Method for constructing transgenic mouse and application thereof

Abstract

The present invention provides a method for constructing transgenic mice and its applications, which relates to the technical field of animal model construction and applications. The technical key points are as follows: The application of G-R mice as transgenic Parkinson's disease mouse models, Alzheimer's disease mouse models, and aging models. This application clarifies the applications of G-R mice in Parkinson's disease, Alzheimer's disease, and aging models, provides new research strategies for the treatment of Parkinson's disease and Alzheimer's disease and the inhibition of aging, and provides research models for further screening drugs for the treatment of Parkinson's disease and Alzheimer's disease and other intervention means research. The transgenic mouse model adopted in this application can be used for further screening research on promoting the survival in dopaminergic neurons and improving the pathological and behavioral changes of Parkinson's disease. It is also an innovative application of this technology in the basic research field of Parkinson's disease, can provide experimental basis for screening new therapeutic drugs or intervention means for Parkinson's disease clinically, and has potential clinical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of animal model construction and application, and particularly relates to a method for constructing a transgenic mouse and its application. Background Art

[0002] Aging is a major risk factor for neurodegenerative diseases such as Alzheimer's Disease (AD) and Parkinson's disease (PD). Brain function gradually declines with aging, manifested as the decline of learning ability, memory ability, attention, decision-making speed, sensory perception (vision, hearing, touch, smell and taste), and motor coordination. With the increase of age, people's cognitive abilities in many aspects decline, including executive ability, working memory (especially task switching) and episodic memory. With the increase of age, the risk of suffering from neurodegenerative diseases is getting higher and higher, among which Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common. The clinical manifestations of PD include motor symptoms such as bradykinesia, resting tremor, muscle rigidity, and non-motor symptoms such as rapid eye movement sleep behavior disorder, constipation, cognitive decline, depression, and anxiety. At present, the specific etiology of PD is still unclear, and there is a lack of effective treatment methods to slow down or prevent the progression of Parkinson's disease. Therefore, if humans want to uncover the etiology of PD and completely cure PD, they must first establish an animal model that can simulate the pathogenesis of human PD, and it is urgent to carry out relevant research on the pathogenic mechanism and treatment methods of PD.

[0003] In the field of PD, there are numerous animal models available for researchers to use, classified according to characteristics such as pathology, pathogenesis, motor and non-motor symptoms, etc. However, for a researcher aiming to address multiple aspects of PD simultaneously, the selection of animal models becomes particularly important, and sometimes 2-3 animal models need to be selected simultaneously to conduct relevant research. The establishment of animal models for Parkinson's disease is currently mainly based on pharmacological, toxin, genetic, and α-synuclein models. The pharmacological model of PD was the earliest developed model and, to a certain extent, promoted the discovery of the symptomatic drug levodopa (L-DOPA). Reserpine and haloperidol administered peripherally were the early PD pharmacological models. Due to their limited duration of effect, relatively long-acting toxin PD animal models are mostly selected at present. There are two main methods that are relatively widely recognized: one is a model made by injecting 6-hydroxydopamine (6-OHDA) into the substantia nigra-striatal system to damage dopamine (DA) neurons in the substantia nigra-striatal system of rats. This modeling requires three-dimensional stereotactic injection, has high technical operation requirements, and has a high failure rate; the other is to use 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to prepare an animal PD model. However, MPTP also has irreparable neurotoxicity to humans, and strict safety control is required during operation. MPTP is metabolized rapidly in animals, and once the drug is stopped, the behavioral dysfunction and substantia nigra damage lesions in mice can recover quickly. Neither 6-OHDA nor MTPT can accurately simulate the pathological process of Parkinson's disease. For example, Lewy bodies do not appear in the above models. In addition, there are rotenone, paraquat, permethrin, and lipopolysaccharide. These toxin models will cause rapid loss of dopaminergic cells, leading to motor dysfunction and further behavioral defects. Due to their own limitations, they have various disadvantages.

