12-19
Also published as: CN107586791B; CN107586791A; WO2019080667A1; AU2018357499A1; AU2018102151A4

Abstract

Provided is a method for constructing an ataxia animal model, which comprises knocking out a target sequence on a genome of the cerebellar Purkinje cells of a target animal; the target sequence is the Tmem30a gene. Also provided are an animal model of the cerebellar ataxia constructed by the described method and a method for screening a medicament used for preventing or treating an ataxia disease using the animal model.

Description

METHOD FOR CONSTRUCTING ATAXIA ANIMAL MODEL AND APPLICATION

The application claims priority to Chinese Patent Application No. 201711012105.9 filed on October 26, 2017 and entitled METHOD FOR CONSTRUCTING ATAXIA ANIMAL MODEL AND APPLICATION, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The disclosure relates to the technical field of medical engineering, and in particular to a method for constructing an ataxia animal model and an application.

BACKGROUND

Ataxia is a kind of pathological state that patients are not able to maintain fine gait and complete precise movement in a certain form, and it is possibly caused by any lesion related to cerebellum afferent and efferent ways. Most ataxia are caused by genetic factors, therefore, it is collectively called Hereditary ataxia (HA). HA is a kind of hereditary disease featured by chronic progressive cerebella ataxia. HA exhibits major three characteristics of pathological changes, namely, hereditary background, ataxia representation and cerebellar damage. HA classification has not been clear. Although there are more than 60 reports up to now, there is no uniform and accepted method. Based on pathogenic sites, it is divided into four types: (1) deep sensory disturbance ataxia; (2) cerebellar ataxia; (3) vestibular labyrinth ataxia; (4) cerebral ataxia. Cerebellar ataxia is a major type of HA and belongs to a broad class of nervous system degenerative disease with genetic heterogeneity. Its major symptoms are instability of gait and weakness of limbs with cerebellar symptoms as its outstanding feature, and accompanied with cognitive disorder. There are diversified genetic models of cerebellar ataxia, including autosomal dominant, recessive heredity, X-linkage heredity and other various modes as well as some sporadic cases. At present, more than 30 types of cerebellar ataxia have been found, accounting for about 10%-15% of nervous system hereditary diseases.

The clinical symptom of cerebellar ataxia varies in a wide range, and even shows high heterogeneity in the same family. Moreover, clinical symptom of cerebellar ataxia caused by different disease genes often overlapped. Therefore, it is very difficult to conduct precise clinical classification due to largely overlapping clinical phenotypes. Therefore, to achieve correct diagnosis, cerebellar ataxia must rely on genetic diagnosis. Complicated relationship between the diversified phenotypes and numerous disease genes causes serious problems for clinical treatment. In addition, there is no specific effective therapeutic method to cerebellar ataxia. The main reason is lack of detailed pathogenesis studies. In the future, the diagnosis and treatment of cerebellar ataxia depends on discovery of disease genes and mechanistic studies.

Suitable animal models for ataxia research are vital to mechanistic studies and therapeutic development. However, currently available animal models cannot meet the rising demand.

In view of this, this disclosure is proposed herein.

i

SUMMARY

An objective of the disclosure lies in including but not limited to provide a method for constructing an ataxia animal model, namely, a cerebellar ataxia animal model of specifically knocking out Tmem30a gene from cerebellar Purkinje cells is constructed by the construction method, thus studying functions thereof in cerebellar Purkinje cells.

Another objective of the disclosure lies in including but not limited to provide an ataxia animal model obtained by the above construction method.

Another objective of the disclosure lies in including but not limited to provide an application of the ataxia animal model obtained by the above construction method in ataxia study.

The disclosure is achieved below:

A method for constructing an ataxia animal model, knocking out a target sequence on a genome of a cerebellar Purkinje cell of a target animal;

The target sequence is a Tmem30a gene.

An application of the ataxia animal model obtained by the above method for constructing an ataxia animal model is applied in ataxia study.

