CN112111529A - Neurodegenerative disease animal model and establishment and application thereof - Google Patents

Abstract

The invention relates to a neurodegenerative disease animal model and establishment and application thereof. Specifically, the invention provides a preparation method of a non-human mammal neurodegenerative disease animal model, which comprises the following steps: (a) providing a cell of a non-human mammal, and performing point mutation on the Uchl1 gene in the cell to obtain a Uchl1 gene point-mutated cell; (b) preparing an animal model with Uchl1 gene point mutation by using the Uchl1 gene point mutation cell obtained in the step (a); wherein, in the animal model, the Uchl1 gene point mutation is homozygous. The mouse model of the invention is highly similar to human parkinson's disease. The Parkinson disease drug screening platform based on the model animal can be used for screening new drugs and developing other treatment methods.

Description

Neurodegenerative disease animal model and establishment and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a neurodegenerative disease animal model and establishment and application thereof.

Background

Neurodegenerative diseases include mainly Parkinson's Disease (PD), Alzheimer's Disease (AD), Huntington's Disease (HD), and so on, and the neuron function is abnormal or even lost mainly due to abnormal neuron metabolism or nutritional disorder. The common characteristics of the method comprise that the insolubility of protein is increased, and abnormal protein is deposited; abnormal microtubule dynamics leads to aggregation disorder; ③ loss of brain and spinal cord neuronal cells.

Parkinson's Disease (PD) is a neurodegenerative disease seriously harming the health of the middle-aged and elderly people and has various clinical manifestations including resting tremor, bradykinesia, increased muscle tone, dyskinesia and the like^[1]Additional non-motor symptoms often accompany it, including cognitive dysfunction and dementia. Parkinson's disease is the second largest neurodegenerative disease, the prevalence rate of people over 50 years old is 1-2%, and the prevalence rate tends to increase with the progress of social aging. However, since the cause of PD is unknown, and it is clinically only symptomatic treatment and does not prevent the progress of the disease, parkinson's disease becomes one of the major causes of disability in the elderly.

One is a model which is made by injecting 6-hydroxy dopamine (6-0HDA) into a nigrostriatal system to damage Dopamine (DA) nervoussoirs of a rat nigrostriatal-striatal system, the modeling needs three-dimensional directional injection, the technical operation requirement is higher, and higher failure rate exists; the other is to use 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to prepare primate models such as monkeys and mouse models, but MPTP also has irreparable neurotoxicity to human beings and requires strict safety control in operation. And both methods can not accurately simulate the pathological process of the Parkinson disease, for example, Lewy bodies (MPTP) do not appear in the MPTP model, so that the application of the models is greatly limited due to the defects of the currently used Parkinson disease models.

Therefore, there is an urgent need in the art to develop a new non-human mammal that effectively mimics neurodegenerative pathology.

Disclosure of Invention

The object of the present invention is to provide a novel non-human mammalian model that effectively mimics neurodegenerative disorders. The animal model can simulate a clinical non-human mammal neurodegenerative model, and is expected to be widely applied to treatment technologies for treating neurodegenerative diseases and screening and evaluation of medicines.

In a first aspect, the present invention provides a method for preparing an animal model of neurodegenerative disease in a non-human mammal, the method comprising the steps of:

(a) providing a cell of a non-human mammal, and performing point mutation on the Uchl1 gene in the cell to obtain a Uchl1 gene point-mutated cell;

(b) preparing an animal model with Uchl1 gene point mutation by using the Uchl1 gene point mutation cell obtained in the step (a);

wherein, in the animal model, the Uchl1 gene point mutation is homozygous.

In another preferred embodiment, the neurodegenerative disease is selected from the group consisting of: parkinson's Disease (PD), Alzheimer's Disease (AD) and Huntington's Disease (HD).

In another preferred embodiment, the neurodegenerative disease is parkinson's disease.

In another preferred example, the Uchl1 gene point mutation comprises: the amino acid I at the 93 th position of the UCHL1 protein is mutated into 19 other amino acids except for I to form point mutation.

In another preferred embodiment, the other 19 amino acids are Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V).

In another preferred embodiment, amino acid I at position 93 of the UCHL1 protein is mutated to Ala or Phe.

In another preferred example, the Uchl1 gene point mutation is the mutation of I at position 93 to M.

In another preferred example, the Uchl1 gene point mutation is the mutation of I at position 93 in SEQ ID No. 1, 2 or 3 to M.

In another preferred embodiment, the non-human mammal is a rodent or primate, preferably including a mouse, rat, rabbit, monkey.

In another preferred example, in the step (b), the method comprises the steps of:

(b1) preparing an animal model of the chimeric Uchl1 gene point mutation, and then obtaining a heterozygous Uchl1 gene point mutation animal model by hybridization; and

(b2) and obtaining a homozygotic Uchl1 gene point mutation animal model by hybridizing the heterozygotic Uchl1 gene point mutation animal model.

In another preferred example, the method comprises:

(1) constructing Uchl1 gene I93M targeted shearing plasmid by using CRISPR-CAS9 technology, and replacing with homologous mutation DNA sequence of a screening marker to obtain Uchl1-I93M point mutation positive monoclonal non-human mammal embryonic stem cells;

(2) preparing a chimeric non-human mammal by using the Uchl1 gene point-mutated non-human mammal embryonic stem cell clone strain obtained in the step (1);

(3) mating and breeding the chimeric non-human mammal obtained in the step (2) and a normal wild non-human mammal, and screening heterozygote non-human mammal with Uchl1 gene point mutation in the offspring;

(4) obtaining a Uchl1-I93M point-mutated homozygous non-human mammal by mating the heterozygous non-human mammals obtained in step (3) with each other, thereby obtaining a Uchl1-I93M point-mutated non-human mammal model.

In another preferred embodiment, the screening marker is a neo gene.

In another preferred embodiment, the non-human mammal model of the Uchl1-I93M point mutation obtained in step (2) has one or more of the following characteristics compared to a wild-type control animal:

exhibit parkinson-like behavior;

increased nuclear synapsin expression in the corresponding region of the brain; and/or

The protein expression of Uchl1 in the corresponding region of the brain is reduced.

In another preferred embodiment, said parkinson-like behavior is selected from the group consisting of: a decreased level of spontaneous activity; the active movement ability is reduced, and the high platform fear is achieved; or a combination thereof.

In a second aspect of the invention there is provided the use of a non-human mammalian model prepared by a method according to the first aspect of the invention, as an animal model for studying parkinson's disease.

In a third aspect of the invention there is provided the use of a non-human mammalian model prepared by a method according to the first aspect of the invention to screen or identify substances (therapeutic agents) that may reduce or treat parkinson's disease.

In a fourth aspect of the invention there is provided a non-human mammalian model prepared by a method according to the first aspect of the invention.