[0004] Currently, PD genetic animal models include overexpression gene models, transgenic models, gene knockout, and gene mutation models, etc. With the discovery of several familial forms of PD, researchers have established animal models that simulate PD through gene mutations. Models with a clear connection to familial and sporadic cases are: α-synuclein (SNCA), leucine-rich repeat kinase 2 (LRRK2), and glucocerebrosidase (GBA). However, in these transgenic models, the complete characteristics of PD are rarely reproduced, and they often differ greatly from the human situation, and nerve damage in regions such as the substantia nigra and striatum is rarely observed.

[0005] The deficiencies of the above-mentioned model greatly limit its application. There is an urgent need in this field to develop a more reliable animal model of Parkinson's disease. The PD gene animal model should better simulate human PD and possess the following basic characteristics: detectable motor defects of PD, able to reflect the selective and progressive DAergic neuron damage that occurs with age, the formation of Lewy bodies, and the PD animal model should also have a relatively short disease course cycle, facilitating the economic and rapid study of the etiological and pathological mechanisms and drug screening, revealing the molecular mechanism of DAergic neuron death in PD and discovering potential therapeutic targets. Summary of the Invention

[0006] The object of the present invention is to provide a new application of a transgenic mouse model, enabling it to be applied to basic research related to aging, Alzheimer's disease, and Parkinson's disease and preclinical screening of therapeutic drugs.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] Application of G-R mice as a transgenic Parkinson's mouse model, an Alzheimer's mouse model, and an aging model, wherein the G-R mice are constructed by a method of CRISPR targeting the sequence information of CAG-LoxP-ZsGreen-Stop-LoxP-tdTomato at the H11 locus on chromosome 11 of the C57BL / 6 mouse genome.

[0009] Preferably, the complete sequence information of CAG-LoxP-ZsGreen-Stop-LoxP-tdTomato is as shown in SEQ ID NO: 1.

[0010] Preferably, the application of G-R mice as a transgenic Parkinson's mouse model in screening drugs for treating Parkinson's and studying other intervention means.

[0011] Preferably, the application of G-R mice as a transgenic Parkinson's mouse model in screening for survival in dopaminergic neurons, improving Parkinson's pathology and behavioral changes.

[0012] Preferably, the method for constructing G-R mice is as follows: loxP sites are buried on both sides of ZsGreen, and then the green fluorescent protein ZsGreen is combined with the red fluorescent protein tdTomato and simultaneously knocked into the murine H11 gene locus.

[0013] Preferably, the application of G-R mice in tracing the specific Cre expression position.

[0014] Through specific verification experiments such as behavioral science, molecular biology, and typical PD-like changes in pathology, this application has clarified the application of G-R mice in Parkinson's disease, Alzheimer's disease, and aging models, providing new research strategies for the treatment of Parkinson's disease and Alzheimer's disease and the inhibition of aging, and providing research models for further screening drugs and other intervention means for the treatment of Parkinson's disease and Alzheimer's disease. The transgenic mouse model used in this application can be used for further screening of research on promoting the survival of dopaminergic neurons, improving the pathological and behavioral changes of Parkinson's disease. It is also an innovative application of this technology in the basic research field of Parkinson's disease, which can provide experimental basis for screening new therapeutic drugs or intervention means for Parkinson's disease clinically and has potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a related drawing of Example 1 of the present invention, used to show a schematic diagram of the construction strategy of the G_R transgenic mouse vector;

[0016] Figure 2 It is a related drawing of Example 2 of the present invention. Among them, (a) is used to show the construction strategy of G_R mice and the schematic diagram of off-target detection; (b) is used to show the detection results of off-target sites in homozygous transgenic (C15) and heterozygous transgenic (G_R HE) mice by combining LTA-PCR and next-generation sequencing systems and analyzing with an integration site analysis system; (c) is used to show the results of using primer Top integration site, primer Tail integration site, and WGS sequencing data;

[0017] Figure 3 It is a related drawing of Example 3 of this application, used to show the gross appearance, body weight, and survival rate changes of G_R mice and C57BL / 6 control mice with the increase of months. Among them, (a) shows the gross appearance of G_R mice and C57BL / 6 control mice at 2 months, 6 months, and 12 months; (b) shows the comparison chart of the body weights of G_R mice and C57BL / 6 control mice at 2, 4, 6, 8, 10, and 12 months (n = 12); (c) shows the survival curves of G_R mice and C57BL / 6 control mice (n = 100).