The ataxia animal model obtained by the above method for constructing an ataxia animal model is applied in screening drugs for preventing or treating ataxia diseases.

Based on an ataxia animal model, the Tmem30a gene on the genome of the cerebellar Purkinje cells of the target animal is knocked out.

The disclosure has the following beneficial effects:

Based upon the method for constructing an ataxia animal model provided by the disclosure, a cerebellar ataxia animal model may be constructed by specifically knocking out a target sequence on a genome of a cerebellar Purkinje cell of a target animal. The animal model shows typical characteristics of cerebellar ataxia: instability of gait, cerebellar atrophy, progressive apoptosis of Purkinje cells, etc. The model may be used for studying cerebellar ataxia to provide basis for the discovery of cerebellar ataxia genes and the exploration of pathogenesis.

BRIEF DESCRIPTION OF THE DRAWINGS

To describe the technical solution of embodiments of the disclosure, the drawings required in the embodiments are described briefly, it should be understood that the following drawings merely show some embodiments of the disclosure, therefore, these are not considered to define the scope, other related drawings may be obtained by those skilled in the art based on those drawings without creative efforts.

FIG. 1 is a diagram showing a route for constructing a specific cerebellar Purkinje cell Tmem30a knockout model provided by embodiments of the disclosure and identification of the knocked-out mouse genotype.

FIG. 2 is a schematic diagram showing the construction of tdTomato reporter genes in cerebellar Purkinje cells provided by embodiments of the disclosure (in the figure: tdTomato is a reporter gene Tomato of a red transgenic fluorescent protein. There is a terminator sequence in front of an initial codon of the red fluorescent protein; both ends of the terminator carry aligned LoxP loci, and when Cre enzyme is present, the terminator is deleted, the red fluorescent protein may be expressed and Cre positive cells are labelled red).

FIG. 3 shows immunofluorescent staining (Immunohistochemistry, IHC) provided by the embodiments of the disclosure, indicating that Pcp2-Cre is specifically expressed in mouse cerebellar Purkinje cells (in the figure: GCL: Granular cell layer, granular cell layer; PCL: Purkinje cell layer, Purkinje cell layer: ML: Molecular layer, molecular layer. Calbindin is a protein expressed in Purkinje cells).

FIG. 4 verifies the reduced expression level of Tmem30a protein in knocked-out mouse cerebellum by Western blot (Western blot, WB) provided by the embodiments of the disclosure (in the figure: WT: Wildtype, wild-type control: KO: Knockout, gene knockout mutation; Cerebrum: cerebrum; Cerebellum: cerebellum).

FIG. 5 shows a picture of the Purkinje cell specific Tmem30a knockout mouse provided by the embodiments of the disclosure when its tail is lifted to curl up hind legs (in the figure: 6mo refers to 6 month-age.)

FIG. 6 shows a picture of cerebellar atrophy of the Tmem30a KO mouse provided by the embodiments of the disclosure (in the figure: lOmo refers to 10 month-age.)

FIG. 7 shows a result of paraffin section staining provided by the embodiments of the disclosure.

FIG. 8 shows progressive decrease of cerebellar Purkinje cells of the Tmem30a KO mouse provided by the embodiments of the disclosure.

FIG. 9 is a diagram showing immunohistochemical staining of endoplasmic reticulum stress marker proteins Chop and Pdi in the Tmem30a KO cerebellum provided by the embodiments of the disclosure; (In the figure, A shows the expression of the endoplasmic reticulum stress marker proteins C/EBP homologous protein (Chop) in Tmem30a KO cerebellum slices. The arrowhead indicates cells occurring endoplasmic reticulum stress. It can be seen that Chop protein expressing cells in the Tmem30a KO cerebellum increase significantly. In the figure, B shows the expression of the endoplasmic reticulum stress marker proteins Protein Disulfide Isomerase (Pdi) in Tmem30a KO cerebellum slices. The arrowhead indicates cells occurring endoplasmic reticulum stress. It can be seen that Pdi expressing cells in the Tmem30a KO cerebellum increase significantly. In the figure, C is quantification of Chop and Pdi protein expression levels in panel A and B).