In another preferred embodiment, the non-human mammalian model is homozygous for the Uchl1 gene point mutation.

In another preferred embodiment, the animal model has significantly increased nuclear synaptophysin expression in the midbrain and/or hindbrain regions of the brain (about 50% or more above wild-type).

In a fifth aspect of the invention, there is provided a method of screening for or identifying potential therapeutic agents for treating or ameliorating parkinson's disease, comprising the steps of:

a. administering a candidate substance to the non-human mammalian model prepared by the method of the first aspect; and

b. performing behavioral analysis on the behavior of the animal model, and comparing the behavior with a control group;

wherein, if the behavior characterizing a neurodegenerative disease is improved in an animal model administered with the candidate substance compared to a control, the candidate substance is indicative of a potential therapeutic agent for a neuropsychiatric disease.

In another preferred example, the behavioral analysis includes: a roller test, an elevated plus maze test, or a combination thereof.

In another preferred embodiment, the analysis comprises evaluating the following criteria: open arm dwell time, total creep time, or a combination thereof.

In another preferred embodiment, the candidate substance is selected from the group consisting of: small molecule compounds, proteins, nucleic acids, mirnas, antibodies, or combinations thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows that CRISPR-CAS9-SgRNA plasmid with directional DNA cutting function is successfully constructed. Wherein FIG. a shows a partial DNA base sequence of mouse Uchl1 gene exon region No. 4, and the position of the corresponding SgRNA sequence. FIG. b shows that CRISPR-CAS9-SgRNA plasmid transfected in NIH-3T3 cell can be cut at the DNA target of Uchl1 gene corresponding to I93M.

FIG. 2 shows that Uchl1-I93M point mutation positive monoclonal stem cells are successfully obtained. Wherein the content of the first and second substances,

FIG. a is a linearized electrophoretogram of plasmid pEASY-Blunt3-Uchl 1-I93M-PGK-NEO;

panel b is a primer identification electrophoretogram, wherein u1 represents ES-S1/PGK-AS primer pair, u2 represents ES-S1/NEO-AS primer pair, u3 represents ES-S2/PGK-AS primer pair, u4 represents ES-S2/NEO-AS primer pair, d1 represents PGK-S/ES-AS1 primer pair, d2 represents PGK-S/ES-AS2 primer pair, d3 represents NEO-S/ES-AS1 primer pair, and d4 represents NEO-S/ES-AS2 primer pair;

c is the electrophoresis chart of the mixed genome DNA of each horizontal row of the 96-well plate A-G;

d is genome DNA electrophoresis of A row and G row of each well of 96-well plate;

e is a positive monoclonal electrophoretogram;

the f picture and the g picture are positive monoclonal sequencing peak pictures, and the purple frame is a mutation site.

h is a sequence comparison diagram of the two-round sequencing results, wherein a brown arrow represents a sequencing sequence, a blue square represents an exon number 4 of the UCHL1 sequence, a green line represents a mutation codon ATG, a white arrow represents a PGK promoter sequence, a light green arrow represents a NEO sequence, and two light blue arrows represent a Loxp site.

FIG. 3 shows morphology of Uchl1 gene mutation mice. The point mutation homozygous mice (HOM) have no significant morphological differences compared to Heterozygous (HET) and Wild (WT) types. Chimeric hair color, partially brown (129 mouse hair color characteristics) indicated that the mouse embryonic stem cell-derived phenotype has integrated into the C57BL/6 mouse embryo.

FIG. 4 shows the genotyping of Uchl1 gene point mutant mice. Panel A, lane D depicts the D2000 component standard; w (WT) denotes the wild type; e denotes Heterozygote (HET); o denotes homozygote type (HOM). FIG. B, sequencing results of HOM mouse-derived DNA fragments.

FIG. 5 shows the observation of paraffin sections HE staining of mouse brain tissue. Wherein WT represents a wild type, HET represents a heterozygous type, and HOM represents a homozygous type in the photograph name; HE represents the staining pattern; and m is the midbrain region when reaching the standard, and h is the hindbrain region.

FIG. 6 shows the Western blot detection of the Uchl1 protein level in the Uchl1 gene point-mutated mouse brain tissue protein extract. Where HOM indicates homozygous for the point mutation, HET is heterozygous and WT is wild.

FIG. 7 shows microscopic observations of paraffin sections of mouse brain tissue after HE staining. Wherein WT represents a wild type, HET represents a heterozygous type, and HOM represents a homozygous type in the photograph name; uchl1 represents immunohistochemical detection of the corresponding protein, while Neg represents a de-immunohistochemical negative control; whereas m is the midbrain in the standard, h is the hindbrain region and mc is the coronal slice of the midbrain region to distinguish it from the other sagittal slices.

FIG. 8 shows microscopic observations of paraffin sections of mouse brain tissue after HE staining. Wherein WT represents a wild type, HET represents a heterozygous type, and HOM represents a homozygous type in the photograph name; syn represents immunohistochemical staining for synuclein, and Neg represents a de-immunohistochemical negative control; whereas m is the midbrain when it comes to standard, h is the hindbrain region and mc is the coronal section of the midbrain region to distinguish it from other atypical sections.

FIG. 9 shows microscopic observations of paraffin sections of mouse brain tissue after HE staining. Wherein WT represents the wild type and HOM represents the homozygous type in the photograph name; HE represents the staining pattern; while m is for mid-brain and mc represents a coronal slice of the mid-brain region to distinguish it from other sagittal slices.

FIG. 10 shows a statistical analysis of the degree of difference in gene expression. The abscissa represents the high-expression gene of the brain tissue of a wild-type (WT) mouse, and the ordinate represents the high-expression gene of the brain tissue of a point mutation homozygous mouse (HOM). In the figure, red dots indicate up-regulated genes with significantly different expression of HOMs with reference to WT, and green dots indicate down-regulated genes with significantly different expression.

FIG. 11 shows a statistical analysis of the degree of difference in gene expression. The abscissa represents a high-expression gene in brain tissue of a wild-type (WT) mouse, and the ordinate represents a high-expression gene in brain tissue of a point mutation heterozygous mouse (HHET). Red dots in the figure indicate up-regulated genes significantly differentially expressed by HET and green dots indicate down-regulated genes significantly differentially expressed by WT.

FIG. 12 shows a scatter plot of the signal pathway enrichment of the differential gene KEGG in mice homozygous for the Uchl1 gene point mutation versus wild type. Wherein the abscissa represents the enrichment factor, the ordinate represents the name of the pathway concerned, the size of the dot represents the number of differentially expressed genes in the pathway, and the color of different dots corresponds to different q-value ranges. It can be seen from the figure that the neuroactive ligand-receptor interaction signaling pathway (mmu04080), cytokine-cytokine receptor interaction (mmu04060), and morphine addiction substance dependent pathway (mmu05032) are enriched to a greater extent and are statistically significant (p 0.00), which is of interest.