[0018] Figure 4 It is a related drawing of Example 4 of this application, used to show the statistical results of the falling time on the rotarod (a), the total distance on the rotarod (b), the turning-around time on the climbing pole (c), and the total time on the climbing pole (d) of G_R mice and C57BL / 6 control mice at 2, 4, 6, 8, 10, and 12 months. Two-way ANOVA was used, and the values are expressed as mean ± SEM (n = 12), ns means no difference, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0019] Figure 5 These are the relevant drawings in Example 5 of this application. (a) is used to show the walking gait diagrams and 3D left hind paw pressure distribution diagrams of G_R mice and C57BL / 6 control mice at 2, 4, 6, and 8 months old in the Catwalk experiment; it is used to show the statistical results of gait analysis parameters, such as the statistical results of walking speed (b), swing phase (c), contact area (d), and average intensity (e). Two-way ANOVA was used, and the values are expressed as mean ± SEM (n = 10), ns means no difference, **p < 0.01, ****p < 0.0001.

[0020] Figure 6 These are the relevant drawings in Example 6 of this application, which are used to show the statistical results of platform latency (a), platform residence time (b), number of platform crossings (c), and quadrant residence time (d) of G_R mice and C57BL / 6 control mice at 2, 4, 6, and 8 months old in the Morris water maze experiment; the trajectory diagrams of 6-month-old C57BL / 6 control mice (e) and G_R mice (f) in the maze. Two-way ANOVA was used, and the values are expressed as mean ± SEM (n = 10), ns means no difference, *p < 0.05, **p < 0.01, ****p < 0.0001.

[0021] Figure 7 These are the relevant drawings in Example 7 of this application. (a) is used to show the number of escape failures of G_R mice and C57BL / 6 control mice at 2, 4, 6, and 8 months old in the shuttle box experiment; (b) is used to show the active avoidance reaction time of G_R mice and C57BL / 6 control mice at 2, 4, 6, and 8 months old in the shuttle box experiment. Two-way ANOVA was used, and the values are expressed as mean ± SEM (n = 10), ns means no difference, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0022] Figure 8 These are the relevant drawings in Example 8 of this application. Among them, (a) is used to show the movement trajectory diagrams of C57BL / 6 control mice and G_R mice in the open field experiment; (b) is used to show the movement time in the central area; (c) is used to show the number of times in the central area per 10 minutes. Two-way ANOVA was used, and the values are expressed as mean ± SEM (n = 10), ns means no difference, *p < 0.05, ***p < 0.001, ****p < 0.0001.

[0023] Figure 9These are the relevant drawings in Example 9 of this application. (a) is used to show the immunofluorescence staining results of the specific marker tyrosine hydroxylase (TH, white) and pathological α-syn (ps129α-syn, red) of dopaminergic neurons in the substantia nigra of the midbrain of 2-, 6-, and 10-month-old G_R mice and C57BL / 6 control mice. Green represents the GFP carried by SKPs-DA neurons, Bar = 500 μm; (b) is used to show the semi-quantitative analysis of TH in the substantia nigra of the midbrain of G_R mice and C57BL / 6 mice using Image J; (c) is used to show the detection of pathological ps129α-syn expressed in the midbrain of 2-, 4-, 6-, and 8-month-old G_R mice and C57BL / 6 mice by Western Blot and the semi-quantitative analysis using Image J. Two-way ANOVA was used, and the values are expressed as mean ± SEM (n = 6), ****p < 0.0001.

[0024] Figure 10 These are the relevant drawings in Example 9 of this application, used to show the immunohistochemical staining results of the olfactory bulb, cerebral cortex, midbrain, hippocampus, and small intestine of 10-month-old G_R mice and C57BL / 6 control mice. The arrow with a tail indicates Lewy body (LB), scale bar = 100 μm.

[0025] Figure 11 These are the relevant drawings in Example 10 of this application, used to show the 10% brain homogenate of the midbrain, hippocampus, and olfactory bulb of 2-month-old G_R mice and C57BL / 6 control mice as the seed protein for the experiment, and perform real-time quaking-induced conversion experiment (RT-QuIC). Detailed implementation manners

[0026] The present invention will be further described in detail below in combination with specific embodiments.