DETAILED DESCRIPTION

To describe the objectives, technical solutions and advantages of the embodiments of the disclosure more clearly, the technical solutions in the embodiments of the disclosure will be described more clearly and integrally here. Any non-indicated in the embodiments shall be performed in accordance with conventional conditions or the conditions recommended by the manufacturer. Any reagent or instrument without manufacturer shall be conventional products commercially purchased available.

The method for constructing an ataxia animal model and application of the embodiments of the disclosure will be described in detail.

The typical neuropathologic change of cerebellar ataxia is cerebellar atrophy, degenerative change and loss of Purkinje cells. Purkinje cell (PC) is a neuron capable of achieving efferent impulse sent from cerebellar cortex. PC axon reaches to deep cerebellum nucleus after through a granular layer and white matter, and it plays an important role in motor coordination.

ATP8A2 (a P4-ATP enzyme) gene mutations may cause cerebellum dysfunction, and patients suffer a symptom of cerebellar ataxia, indicating that the activity of the P4-ATP enzyme is closely related to cerebellar ataxia. P4-ATP enzyme is a kind of enzyme with phospholipid flippase activity in cytomembrane, also called phospholipid flippase, and it plays an important role in keeping the distribution of bilateral phospholipids in cytomembrane asymmetry. The asymmetrical distribution of the phospholipids at both sides of the cytomembrane is crucial to maintain stable membrane micro environment, exertion of membrane protein functions, transport of cell vesicae, cell polarity, cell apoptosis, etc. Some of the phospholipids are collectively distributed on one side of cytoplasm, e.g., PE and PS, and such asymmetrical distribution depends on the activity of the P4-ATP enzyme in cytomembrane. The genome of mammals encodes 14 different P4-ATP enzymes, and the defect of the P4-ATP enzyme may cause various diseases, indicating its great importance in vivo.

Cdc50 family (Cdc50a, b, c, also called Tmem30a, b, c) may interact with P4-ATP enzymes, and it is essential to the correct folding and subcellular localization of P4-ATP enzymes, therefore, it is regarded as β subunits of P4-ATP enzymes. There are 14 kinds of P4-ATP enzymes in animal's body, indicating that the enzymes have redundant functions and interact with each other in tissues; therefore, it is difficult to research the functions.

But there are only three β subunits, and most of P4-ATP enzymes interact with Tmem30a, therefore, to study the functions of P4-ATP enzymes and specify the pathogenesis of cerebellar ataxia, studying β-subunit Tmem30a is the best choice.

Based on the above facts, the discloser of the disclosure constructs a specific cerebellar Purkinje cell Tmem30a knockout mouse model, thus providing a basis for the intensive study on its pathogenesis of cerebellar ataxia.

In one aspect, embodiments of the disclosure provide a method for constructing an ataxia animal model, knocking out a target sequence on a genome of a cerebellar Purkinje cell of a target animal; where the target sequence is the Tmem30a gene.

Further, in some embodiments of the disclosure, the target sequence is an exon sequence of the Tmem30a gene.

The Tmem30a gene has 4 exons (i.e., No.l exon, No.2 exon, No.3 exon, No.4 exon), therefore, any one or more of the exons can be knocked out to make the Tmem30a gene inactive, thus achieving knockout.

Further, in some embodiments of the disclosure, the target animal is selected from any of mouse, rat, dog, monkey and ape.

Further, in some embodiments of the disclosure, the target animal is a mouse, and the sequence of the exon is the sequence of No. 3 exon.

Certainly, in other embodiments of the disclosure, the sequence of No. 1 exon, the sequence of No. 2 exon, the sequence of No. 4 exon, or a combination thereof may be optionally knocked out, which belongs to the protection scope of the disclosure.