FIG. 13 shows the autonomous activity of Uchl1 gene I93M point mutant mice at 12 weeks of age.

FIG. 14 shows the results of suspension test of Uchl1 gene I93M point mutant mice at 12 weeks of age.

FIG. 15 shows the time of on-roll crawl of Uchl1 gene I93M point mutant mice at 12 weeks of age.

FIG. 16 shows the percentage of entry times into the open arms in the test plateau maze of Uchl1 gene I93M point mutant mice at 12 weeks of age.

Figure 17 shows the percentage of time in the open arm in the test plateau maze of Uchl1 gene I93M point mutant mice at 12 weeks of age.

FIG. 18 shows the fertility of Uchl1 gene point mutant mice.

Figure 19 shows the amino acid sequence of human, mouse and rat UCHL 1.

Figure 20 shows the homology of human, mouse and rat UCHL1 protein.

Detailed Description

The inventor constructs Uchl1 gene I93M targeted cutting plasmid by using CRISPR-Cas system through extensive and intensive research, then obtains Uchl1-I93M point mutation positive monoclonal stem cells, and finally obtains UCHL1-I93M point mutation homozygous mouse through transgenic chimeric technology and large-scale screening. The UCHL1-I93M point mutation homozygous mouse successfully established by the invention can spontaneously generate Parkinson-like pathological manifestations and behavioral phenotypes in early adult stages, and the invention is completed on the basis.

Specifically, in the invention, the behavioral tests of a mouse model of Uchl1 gene point mutation homozygote and wild type prove that the animal homozygote of the Parkinson disease model has the advantages of reduced spontaneous activity level, reduced active motor ability, appearance of Parkinson disease-like behavioral phenotype, and the histological immunohistochemistry and immunowestern blot detection of midbrain parts and hindbrain parts show that the nuclear synapsin expression is increased and is simultaneously reduced along with the Uchl1 protein expression.

Term(s) for

As used herein, the term "(PARK 1)" refers to Alpha-synuclein (Alpha-synuclein).

As used herein, the terms "model animal of the invention", "neurodegenerative disease model of the invention", "early-onset neurodegenerative disease model", and the like, are used interchangeably to refer to a homozygous non-human mammal, particularly a rodent (e.g., rat and mouse), having a site-directed mutation of UCHL1 (particularly an I93M point mutation).

As used herein, the terms "point mutation I93M", "point mutation I → M" and the like are used interchangeably and refer to the mutation of the UCHL1 protein from I (Ile) to M (Met) at position 93 corresponding to the amino acid sequence set forth in SEQ ID Nos. 1-3. At the gene level, the mutation was a mutation of cytosine to guanine at position 277 in the UCHL1 gene (C277G).

UCHL1 gene and protein thereof

As used herein, the terms "UCHL 1", "Ubiquitin C-terminal hydrolases L1", "Ubiquitin carboxy-terminal hydrolase L1" are used interchangeably and all refer to a UCHL1 gene or protein component.

Ubiquitin carboxyl-terminal hydrolase L1(Ubiquitin C-terminal hydrolases L1, UCHL1) is highly expressed in toad and mouse tissues specifically in brain and gonad and highly conserved. UCHL1 is mainly present in nerve cells, is a soluble protein, generally consisting of 223aa, with a theoretical molecular weight of approximately 24 kDa. For its cellular functions including deubiquitinase and ubiquitin ligase activity, the function of stabilizing ubiquitin monomers, in combination with the effect of activating cell cycle kinases to regulate the cell cycle. Studies have shown that UCHL1 is involved in different roles, including regulation of nuclear synapsin (α -Synuclein) accumulation in nerve cells.

UCHL1 is present in many different species, and is highly homologous, especially in mammals, also being substantially 223 amino acids in length (table a, table B and fig. 20). The human UCHL1 gene (NC-000004.12) is located on the human genome 4p13 and has a total length of 12043 bp.

TABLE A UCHL1 Gene and protein

Homology of UCHL1 protein in part B of Table

	Human being	Chimpanzee	Kiwi fruit	Mouse	Rat
						Human being	100％
Chimpanzee	100％	100％
						Kiwi fruit	99％	99％	100％
Mouse	96％	96％	95％	100％
						Rat	96％	96％	95％	100％	100％

The amino acid sequences of human, mouse and rat UCHL1 are shown in SEQ ID nos. 1, 2 and 3, respectively (fig. 19).

Parkinson's disease

The prominent pathological changes in parkinson's disease are degenerative death of Dopaminergic (DA) neurons of the midbrain, a significant reduction in the striatal DA content, and the appearance of eosinophilic inclusions, Lewy bodies, within the cytoplasm of the substantia nigra residual neurons. Clinically, the symptoms are static tremor, bradykinesia, muscular rigidity and gait disorder.

As used herein, the term "parkinson-like phenotype," includes, but is not limited to, the pathological degenerative death of Dopaminergic (DA) neurons of the midbrain, a significant reduction in striatal DA content, and the presence of eosinophilic inclusions, Lewy bodies, within the cytoplasm of surviving neurons of the substantia nigra. Resting tremor, bradykinesia, myotonia, and postural gait changes occur in behavior.

The term "animal model of parkinson's disease", as used herein, generally refers to a model animal that exhibits one or more parkinson-like phenotypes, used to mimic the spontaneity or inducibility of parkinson's disease.

Dominant inheritance

Dominant inheritance is controlled by a dominant gene, and on homologous chromosomes, two homotypic dominant genes exist in pairs, or when dominant and recessive genes exist in allelic genes, phenotypes are shown. This mode of inheritance is known as dominant inheritance.

The genetic mode is that the phenotype of the heterozygote is between the phenotype of the dominant homozygote and the recessive homozygote, namely, the role of the recessive gene is expressed to a certain extent in the heterozygote, and the genetic mode is called incomplete dominant inheritance.

Studies have shown that Parkinson's disease symptoms are observed in individuals heterozygous for a UCHL1-I93M point mutation.

However, in the present invention, the present inventors have unexpectedly found that although heterozygous mice with a UCHL1-I93M point mutation do not exhibit a parkinson's disease-like phenotype, homozygous mice with a UCHL1-I93M point mutation may pathologically and behaviorally exhibit a parkinson's disease-like phenotype and may be able to express a parkinson's disease-like phenotype at an earlier stage. This suggests that the homozygous mouse (or other rodent) model of UCHL1-I93M point mutation is a valuable non-human animal model of Parkinson's disease.

Animal model

Animal models of human diseases (animal models of human diseases) refer to animals with human disease-mimicking manifestations established in various medical science studies, and are classified into spontaneous animal models and induced or experimental animal models according to the cause of the disease.