[0027] The application of G-R mice in transgenic Parkinson's mouse models, Alzheimer's mouse models, and aging models, and the G-R mice are constructed by the method of CRISPR targeting the CAG-LoxP-ZsGreen-Stop-LoxP-tdTomato sequence information at the H11 locus on chromosome 11 of the C57BL / 6 mouse genome.

[0028] The complete sequence information of CAG-LoxP-ZsGreen-Stop-LoxP-tdTomato is shown as SEQ ID NO: 1.

[0029] This application also provides the application of the G-R mice in screening drugs for treating Parkinson's and other research on intervention means.

[0030] The present application also provides the use of the G-R mouse model in screening for survival in dopaminergic neurons and improving Parkinson's pathology and behavioral changes.

[0031] The following specific verification experiments are used to determine the application of G-R mice in Parkinson's disease.

[0032] Example 1: Establishment of a transgenic Parkinson's disease mouse model:

[0033] The green fluorescent protein ZsGreen was combined with the red fluorescent protein tdTomato and simultaneously knocked into the murine H11 gene locus. Under normal circumstances, mice express ZsGreen throughout the body and can emit a dazzling green light, which can illuminate the morphology and structure of various cells and tissues. Therefore, newborn mice are green all over. However, in the present application, loxP sites were implanted on both sides of ZsGreen. When Cre recombinase is present, ZsGreen in the mouse genome will be deleted, turning on the expression of tdTomato, which can emit a dazzling red light. Since Cre can be expressed in a tissue-specific manner, and mice turn on the red light following the expression time and location of Cre. This model can use the Cre / loxP site-specific recombination system to track and display cell fate, revealing the regeneration, self-renewal, and differentiation processes of specific types of cells during development and disease. The dual-fluorescent reporter gene system provides two fluorescent markers, zsGreene and tdTomato, enabling real-time detection of recombinant / non-recombinant cells. Therefore, this model is also an ideal tool for tracing the location of specific Cre expression. As Figure 1 and Figure 2 Figure a shows the construction strategy of the G-R transgenic mouse vector.

[0034] Example 2: Off-target detection of G-R mice:

[0035] As Figure 2 shown, the specific positions of off-target sites in the genomes of homozygous transgenic (C15) and heterozygous transgenic (G-R HE) mice were analyzed using a combination of LTA-PCR and next-generation sequencing systems and an integration site analysis system, and the sequencing results were verified using primers Top integration site, primers Tail integration site, and WGS sequencing. LTA-PCR is a technique used to amplify and detect unknown genomic sequences flanking the integrated vector DNA. Integration Site Analysis (ISA) is usually used for safety monitoring during the use of cell and gene therapy drugs. The primer information is shown in the following table:

[0036] Table 1 Primer information table

[0037]

[0038]

[0039] The above detection indicates that the G-R mice have single-copy insertion and no off-target sites are generated.

[0040] Example 3: Comparison of gross appearance, body weight, and survival rate of G-R mice and C57BL / 6 control mice with increasing age:

[0041] As Figure 3 shown, the gross appearances of 2-month-old, 6-month-old, and 12-month-old G-R mice and C57BL / 6 control mice were compared respectively. The G-R mice showed PD-like symptoms: significantly lower body weight than that of the control mice of the same age, kyphosis, reduced appetite, and progressive symptoms such as motor retardation, muscle rigidity tremors, and decreased exploratory behavior. Their motor ability and memory significantly declined. The novel Parkinson's disease genetic model mice at 6 to 12 months of age had kyphosis, were emaciated, had sparse hair, and showed obvious signs of aging compared with the C57BL / 6 control mice of the same age. The body weight of C57BL / 6 mice began to increase continuously at 2 months of age and remained above 29 g after 6 months of age, while the body weight of G-R mice continued to be below 25 g, and some were even lower than 20 g, and the survival rate at 12 months was less than 10%.