Further, in some embodiments of the disclosure, the target sequence is knocked out by Cre-loxP knockout technology.

Generally, there are lots of gene knockout technologies, such as CRISPR/Cas9. Therefore, CRISPR/Cas9 or other technological means can be taken to knock out Tmem30a gene in other embodiments, belonging to the protection scope of the disclosure as well.

Certainly, in some embodiments of the disclosure, a conditional Tmem30a gene knockout homozygote mouse mates with a Pcp2-Cre transgenic mouse to obtain a conditional cerebellar Purkinje cell Tmem30a gene knockout mouse.

It should be noted that the obtaining of the conditional Tmem30a gene knockout homozygote mouse can be referred to a Chinese patent application 2017103803265, titled construction method of a conditional pancreatic β cell Tmem30a gene knockout mouse model and application.

In another aspect, embodiments of the disclosure further provide an application of the ataxia animal model obtained by the above method for constructing an ataxia animal model in ataxia study.

Further, in some embodiments of the disclosure, the study is a study on the pathogenesis or pathogenic mechanism of human ataxia.

In a further aspect, the disclosure provides an application of the ataxia animal model obtained by the above method for constructing an ataxia animal model in screening drugs for preventing or treating ataxia diseases.

Still further, in some embodiments of the disclosure, the application includes: a candidate reagent is applied in the ataxia animal model;

if it is detected that one or more of the following phenomena occurs in the ataxia animal model after applying the candidate reagent, it indicates that the candidate reagent may serve as a drug for preventing or treating ataxia diseases:

phenomenon (1): compared with the ataxia animal model without the candidate reagent, the ataxia animal model with the candidate reagent shows: normal or more stable gait, or hind legs do not curl up when its tail is lifted or curling degree is lower;

phenomenon (2): compared with the ataxia animal model without the candidate reagent, the ataxia animal model with the candidate reagent shows: no progressive atrophy occurs in cerebellum or atrophy degree is lower with age;

phenomenon (3): compared with the ataxia animal model without the candidate reagent, the ataxia animal model with the candidate reagent shows: no decrease of Purkinje cells occurs in cerebellum or the decrease scope of Purkinje cells is lower with age;

phenomenon (4): compared with the ataxia animal model without the candidate reagent, the ataxia animal model with the candidate reagent shows: the expression quantity of endoplasmic reticulum stress marker protein CHOP is lower.

In a further aspect, the disclosure provides an ataxia animal model, and the Tmem30a gene in the genome of a cerebellar Purkinje cell of the animal model is knocked out.

Further, in some embodiments of the disclosure, the target animal is mouse, rat, dog, monkey and ape.

Further, in some embodiments of the disclosure, any of No. 1 exon, No. 2 exon, No. 3 exon and No. 4 exon of the Tmem30a gene in the genome of the cerebellar Purkinje cells of the animal model is knocked out or multiple exons are knocked out at the same time.

Further, in some embodiments of the disclosure, the animal model is a mouse, No. 3 exon of the Tmem30a gene in the genome of the cerebellar Purkinje cells of the animal model is knocked out.

In a further aspect, the disclosure provides an application of the above ataxia animal model in ataxia study.

In a further aspect, the disclosure provides a method for screening drugs for preventing or treating ataxia disease, including: applying a candidate reagent to the above ataxia animal model.

Further, in some embodiments of the disclosure, if it is detected that one or more of the following phenomena occurs in the ataxia animal model after applying the candidate reagent, it indicates that the candidate reagent may serve as a drug for preventing or treating ataxia diseases:

Features and performance of the disclosure will be further described in detail with reference to the embodiments.

Embodiment 1

In this embodiment, mouse served as a target animal, set as an example to describe the method for constructing an ataxia animal model provided by the disclosure, specifically as follows.