Spontaneous Animal Models (Spontaneous Animal Models) refer to diseases that occur in natural conditions in experimental animals without any conscious artificial treatment. Including genetic diseases of mutant lines and tumor disease models of inbred lines. The biggest advantage of utilizing the animal disease model to research human diseases is that the occurrence and development of the diseases are similar to the corresponding diseases of human beings, and the diseases are all diseases occurring under natural conditions and have higher application value, but the model is difficult to source.

Techniques for site-directed mutagenesis of a target gene by the CRISPR-CAS9 technique are known in the art, and such conventional techniques can be used in the present invention.

In the present invention, a non-human mammalian model capable of drug-free modeling is provided that can begin spontaneously exhibiting symptoms of Parkinson's disease early, particularly early adulthood (e.g., at 12 weeks of mouse age).

In the present invention, examples of non-human mammals include (but are not limited to): mouse, rat, rabbit, monkey, etc., more preferably rat and mouse.

In the invention, I93M site-directed mutagenesis can be introduced into UCHL1 by a site-directed mutagenesis method, so that a chimeric mouse containing UCHL 1I 93M site-directed mutagenesis is obtained, and a homozygous mouse with UCHL 1I 93M site-directed mutagenesis is further obtained by means of screening, hybridization and the like.

In a preferred embodiment of the invention, the homozygote (or heterozygote) mice obtained by the present invention are fertile and develop normally. In addition, Uchl1-I93M point mutations can be inherited by progeny mice in Mendelian regularity. This indicates that the model animals of the present invention are suitable for large-scale, low-cost production.

Construction of animal models

In a preferred embodiment of the invention, a Uchl1 gene I93M targeted shearing plasmid is constructed by using a CRISPR-Cas system, then Uchl1-I93M point mutation positive monoclonal stem cells are obtained, and finally, UCHL1-I93M point mutation homozygous mice are obtained through transgenic chimeric technology and mass screening.

In a preferred embodiment, the invention provides a method for constructing a mouse Uchl1 gene targeted mutation plasmid, which comprises the following steps:

(1) designing gRNA according to a surrounding DNA sequence of the Uchl1 gene corresponding to the I93M mutation site;

(2) recombined with the sequence of the gene coding CRISPR-CAS9 to form a recombinant plasmid CRISPR-CAS 9-SgRNA;

(3) the plasmid is transfected into a eukaryotic cell, and under the guidance of the transcribed gRNA, Cas9 DNA endonuclease is specifically combined with a gene site corresponding to Uchl1 mutation site I93M to perform DNA double-strand cutting;

(4) the recombinant plasmid CRISPR-CAS9-SgRNA is transfected into NIH-3T3 cells, positive cells are screened through puromycin, genome DNA is extracted, primers are respectively designed on the upstream and downstream of a SgRNA targeting sequence for PCR amplification, and the condition of introducing mutation can be detected.

The technical scheme of the invention has the following main advantages:

(a) the mouse model for the Parkinson disease is a homozygous UCHL1 gene point mutation mouse obtained by breeding through I93M point mutation of UCHL1 gene.

(b) Compared with UCHL1 transgenic mice, the Parkinson disease model mouse of the invention more truly simulates sporadic Parkinson disease discovered clinically.

(c) The Parkinson disease model mouse, namely the homozygous UCHL1 gene I93M point mutation mouse, can start to spontaneously show Parkinson disease physiological symptoms including the increase of the nuclear synapsin expression of corresponding areas of the brain at the early adult stage (12 weeks old).

(d) The Parkinson disease model mouse, namely the homozygous UCHL1 gene I93M point mutation mouse, can spontaneously show Parkinson disease-like behavioral symptoms including active motor ability reduction at the early adult stage (12 weeks old).

(e) In the Parkinson disease model mouse, signal pathways related to interaction of nerve cell ligands and receptors in brain tissues are subjected to regulatory change.

(f) The present invention focuses on the UCHL1 gene of the ubiquitin dependent pathway, which is interrelated to the autophagy pathway, but has its uniqueness. At present, most of the drugs for treating the neurodegeneration are directed to autophagy-related pathways, but no drugs for ubiquitination-related pathways are reported.

(g) Neurodegeneration is associated with senescence, and the Uchl1 gene mutation model is used in senescence studies, not only as a complicating factor of neurodegeneration together with senescence, but also possibly as a cause of senescence itself.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Example 1: construction of mouse Uchl1 gene targeted mutation plasmid

The grnas were first designed using CRISPR-CAS9 gene editing techniques based on the surrounding DNA sequence of the Uchl1 gene corresponding to the I93M mutation site (table 1).

Table 1:

the DNA sequence corresponding to the gRNA, the forward and reverse strands are annealed to form a double strand, and then recombined with the sequence encoding the CRISPR-CAS9 gene to form the recombinant plasmid CRISPR-CAS 9-SgRNA. The plasmid is transfected into a eukaryotic cell, can express Cas9 DNA endonuclease and specific sgRNA, and performs the function of guiding specific DNA sites to perform DNA cutting depending on the sgRNA. The protein expressed by CAS9 is specifically combined with the gene site corresponding to Uchl1 mutation site I93M under the guidance of transcribed gRNA, and DNA double-strand cutting is carried out.

Transfecting NIH-3T3 cells by using a recombinant plasmid CRISPR-CAS9-SgRNA, screening positive cells by puromycin, extracting genome DNA, and respectively designing a primer at the upstream and downstream of a SgRNA targeting sequence for PCR amplification to obtain a product with the total length of about 900 bp. Further sequencing revealed that a doublet appeared at the SgRNA targeting sequence (fig. 1).

The aim of this example was to mutate isoleucine to methionine at position 93 of the mouse Uchl1 protein, i.e., the codon was mutated from ATC to ATG.

To perform site-directed mutagenesis efficiently, sgrnas were screened. Typically, the CAS9 enzyme cleaves at the recognition sequence, i.e., the third base upstream of the PAM sequence. The probability of secondary targeting of SgRNA-1 after sequence point mutation is higher, the enzyme cutting sites corresponding to SgRNA-2 and SgRNA-4 are far away from the target point mutation site, the shearing site corresponding to SgRNA-3 is just positioned at the point mutation site (figure 1), and the CRISPR-CAS9-SgRNA-3 is finally determined to be used as a targeting enzyme cutting vector by combining the factors.