[0042] Example 4: Motor ability test of G-R mice and C57BL / 6 control mice in the rotarod and pole climbing tests:

[0043] As Figure 4 shown, in order to accurately evaluate the changes in the motor ability of mice, in this application, the rotarod test and pole climbing test were performed on G-R mice and C57BL / 6 control mice at 2, 4, 6, 8, 10, and 12 months of age. One week before the experiment, the mice were subjected to adaptive training to reduce the anxiety and exploratory behavior that might cause them to accidentally fall off the pole. The results of the rotarod test showed that the rotarod time of G-R mice was significantly reduced compared with that of C57BL / 6 mice, and the running distance was significantly shortened, indicating that the motor ability of G-R mice decreased significantly with age. Especially after 6 months of age, their motor ability dropped drastically. In the pole climbing test, G-R mice needed more time to turn around and took longer to complete climbing the pole.

[0044] Example 5: Gait test of G-R mice and C57BL / 6 control mice:

[0045] As Figure 5As shown, in this application, a small animal gait analysis system (Catwalk XT) was used to collect the gaits of G-R mice at 2, 4, 6, and 8 months of age, and C57BL / 6 mice of the same age were used as the control group. In the Catwalk experiment, it can be seen from the walking gait map and the left hind foot pressure map that as the age of G-R mice increases, statistical analysis shows that their stride frequency continuously increases, coordination decreases, hind foot pressure gradually decreases, and walking speed gradually decreases. In addition, the distance between their left and right feet significantly increases, the distance between the front and hind feet decreases, and the swing phase increases. The contact area of the hind feet of 8-month-old mice significantly decreases, and the relative pressure of the hind feet also significantly decreases. The motor ability of G-R mice continuously declines with increasing age, especially after 6 months of age.

[0046] Example 6: Memory function of G-R mice and C57BL / 6 control mice was evaluated through a water maze experiment:

[0047] As Figure 6 shown, in this application, Morris water maze experiments were conducted on G-R mice and C57BL / 6 control mice at 2, 4, and 6 months of age. The experiment found that the platform latency of 6-month-old G-R mice was significantly longer than that of C57BL / 6 control mice, the number of times crossing the platform and the platform residence time were significantly reduced, and the quadrant residence time was also significantly decreased. By comparing the trajectory maps of the Morris water maze, it can be clearly seen that the movement trajectory map of C57BL / 6 control mice is linear, indicating that as the number of training sessions increases, they can quickly find the platform and accurately remember the relative position of the platform in the spatial exploration experiment. However, the movement trajectory of G-R mice is still marginal, proving that they do not remember the platform or cannot find the accurate position of the platform in the spatial exploration experiment, indicating that their spatial learning and memory abilities decline, suggesting a significant decrease in the memory and cognitive abilities of G-R mice.

[0048] Example 7: Memory function of G-R mice and C57BL / 6 control mice was evaluated through a shuttle box experiment:

[0049] As Figure 7 shown, by comparing G-R mice and C57BL / 6 control mice, it was found that the reaction time gap between the two groups of mice was not significant at 2 and 4 months of age. After exceeding 6 months of age, the reaction time of G-R mice was longer. In addition, the number of avoidance failures of G-R mice has far exceeded that of C57BL / 6 mice since 2 months of age. During the training process, C57BL / 6 mice can quickly respond and avoid harmful stimuli when the harmful stimuli occur again through familiarizing with the environment and learning and memory after several stimulations, while G-R mice cannot.

[0050] Example 8: G-R mice and C57BL / 6 control mice were evaluated for their emotional states through an open field experiment:

[0051] The open field test is used to detect the spontaneous activity behavior and exploratory behavior of animals in a strange environment, and can evaluate the autonomous behavior, exploratory behavior and tension of animals in a novel environment. As Figure 8 shown, the open field test compared the movement trajectories of G-R mice and C57BL / 6 control mice in the open field, and found that after G-R mice exceeded 6 months of age, the exploratory trajectories in the central area of the open field significantly decreased, and the overall movement trajectories decreased at 8 months of age. Statistical analysis showed that the movement time and central area frequency of G-R mice exploring the central area were significantly reduced compared with C57BL / 6 control mice.