1. A Tmem30a gene (No. 3 exon) conditional knockout homozygote mouse was obtained by reference to a Chinese patent application 2017103803265, titled construction method of pancreatic β cell Tmem30a gene conditional knockout mouse model and application;

2. The conditional Tmem30a gene knockout homozygote mouse mated a Pcp2-Cre transgenic mouse to obtain a conditional cerebellar Purkinje cell Tmem30a gene knockout mouse (FIG. 1A). As shown in FIG. 1A, half of the descendants of the Pcp2-Cre transgenic mouse and conditional Tmem30a gene knockout homozygote mouse carried Pcp2-Cre and Tmem30a conditional knockout at the same time. The animal mated again with the conditional Tmem30a gene knockout homozygote mouse to obtain a specific cerebellar Purkinje cell Tmem30a knockout mutation animal (represented by Tmem30a KO).

Cre enzyme was expressed in cerebellar Purkinje cells of Pcp2-Cre gene to achieve the effect of conditional Tmem30a gene knockout of cerebellar Purkinje cells.

3. The Tmem30a conditional knockout mouse was identified by primers 2: 5’-TGGTTAGGAACACATAGAACAAA-3’ and R2: 5’-CTAAGGGGCTGGTATGGGAAT-3’. Cre genotype was identified by primers CreF: 5’-ATTTGCCTGCATTACCGGTC-3’; Cre-R: 5’-ATCAACTGGTTCTTTTCGG-3’, and results were shown in FIG. IB. Results of FIG. 1 show that (in the figure: WT denotes wildtype mouse, Tmem30a loxp/loxp, Pcp2-Cre denotes conditional cerebellar purkinje cell Tmem30a gene knockout homozygote mouse (abbreviated for Tmem30a KO) to obtain conventional cerebellar Purkinje cell Tmem30a gene knockout homozygote mouse.

Embodiment 2

To determine recombination efficiency of Cre-mediated loxP loci, tdTomato reporter gene was constructed in this embodiment (FIG. 2). Without Cre expression, a STOP gene cassette carrying loxP recombination loci at both sides can prevent the expression of the downstream red fluorescent protein tdTomato. In the presence of Cre, the STOP gene cassette is removed from the Cre specifically-expressed tissues, resulting in the expression of tdTomato. Therefore, the expression pattern of a Cre recombinase may be determined by observing the fluorescence brought by tdTomato expression. An immunohistochemistry was used to observe the specific expression of Cre in Tmem30a KO animal in cerebellar Purkinje cells (as shown in FIG. 3). In FIG. 3: GCL: Granular cell layer, granular cell layer; PCL: Purkinje cell layer; ML: Molecular layer.

Immunohistochemistry:

A mouse was killed by cervical dislocation, then eyeball was taken and fast put into 4% PFA on ice to fix for 15 min, and an incision was cut on the cornea, then fixation was performed continuously on ice. After 2 h, it was washed by a PBS buffer solution, then the eyeball was put into a 30% sucrose solution for dehydration for 2 h, then cornea and lens were cut out under an anatomical lens, embedded by OCT, and rapidly put into a -80°C refrigerator for freezing. OCT embedded eyeball was taken out about 10 min later, and put into a freezing microtome at -25°C for balancing for about 30 min, then sliced. Slice thickness was 12 pm.

After slicing, higher-quality slices were selected and put into an oven for 30 min at 37°C, then an immunohistochemical pen was used to draw a circle on the site with retina tissues, it was washed by PBS for three times to remove OCT, then sealed by 5% NDS (containing 0.25% Triton) for 2 h, primary antibodies were incubated for staying overnight at 4°C. It was washed by PBS for twice in the following day, corresponding fluorescent second antibodies were incubated, then washed by PBS for twice, and slices were sealed for observation.

Embodiment 3

Western blot (WB) was applied to observe the expression of TMEM30A protein in Tmem30a KO mouse.