The sequence of SgRNA-3 is as follows: gcacugcguggaucaacccga (SEQ ID No.:4)

Example 2: preparation of mouse Stem cells with Uchl1 Gene Point mutation

2.1 transfection of mouse embryonic Stem cells

Firstly, PCR amplification is utilized to obtain a mouse Uchl1 gene DNA fragment with point mutation, wherein the design of a primer for introducing a mutation site is shown in Table 2

Table 2:

name (R)	Sequence (5'→ 3')	SEQ ID No.:
			pri-I93M-F	GAAACTCCTGTGGTACCATGGGGTTGATCCACGCAGTGG		13
pri-I93M-R	CCACTGCGTGGATCAACCCCATGGTACCACAGGAGTTTC						14

The Uchl1 fragment with the point mutation sequence and the corresponding DNA fragment of the selection marker PGK-NEO (purchased from Shanghai Renyuan Biotechnology Co., Ltd.) were recombined in pEASY-Blunu3 plasmid (purchased from Beijing Quanjin Biotechnology Co., Ltd.) to form a recombinant plasmid pEASY-Blunu3-Uchl 1-I93M-PGK-NEO. Wherein PGK-NEO is a neomycin resistance gene with a PGK gene promoter, and can be used for screening positive cell clones successfully recombined in cell genome DNA after eukaryotic cells are transfected.

The plasmid CRISPR-CAS9-SgRNA with the function of targeting mutation sites to perform DNA shearing and the plasmid pEASY-Blunu3-Uchl1-I93M-PGK-NEO containing Uchl1 point mutation sequence donor DNA fragments are subjected to conventional amplification through inoculated bacteria to obtain amplified plasmids. The plasmid pEASY-Blunu3-Uchl1-I93M-PGK-NEO is linearized by using restriction endonuclease pvuI, mixed with the targeting shearing plasmid CRISPR-CAS9-SgRNA according to the ratio of 1:5, and dissolved in PBS for standby after ethanol precipitation and purification.

Mouse embryonic stem cells (purchased from Shanghai kernel-derived Biotechnology Co., Ltd.) were transfected by electroporation, and inoculated in a gradient in a 10 cm-diameter culture dish, and the culture medium containing G418 was used to select the monoclonal antibodies expressing resistance, and when the cells grew to the extent that they were visible to the naked eye, they were picked up in a 96-well plate and cultured and identified, and only 2 of the picked-up monoclonal antibodies were identified as positive by one round of identification (FIG. 2).

2.2 screening of Positive clones

Donor DNA was linearized (as in fig. 2a), and under sgRNA guidance, expressed Cas9 resulted in mouse stem cells with a gene point mutation corresponding to the Uchl1 protein I93M mutation.

All mixed clones were tested by PCR and the genome was found to have the inserted sequence (FIG. 2 b). The DNA sequence of the mixed clones was examined as indicated and the target point mutation was found in columns A and G (FIG. 2 c). Further examination of each clone in columns A and G revealed that insertion of the A11 and G2 sequences was possible with point mutations. Gene validation, with the corresponding insertion sequence (fig. 2 d). Positive clones were sequenced and found that the A11 clone had the correct coding sequence (FIG. 2f), while the G2 clone had a frame shift mutation (FIG. 2G).

Thus, the a11 clone was selected for subsequent manipulation.

2.3 preparation of chimeric mice

The embryonic stem cell clone A11 of 129 mice was selected and microinjection experiments of C57BL/6J mouse embryos were performed. The chimeric embryo injected by stem cell micro-injection is transplanted to the uterus of a pseudopregnant D3 generation pregnant mouse, so that the chimeric mouse is obtained by breeding.

In this example, genomic recombination changes of the obtained stem cells, in addition to the construction of the I93M point mutation in the exon 4 coding region, a Neo gene sequence driven by the PGK gene promoter was inserted into the intron (fig. 2 h). Loxp sequences at both ends are used to excise the marker gene by means of Cre transgenic mice during mouse breeding.

As a result: by combining the CRISPR-CAS9 technology with the classical homologous recombination technology, a UCHL1-I93M point mutation transgenic chimeric mouse model is successfully obtained.

Example 3: breeding and identification of mice carrying Uchl1 Gene Point mutation

3.1 Breeding of Uchl1 Gene Point-mutated mice

In this example, the obtained chimeric mouse male was further used to cross with the C57BL/6J mouse female, thereby obtaining F1 generation heterozygous mice. If the E14 embryonic stem cells were successfully chimerized into the germ cells, the resulting F1 mouse hair color was brown.

Because of the need for selection by homologous recombination, LoxP-PGK-NEO-LoxP element was introduced into the sequence, and although this element was inserted into the intron region not involved in gene coding, in order to prevent the occurrence of the situation where the intron regulates gene expression, F1 generation mice were mated with cre mice to remove the PGK-NEO element, leaving only one Lox P site. After removal of the insertion elements from the F1 mice, mating with C57BL/6 mice was continued to expand the number of F1 mice, after which F1 mice were selfed.

The results are shown in FIG. 3. Through identification, UCHL1-I93M point mutation homozygous mice were obtained to further study the phenotype of UCHL1-I93M in neurodegeneration.

3.2 genotyping of Uchl1 Gene Point-mutated mice

Proteinase K and DNA lysates were mixed at 1: the ratio of 200 was configured to mix the lysates. The toes or tails of the mice were cut, placed in a 1.5ml volume EP tube, added to 200ul of the mixed lysate, and lysed in an oven at 55 ℃ overnight. The next day the EP tube was boiled in 100 ℃ boiling water for 5 minutes to denature and inactivate the protease. After taking out, the DNA template was centrifuged at 12000rpm for 5 minutes and the supernatant was used as a DNA template. And (3) carrying out PCR amplification on the extracted DNA template to obtain a specific DNA fragment. The reaction conditions are

Pre-denaturation at 94 ℃ for 5 min. Denaturation at 94 ℃ for 30 sec; annealing at 60 ℃ for 30 sec; the elongation was 72 ℃ for 36s for 30 cycles. Fully extending at 72 ℃ for 5 min. The primer sequences are shown in Table III.

Table 3:

name (R)	Sequence of	SEQ ID No.:
			pri-S1	AAGCAGACCAUCGGAAAC
pri-PN-AS1	CAGACUGCCUUGGGAAAA
			pri-As1	CCAGCCAAAAUUAGAGCC

Expected amplified fragment length:

wild type, 373bp

Homozygous, 516bp

The reaction for the PCR amplification reaction also consisted of:

composition (I)	Volume of	Final concentration
			2×Uaq PCR MasuerMix	10μL		1×
Primer S1	0.5μL	0.25μM
			Primer PN-AS1	0.5μL	0.25μM
Primer AS1	0.5μL	0.25μM
			DNA template	1μL	——
RNaseFree ddH2O	8μL	——

After the PCR amplification reaction, the reaction solution containing the DNA fragment was separated and detected by 2% agarose gel electrophoresis. Electrophoresis conditions were 120V for 40 min. The reference DNA molecular weight standard was D2000 (available from Tiangen Biochemical technology, Beijing) Ltd.).