[0052] Example 9: Pathological changes in the midbrain of G-R mice and C57BL / 6 control mice:

[0053] In addition to motor symptoms such as bradykinesia, resting tremor, muscle rigidity, and non-motor symptoms such as cognitive decline, depression, and anxiety, PD also has very important pathological changes, including irreversible death of dopaminergic neurons in the substantia nigra of the midbrain and the appearance of Lewy bodies (LB).

[0054] As Figure 9 shown, G-R mice and C57BL / 6 mice of different months of age were taken respectively, and it was found by immunofluorescence staining that the expression of tyrosine hydroxylase (TH), a specific marker of dopaminergic neurons in G-R mice, gradually decreased with age, and the expression level decreased by more than 70% at 10 months of age, while the marker of pathological α-syn, ps129-syn, increased continuously. Experiments have proved that with age, the dopaminergic neurons in the substantia nigra of the midbrain of G-R mice continuously die, and Lewy bodies (LB) appear in the mouse brain tissue, especially in the substantia nigra of the midbrain, while C57BL / 6 mice are normal.

[0055] As Figure 10 shown, the brain tissue and small intestine of 10-month-old G-R mice and C57BL / 6 control mice were taken respectively, and ps129-syn was detected by immunohistochemistry. It was found that Lewy bodies (LB) appeared in the olfactory bulb, midbrain, hippocampus, cortex and even small intestine of 10-month-old G-R mice, while the pathological examination of C57BL / 6 control mice was negative. It was further confirmed that ps129-syn existed in G-R mice and damaged the dopaminergic neurons in the substantia nigra of the midbrain.

[0056] Example 10: Application of real-time quaking-induced conversion (RT-QuIC) in the early diagnosis of PD in G-R mice:

[0057] Real-time quaking-induced conversion (RT-QuIC) is an in vitro experiment that amplifies pathological α-syn in large quantities through continuous cycles of shaking and incubation, and reflects the amount of protein in real time through thioflavin T fluorescence labeling, so as to detect the pathologically folded α-syn in cerebrospinal fluid. It has the characteristics of high throughput, high speed, high sensitivity, and high specificity.

[0058] Research has shown that the highly sensitive and specific RT-QuIC can detect pathological α-synuclein in cerebrospinal fluid and skin, providing strong support for the early diagnosis of PD. The plasmid expressing murine α-synuclein was transformed into Escherichia coli and highly expressed. Then, the Escherichia coli was collected, and the supernatant was obtained by breaking the cell wall, followed by affinity chromatography and molecular sieve concentration to purify the α-synuclein protein. The purified α-synuclein protein was used as the substrate for seed amplification, and the RT-QuIC system was configured.

[0059] The results are as Figure 11 shown. Brain homogenates from G-R mice and C57BL / 6 control mice were used as seeds for the RT-QuIC experiment. The results of the brain homogenates of G-R mice were positive, demonstrating the presence of pathological α-synuclein in the brain tissues of G-R mice, including the midbrain, hippocampus, and olfactory bulb.

[0060] Aging is a major risk factor for neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Therefore, studying the biological mechanisms of aging and how it is interrelated with the progression of neurodegenerative diseases is a research field worthy of in-depth exploration. A large amount of evidence indicates that brain aging markers act upstream of the disease-defined Aβ plaques and pTau neurofibrillary tangles. On the other hand, the aggregation of Aβ and pTau can cause oxidative stress, Ca2+ homeostasis disorders, mitochondrial dysfunction, and other aging markers in neurons. During the occurrence of PD, the main aging process leads to the accumulation of neurotoxic forms of α-synuclein in cells. Conversely, the accumulation of α-synuclein exacerbates the aging process and leads to neuronal dysfunction and apoptosis. PD is a typical disease in which the molecular pathogenesis exacerbates aging characteristics. The overexpression of mutant or wild-type α-synuclein is sufficient to cause signs of aging in young mice, including the accumulation of oxidized modified proteins, DNA damage, mitochondrial dysfunction, microglial activation, and impaired autophagy.