WB:

(1) Retina tissues of wildtype and mutant mice were respectively separated, placed into a 1.5 ml centrifugal tube, and added 200 μΐ protein lysate RIPA;

(2) Retina tissues were broken by ultrasonication and disintegrated on ice for 20 min;

(3) 16000 g were centrifuged for 10 min at 4°C, supernatant was taken and transferred to another clean centrifugal tube, 50 μΐ protein loading solution was added for mixing well, then heated for 5 min at 95°C;

(4) After cooling, 20 μΐ samples were respectively taken for polyacrylamide gel electrophoresis (SDS-PAGE) at 160V to separate the protein;

(5) At the end of SDS-PAGE, proper-size nitrocellulose membrane was trimmed as required, and filter paper, glue, nitrocellulose membrane and filter paper were assembled in sequence. Air bubbles were removed, membrane transfer was conducted by 0.28 A current for 2 h in a membrane groove in an ice-water bath;

(6) At the end of membrane transfer, the nitrocellulose membrane was washed by pure water, aired and labeled. Then 8% skim milk was used for blocking for 2 h;

(7) At the end of blocking, a certain amount of primary antibodies were diluted in a blocking buffer according to certain proportion (operation manual of the antibody), incubated at 4°C overnight.

(8) Primary antibodies were removed, the membrane was washed by a IxTBST buffer solution for 4 times 10 min each time. Proper secondary antibodies were selected according to the source of the primary antibodies. IxTBST was used to dilute the HRP-labeled secondary antibodies, and secondary antibody was incubated on a shaker for 2 h at room temperature.

(9) At the end of secondary antibodies incubation, the membrane was washed by IxTBST for 3 times 10 min each time. Protein expression was detected by a Thermo ELC kit. The instrument used was a chemiluminiscence gel-imaging system from Bio-Rad.

Results were shown in FIG. 4. In the figure, WT denotes wildtype mouse, KO denotes Tmem30a KO mouse. Cerebrum denotes brain tissues, Cerebellum denotes cerebellar cells. WB has proved that Tmem30a protein is knocked out in cerebellar Purkinje cells of the Tmem30a KO mouse (FIG. 4). Due to the existence of granular cells and glial cells in cerebellum, Tmem30a in those two cells were not knocked out. Tthe expression quantity of Tmem30a in cerebellum decreased partially.

Based upon the above results, the specific cerebellar Purkinje cell Tmem30a knockout mouse model Tmem30a KO may be provided.

Embodiment 4

Study on cerebellar ataxia by the constructed Tmem30a gene knockout mouse model

Tmem30a KO mouse suffered form cerebellar ataxia

By an observation of Tmem30a KO behaviors and Hind-limb clasping test, it indicated that Tmem30a KO knockout mouse suffered the symptom of cerebellar ataxia. Specific performance was instability of gait and curled hind legs after its tail was lifted (FIG. 5). 6mo in FIG. 5 denoted 6 month-age.

Tmem30a KO mouse suffered cerebellar progressive atrophy

Cerebellum of the knockout mouse was dissected to find that the knockout mouse suffered cerebellar progressive atrophy with age (FIG. 6, in the figure: WT denoted a wildtype control, KO denoted a Purkinje cell knockout animal; lOmo denoted 10 month-age; Cross-sectional area of cerebellum denoted vertical cross-sectional area of cerebellum, and the vertical cross-sectional area of cerebellum was merely 50% of the WT mouse at 10 month-age. Paraffin section was performed to the cerebellum of the mouse at different ages, H&E staining was conducted to the section to find that the molecular layer of cerebellum thinned gradually and granular cells decreased. In FIG. 7, WT denoted a wildtype control, KO denoted a Tmem30a Purkinje cell knockout animal; P42 denoted 42 lOmo denoted 10 days of age; 5mo denoted 5 month-age, 12mo denoted 12 days of age.