The separated target band was recovered by agarose gel DNA recovery kit gel (Tiangen Biochemical technology Co., Ltd., Tiangen, Beijing). The recovered DNA fragment was ligated to pMD-18U plasmid (purchased from Toyo Boseki Co.), ligated to UOP10 competent cells (purchased from Tiangen Biochemical technology Ltd.), shaken for single cloning, amplified, extracted, and sequenced by Pennoson.

The results are shown in FIG. 4. Three mice, homozygous, heterozygous and wild, were differentiated by PCR amplification of the DNA fragment length for the Uchl1 gene point mutation (FIG. 4A). The relevant genomic region was found by sequencing, and the I93M point mutation of the Uchl1 gene was obtained (fig. 4B), while the other sites were not mutated.

Example 4: morphological change of cells in brain tissue of Uchl1 gene point mutation mouse

Mouse brains fixed with 4% paraformaldehyde were processed by conventional paraffin embedding and sectioning, with a section thickness of 6 μm. Paraffin sections were xylene dewaxed and transferred to distilled water after passing through an alcohol gradient. After routine hematoxylin and eosin staining (HE), the neutral gum seals were observed. Typically, basophilic structures are stained blue and eosinophilic structures are stained red.

Immunohistochemistry results showed no significant difference in histology at midbrain and hindbrain sites between the Uchl1 point mutant Homozygote (HOM) and wild type (WU) and Heterozygote (HEU) (FIG. 5).

Example 5: content change of Uchl1 protein in point mutation mouse brain tissue protein extract

Mouse brain tissue was cut lengthwise in half and weighed in 1.5ml centrifuge tubes, and 20. mu.l of 0.1M PMSF was added to a final concentration of 1mM per 2ml of cell lysate at the present time. Mu.l of cell lysate (Biyuntian, p0013) was added per 20mg of tissue. The homogenizer breaks up the tissue and then carries out ultrasonic treatment to ensure that the lysate is fully contacted with the tissue and is cracked for 30min on ice. Centrifuge at 12000rpm, 4 ℃ for 30 min. The supernatant was taken, added to the loading buffer to a final concentration of 1 ×, and boiled at 100 ℃ for 5 min. Subpackaging and freezing at-20 ℃.

The proteins in the mouse brain tissue protein extract were separated by vertical electrophoresis (SDS/PAGE) using a 10% denaturing polyacrylamide gel. After loading, firstly carrying out low-pressure (80V) electrophoresis for 20min for carrying out lamination gel aggregation, and then carrying out constant-pressure electrophoresis for 1 hour at 120V for carrying out strip separation on the protein. Wet electrotransfer on nitrocellulose membrane, ponceau red staining for 10min, marker position with pencil, then Wesuern analysis using antibody. When the immunoblotting detection is carried out, firstly, the blotting membrane is washed by UBSU for 3 multiplied by 5min to remove ponceau staining, and then 6% skimmed milk powder is sealed in a closed liquid for 5 hours at a constant temperature.

Uchl1 antibody was diluted with 5% BSA in UBSU solution to prepare a primary antibody reaction solution. Uchl1 rabbit mab, dilution factor 1:1000, purchased from Cell Signaling Utechnology, Inc. acuin murine mab was diluted with 6% skim milk UBSU solution and used as reference. Acuin mab was purchased from Prouein Uech corporation at a dilution factor of 1: 4000. The blotting membrane was placed in the primary antibody reaction solution and incubated overnight at 4 ℃ with shaking. Rewarming for 1h at room temperature, washing with UBSU for 3 × 5min, respectively, diluting with 1:4000 times of UBSU for two antibodies, incubating for 1h at room temperature, and washing with UBSU for 3 × 5 min. And exposing the antigen-antibody complex labeled by the enzyme-labeled antibody in ECL luminescent liquid to present a strip with specific strength.

The results are shown in FIG. 6. The electrophoresis behavior of the mutant Uchl1 protein in the brain tissue of a mouse Homozygous (HOM) with Uchl1 gene point mutation is not obviously changed, but the content of the mutant Uchl1 protein is reduced to a certain extent compared with that of a wild type (WU) and a heterozygous type (HEU).

Example 6: immunohistochemical detection of Uchl1 in brain tissue of Point-mutant mice

Mouse brains fixed with 4% paraformaldehyde were processed by conventional paraffin embedding and sectioning to a slice thickness of 6 μm. Paraffin sections were xylene deparaffinized and transferred to 0.01M PBS after passing through an alcohol gradient. Then, the cells were immersed in 0.01M sodium citrate buffer solution (pH6.0) and boiled in a microwave oven for 5min to expose the antigen. Re-immersion in PBS to make fresh 3% H₂O₂The solution was treated for 10 minutes to eliminate the effect of endogenous peroxidase. In the immune reaction process, firstly, primary antibody reaction liquid is added dropwise according to a certain dilution. The different primary antibodies were diluted according to the guidelines provided by the supplier. Uchl1 rabbit mab was added at a ratio of 1: at a ratio of 400, diluting the mixture in a blocking solution consisting of 10% goat serum to form Uchl1 rabbit monoclonal antibody reaction solution. For the recognition of primary anti-IgG, the reaction solution was incubated for 1h at 37 ℃ with a biotin-labeled rabbit secondary antibody (1:200), and then biotin was specifically bound with Peroxidase-labeled streptavidin Surapuvidin-Peroxidase (1: 200). The peroxidase-labeled antigen-antibody complex was developed by a conventional method with the aid of a DAB substrate for 2.5min, and washed with double-distilled water for 2X 2min to terminate the color development reaction. Immunohistochemically developed sections were mounted with the aid of conventional Canadian gum. Under a microscope, a brown precipitate indicates a positive reaction.

Neurons of the substantia nigra pars compacta (SNpc) contain melanin, and thus in brain sections, these neurons appear black. These neurons have long and thick dendrites, and many ventral dendrites project to the substantia nigra reticula. The midbrain, outside the substantia nigra pars compacta, also has a number of similar neurons distributed throughout. All of these melanin-containing neurons project through the nigrostriatal pathway (nigrostriatal pathway) to the striatum, delivering a neurotransmitter called dopamine.

The results showed that Uchl1 was also expressed in the striatum, particularly in the ventral thalamus nucleus (venous nucleus of uhalamus), in addition to being highly expressed in substantia nigra neurons (FIG. 7). Further, observation of the cerebellar region revealed that the nuclei (Parabrachial nuclei and the like) in the lower part of the cerebellum were highly expressed. Compared with wild type and heterozygote type, the expression of the homozygote type at the corresponding part is obviously reduced.