[0061] Parkinson's disease is a central nervous system degenerative disease with a high incidence in the elderly population. The medical community believes that the cause of Parkinson's disease is due to the decline in the function and the reduction in the number of dopamine neurons in the central nigrostriatal region, resulting in insufficient secretion of the excitatory neurotransmitter dopamine. However, the cause and pathogenesis of the degenerative changes of Parkinson's nerve cells are not yet clear, but aging is undoubtedly one of the important inducing factors of Parkinson's disease. Therefore, exploring the role of aging factors in the occurrence and development of Parkinson's disease is crucial for understanding Parkinson's disease. Examples 3-8 above demonstrated that G-R mice had obvious aging manifestations.

[0062] Parkinson's disease (PD) is a neurodegenerative disease commonly found in middle-aged and elderly people. The main symptoms include asymmetric resting tremors, bradykinesia, postural instability, and stiffness. Parkinson's disease patients often experience cognitive impairment during the disease process and may develop dementia symptoms in the late stage. Parkinson's disease dementia (PDD) is a common complication of Parkinson's disease, seriously affecting the health and quality of life of patients. Parkinson's disease is a degenerative disease related to aging. Until now, the specific mechanism of degeneration remains unclear in medicine. However, current research shows that the onset of Parkinson's disease is related to multiple factors, including age growth, social factors, environmental factors, exposure to toxic substances, genetic factors, stress, and unhealthy lifestyles. When various factors cause the secretion of dopamine in the substantia nigra in the brain to fail to meet the normal physiological needs of the human body, Parkinson's disease will occur.

[0063] Regarding the clinical manifestations, Parkinson's disease and Alzheimer's disease have the following different manifestations:

[0064] Parkinson's disease:

[0065] Clinically, the main symptoms are motor symptoms such as tremors, slow movements, body stiffness, and abnormal gait. At the same time, there will also be non-motor symptoms such as reduced sense of smell, constipation, sleep disorders, and excessive sweating. And in the middle and late stages, symptoms such as memory decline, hallucinations, and dementia may occur.

[0066] Examples 3-5 and 8-9 in this application demonstrate the application of G-R mice in Parkinson's disease in this application.

[0067] Alzheimer's disease:

[0068] In the early stage, Alzheimer's disease patients will experience memory decline (such as forgetting what was said later after hearing it), language disorders (such as failing to express oneself properly), and cognitive function disorders (such as starting to get lost). In the late stage, there will be mental and behavioral disorders such as difficulty in moving and incontinence, but there will be no motor symptoms like those in Parkinson's disease.

[0069] Examples 6-8 in the above embodiments demonstrate the application of G-R mice in Alzheimer's disease in this application.

[0070] In summary, through specific verification experiments such as typical PD-like changes in behavioral science, molecular biology, and pathology, this application has clarified the application of G-R mice in Parkinson's disease and Alzheimer's disease, providing new research strategies for the treatment of Parkinson's disease and Alzheimer's disease, and providing a research model for further screening of therapeutic drugs and other intervention means for Parkinson's disease and Alzheimer's disease; the transgenic mouse model used in this application can be used for further screening of research on promoting survival in dopaminergic neurons and improving the pathological and behavioral changes of Parkinson's disease. It is also an innovative application of this technology in the basic research field of Parkinson's disease, which can provide experimental evidence for screening new therapeutic drugs or intervention means for Parkinson's disease clinically and has potential clinical application value.

Claims

1. The use of GR mice as a transgenic Parkinson's mouse model is characterized by: The GR mouse was constructed by inserting a CAG-LoxP-ZsGreen-Stop-LoxP-tdTomato sequence into the chr11_3195375:chr11_3195392 site of chromosome 11 H11 of the C57BL / 6 mouse genome through CRISPR; The complete sequence information of the CAG-LoxP-ZsGreen-Stop-LoxP-tdTomato is shown in SEQ ID NO:

1.

2. The use of the GR mouse according to claim 1 as a transgenic Parkinson's mouse model, characterized in that: The GR mouse is used as a transgenic Parkinson's mouse model in the research of screening therapeutic drugs or intervention methods for Parkinson's disease.

3. The use of the GR mouse according to claim 1 as a transgenic Parkinson's mouse model, characterized in that: The GR mouse is used as a transgenic Parkinson's mouse model in the study of screening drugs that improve Parkinson's pathology.

Images