Tmem30a KO mouse suffered progressive decrease of cerebellar Purkinje cells

An analysis on the immunohistochemical staining by frozen section were conducted to the knockout mouse, Purkinje cell marker protein Calbindin-D28K was stained to find that Purkinje cells in cerebellum decreased gradually, and almost all Purkinje cells in the knockout mouse died on the 42^nd day after birth (FIG. 8). In FIG. 8: Calbindin denoted calcium binding protein, a marker protein of Purkinje cells, DAPI denoted 4', 6-diamidino-2-phenylindole, and a fluorochrome capable of binding DNA strongly, usually used in fluorescent microscope observation. P16 denoted 16 days of age, P25 denoted 25 days of age, P30 denoted 30 days of age and P42 denoted 42 days of age.

Tmem30a KO appeared endoplasmic reticulum stress (ER stress) in cerebellar Purkinje cells

To explore the death cause of Purkinje cells, frozen section was performed to the knockout mouse for immunohistochemical staining analysis. Tmem30a is associated with intracellular transport, therefore, its deletion may cause the disorder of intracellular vesicle, resulting in endoplasmic reticulum stress. Therefore, endoplasmic reticulum stress marker protein CHOP and PDI staining was conducted to cerebellum freezing slices to find that the expression quantity of Purkinje cells CHOP and PDI of the Tmem30a KO mouse increased obviously on the 20th day after birth (FIG. 9). In FIG. 9: GCL: Granular cell layer; PCL: Purkinje cell layer; ML: Molecular layer; P20 denoted 20 days of age. Compared with the wildtype control, the expression of endoplasmic reticulum stress protein marker CHOP in Tmem30a KO cerebellar cells increased by nearly 6 times, and PDI increased by nearly 4 times (FIG. 9). Tmem30a knockout would cause endoplasmic reticulum stress. After appearing endoplasmic reticulum stress, related pathway of cells apoptosis was activated, resulting in final apoptosis.

To sum up, in the embodiments of the disclosure, the specific cerebellar Purkinje cell Tmem30a gene knockout mouse model is constructed, and the animal model shows typical characteristics of cerebellar ataxia: instability of gait, cerebellar atrophy, progressive apoptosis of Purkinje cells, etc. The model may be used in the study of cerebellar ataxia.

The mentioned above are merely preferred embodiments of the disclosure, not employed to limit the disclosure, various modifications and changes of the disclosure are available to those io skilled in the art. Any modification, equivalent replacement, improvement, etc. made according to the spirit and principle of the disclosure shall be included within the protection scope of the disclosure.

Industrial applicability: By the method for constructing an ataxia animal model provided by the disclosure, a specific Tmem30a knockout ataxia animal model of pancreatic β cells in cerebellar Purkinje cells may be constructed, and the model shows typical ataxia features; the mouse model may be used in the study of ataxia or screening drugs for treating or preventing ataxia and other fields, thus providing a model basis to further know the pathogenesis of ataxia and screen ataxia drugs.

Claims

What is claimed is:

1. A method for constructing an ataxia animal model, characterized by knocking out a target sequence on a genome of a cerebellar Purkinje cell of a target animal; wherein the target sequence is the Tmem30a gene.
2. The method for constructing an ataxia animal model according to claim 1, wherein the target sequence is an exon sequence in the Tmem30a gene.
3. The method for constructing an ataxia animal model according to claim 2, wherein the target animal is selected from any of mouse, rat, dog, monkey and ape.
4. The method for constructing an ataxia animal model according to claim 3, wherein the target animal is mouse, and the exon sequence is a No. 3 exon sequence, preferably, wherein a conditional Tmem30a gene knockout homozygote mouse mates a Pcp2-Cre transgenic mouse to obtain a conditional cerebellar Purkinje cell Tmem30a gene knockout mouse.
5. An application of the ataxia animal model obtained by the method for constructing an ataxia animal model according to any one of claims 1-4 in the study of ataxia.