Example 7: immunohistochemical detection of nuclear synapsin in brain tissue of point-mutant mice

Mouse brains fixed with 4% paraformaldehyde were processed by conventional paraffin embedding and sectioning to a slice thickness of 6 μm. Paraffin sections were xylene deparaffinized and transferred to 0.01M PBS after passing through an alcohol gradient. Then, the cells were immersed in 0.01M sodium citrate buffer solution (pH6.0) and boiled in a microwave oven for 5min to expose the antigen. Re-immersion in PBS to make fresh 3% H₂O₂The solution was treated for 10 minutes to eliminate the effect of endogenous peroxidase. In the immune reaction process, firstly, primary antibody reaction liquid is added dropwise according to a certain dilution. For the detection of the nuclear synapsin, the rabbit monoclonal antibody is adopted and diluted according to the proportion of 1: 200. For the recognition of primary anti-IgG, the reaction solution was incubated for 1h at 37 ℃ with a biotin-labeled rabbit secondary antibody (1:200), and then biotin was specifically bound with Peroxidase-labeled streptavidin Surapuvidin-Peroxidase (1: 200). Peroxidase-labeled antigen-antibody complexes were developed with the aid of DAB substrates using conventional methods. The color development was carried out for 2min, and the reaction was stopped by washing with double-distilled water for 2X 2 min. Immunohistochemically developed sections were mounted with the aid of conventional Canadian gum. Under a microscope, a brown precipitate indicates a positive reaction.

The results are shown in FIGS. 8 and 9. In comparison with Uchl1, the distribution of the synuclein in the brain is similar (FIG. 8), but is predominantly distributed in cells close to synaptic terminals, with a negative correlation with the distribution of Uchl1 protein (FIG. 9).

Example 8: mRNA expression profiling changes in brain tissue of point-mutant mice

Taking a mouse brain tissue total RNA sample to perform transcriptome sequencing analysis, and performing high-throughput sequencing on each sample by adopting a double-end sequencing mode of an illumina Hiseq sequencing platform.

The results show that the Uchl1 gene point mutation can cause a plurality of differences in gene expression of mice (FIGS. 10 and 11), possibly cause the signal pathway related to the interaction of nerve cell ligand and receptor to have regulatory change, and possibly serve as a molecular mechanism of inducing Parkinson's disease by Uchl1 gene mutation (FIG. 12).

Example 9: autonomic activity counting

The flour is placed in a big mouse cage and flattened by paper, then a grid (the length of the cage bottom is 30cm, the width is 24cm) of 6cm multiplied by 6cm is placed in the big mouse cage, a mouse is placed in the big mouse cage from one corner of the mouse cage, the number of the moving grids and the standing times of the mouse within 3 minutes are observed, and the conscious moving motility of the mouse is mainly tested.

As a result: as shown in fig. 13, the I93M point mutation (homozygous and heterozygous) of the Uchl1 gene tended to decrease in the active mobility of mice at 12 weeks of age compared to the wild type, but the difference was not statistically significant. In the process of detecting the autonomous activity of the mouse, the standing times of the mouse are counted, and the innervation capability of the hind limb of the mouse is reflected.

Example 10: suspension test

The mice were placed on a hanging bar (hanging bar 45cm long, 19cm high, 1.5mm diameter), the mice hanging time was recorded and scored, with the scoring rules: the score is 0-4 s,1 score is 5-9 s, 2 score is 10-14 s, 3 score is 15-19 s, 4 score is 20-24 s, 5 score is 25-29 s, and 6 score is more than 30 s. The time interval for each test was one minute. Mainly tests the coordination and endurance of the limbs of the mouse.

Results as shown in fig. 14, the score analysis of the results of the suspension experiment at 12 weeks of age showed no significant difference between the mutant mice (heterozygous and homozygous) and the wild mice, indicating that the holding ability of the limbs of the mice was also able to maintain their own body weight.

Example 11: roller test

The mice were placed in the middle of a roller (roller length 30cm, diameter 6cm, rotation speed 20r/min) and observed for whether the mice could follow the rolling axis in 2 minutes and for the duration. Mice were tested primarily for motor balance ability. The crawling time of the mutant mice on the rolling shaft reflects the self-crawling capability of the mutant mice in response to the rolling shaft so as to prevent falling.

The results are shown in fig. 15, and unexpectedly, the duration of crawling was significantly shorter in homozygous mice with I93M point mutation in Uchl1 gene (HOM vs WU, p 0.024; HOM vs HEU, p 0.032) compared to wild-type and heterozygous. This indicates that the active crawling ability of the point mutation homozygous mouse (HOM) is weakened, and especially reflects that the limbs, especially hind limbs, of the homozygous mouse are reduced in movement coordination, which is a precursor of involuntary tremor and represents the limb discordance symptom of parkinson's disease.

Example 12: elevated maze experiment

The mice were placed on one end of the open arm area of the elevated maze with the head facing outward (maze (65 cm. times.5 cm) comprising 3 parts: two opposite open arm areas (30 cm. times.5 cm), two opposite closed arm areas (30 cm. times.5 cm. times.30 cm), central area (5 cm. times.5 cm.) cross maze 50cm from the ground), observed for 5 minutes for open arm times (OE), open arm times (open arm, OU), closed arm times (CE), closed arm times (close arm, CE), closed arm times (cleararm, CU), downward probing times, standing times on closed arms. The experimental indices were based on the percentage of the number of entries into the open arm (OE%) and the percentage of the residence time in the open arm (OU%). Downward exploration times of the open arms and the central platform region reflect exploration behaviors in the unprotected region, and represent curious exploration of the animal on strange environments or escape seeking due to fear; the total number of entries into the open and closed arms reflects the animal's motor ability. After the experiment of each animal is finished, the excrement is removed, 70% ethanol is sprayed on the bottom of the box, and clean gauze is used for wiping the excrement to be dry, so that the influence of the residual smell of the previous animal on the experiment is avoided.

The results are shown in FIGS. 16 and 17, and the number of times and the time of entry into the open arm were higher in both the wild type (WU) and the heterozygous type (HEU) in the point mutation Homozygous (HOM) mice. The analytical reasons suggest that it may be associated with fewer escape of the HOM mice from the open arms (fig. 17). This suggests that point mutation Homozygous (HOM) mice have a higher fear, but at the same time have a weaker active motor capacity. This is consistent with the following symptoms appearing in PD patients: the limbs do not move in harmony and present anxiety and fear.

Example 13: fertility of Uchl1 gene point mutation mouse

In this example, statistical analysis of fertility was performed in point mutation Homozygous (HOM) mice, Heterozygous (HEU) mice and wild type (WU).

The results are shown in FIG. 18: the litters of Heterozygous (HEU) mouse Heterozygous (HEU) mating of Uchl1 gene I93M can reach 11 (6.22 +/-3.42 per litter), and the litters of homozygous type can reach 9 (5.50 +/-2.31 per litter), and the litters have no statistically significant difference (p is 0.470). This indicates that homozygous mouse fertility is not significantly affected by C57BL/6J fertility.

Discussion of the related Art

Because the cause of neurodegenerative diseases such as PD is unknown, the symptomatic treatment is difficult in clinic. In addition, drug development cannot be effectively performed due to the lack of an effective animal model that spontaneously develops similar clinical symptoms (particularly an animal model that exhibits clinical symptoms at an earlier stage).

It has been reported that 14 or more mutations in genes may be associated with Parkinson's disease. Some researches report that pathogenic genes mainly comprise alpha-Synuclein, UCH-L1 and LRRK2 are involved in autosomal dominant inheritance of PD, and DJ-1 and PINK1 genes are involved in recessive inheritance of PD.

Although there are several animal models of transgenic mouse Parkinson's disease including animal models based on Alpha-synuclein, LRRK2, DJ-1, etc., it is difficult to satisfy them because the major clinical symptoms of neurodegenerative diseases are not spontaneously and early manifested.

In addition, the reported transgenic mice basically depend on universal strong promoters, are randomly inserted into genomes, often have unnecessary markers, and the insertion sites influence the expression of other genes, and sometimes have expression quantity far exceeding the natural condition, so that the application of the obtained animal model in drug screening and evaluation is limited.

Meanwhile, for theoretical research, due to the existence of various unknown factors of genetic background change, the analysis of the action mechanism of the target gene is influenced. In addition, although the transgenic mouse model can introduce mutant genes, wild-type genes still exist, and the transgenic mouse model is not suitable for simulating clinical research on the genetic characteristics of pathogenic genes.

Previous studies found 1 cytosine to guanine mutation in the UCHL1 gene in 1 PD family (C277G), this missense mutation resulted in replacement of isoleucine at position 93 of the UCHL1 protein by methionine (I93M), and 4 PD patients appeared in 7 family members of this family, being autosomal dominant; the clinical manifestations of all patients are similar to sporadic PD, resting tremor is the first phenotype, and then the progression is rigidity, bradykinesia and gait abnormality, and levodopa treatment is shown to be effective.

However, in a transgenic mouse model constructed by introducing the exogenous gene of UCHL 1I 93M into a normal mouse, although the exogenous gene of UCHL 1I 93M was introduced, such transgenic mouse could not effectively exhibit clinical symptoms of parkinson's disease, particularly behavioral symptoms, although the randomly inserted gene was overexpressed (this may be because the original wild-type UCHL1 was still expressed). In addition, the random insertion of the genome in the transgenic mouse model not only can cause damage to other gene loci, but also is inconsistent with the genetic variation of clinical diseases.

The inventor firstly utilizes the Uchl1 point mutation (I93M) homozygous mouse constructed by the CRISPR-Cas system to truly simulate clinically-found human genetic diseases for the first time, and is expected to establish a novel mouse model of neurodegenerative diseases with wide application value.

Through comprehensive evaluation, the animal model mainly reflects the pathogenesis characteristics of the Parkinson disease and can be used as an animal model for screening drugs for treating the Parkinson disease. Meanwhile, the method is expected to be used for researching the action relationship between UCHL1 and other pathogenic genes of the Parkinson disease so as to further analyze the pathogenesis of the Parkinson disease.

The inventor utilizes the transgenic technology assisted by the advanced CRISPR-Cas9 to construct a spontaneous and early-onset Parkinson disease non-human mammal model for the first time. Uchl1 has a point mutation in this model. The disease model disclosed by the invention can be used for carrying out body level function analysis on UCHL1 so as to determine the etiology and pathogenesis of UCHL1 in Parkinson's disease, and has important scientific significance. In addition, the animal model is expected to lay a foundation for developing new prevention and treatment technologies and medicines by carrying out medicine treatment on the animal model.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Claims (10)

1. A method for preparing an animal model of neurodegenerative disease in a non-human mammal, comprising the steps of:

(a) providing a cell of a non-human mammal, and performing point mutation on the Uchl1 gene in the cell to obtain a Uchl1 gene point-mutated cell;

(b) preparing an animal model with Uchl1 gene point mutation by using the Uchl1 gene point mutation cell obtained in the step (a);

wherein, in the animal model, the Uchl1 gene point mutation is homozygous.

2. The method according to claim 1, wherein the Uchl1 gene point mutation comprises: the amino acid I at the 93 th position of the UCHL1 protein is mutated into 19 other amino acids except for I to form point mutation.

3. The method of claim 1, wherein the Uchl1 gene point mutation is the mutation of I at position 93 to M.

4. The method according to claim 1, wherein the non-human mammal is a rodent or primate, preferably comprising a mouse, rat, rabbit, monkey.

5. The method of claim 1, wherein in step (b), comprising the steps of:

(b1) preparing an animal model of the chimeric Uchl1 gene point mutation, and then obtaining a heterozygous Uchl1 gene point mutation animal model by hybridization; and

(b2) and obtaining a homozygotic Uchl1 gene point mutation animal model by hybridizing the heterozygotic Uchl1 gene point mutation animal model.

6. The method according to claim 1, characterized in that it comprises:

(1) constructing Uchl1 gene I93M targeted shearing plasmid by using CRISPR-CAS9 technology, and replacing with homologous mutation DNA sequence of a screening marker to obtain Uchl1-I93M point mutation positive monoclonal non-human mammal embryonic stem cells;

(2) preparing a chimeric non-human mammal by using the Uchl1 gene point-mutated non-human mammal embryonic stem cell clone strain obtained in the step (1);

(3) mating and breeding the chimeric non-human mammal obtained in the step (2) and a normal wild non-human mammal, and screening heterozygote non-human mammal with Uchl1 gene point mutation in the offspring;

(4) obtaining a Uchl1-I93M point-mutated homozygous non-human mammal by mating the heterozygous non-human mammals obtained in step (3) with each other, thereby obtaining a Uchl1-I93M point-mutated non-human mammal model.

7. Use of a non-human mammalian animal model prepared according to any one of claims 1 to 6, as an animal model for studying neurodegenerative diseases.

8. Use of a non-human mammalian animal model prepared according to any one of claims 1 to 6, wherein the model is used to screen or identify substances (therapeutic agents) that can reduce or treat neurodegenerative diseases.

9. A method of screening or identifying potential therapeutic agents for treating or ameliorating a neurodegenerative disease comprising the steps of:

a. administering a candidate substance to the non-human mammalian model prepared by the method of claim 1; and

b. performing behavioral analysis on the behavior of the animal model, and comparing the behavior with a control group;

wherein, if the behavior characterizing a neurodegenerative disease is improved in an animal model administered with the candidate substance compared to a control, the candidate substance is indicative of a potential therapeutic agent for a neuropsychiatric disease.

10. The method of claim 9, wherein said behavioral analysis comprises: a roller test, an elevated plus maze test, or a combination thereof.

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