GuideRNA of targeted mouse Gba gene and construction method and application of Parkinson mouse model

Abstract

The invention discloses guideRNA (Ribonucleic Acid) of a targeted mouse Gba gene, the guideRNA is designed according to the eleventh exon of a mouse Gba-202 transcript, and the nucleotide sequence of the guideRNA is as shown in SEQ ID No. 1. The invention also discloses a construction method of the Parkinson's mouse model and application of the construction method in research on pathogenesis of primary Parkinson's disease. The invention provides an innovative and reliable construction method of the Parkinson's mouse model through the combined action of a PD risk gene Gba mutant mouse and a high milk diet dangerous environment, and the constructed Parkinson's mouse model can better simulate the occurrence and development process of PD, conforms to the pathological progress of PD progressive aggravation, can monitor the early change of PD, and has a good application prospect. And the model can dynamically quantify the dyskinesia and pathological progress of the PD, and can provide a research basis for different stages of PD.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a guideRNA targeting the mouse Gba gene and a method for constructing and applying a Parkinson's disease mouse model. Background Technology

[0002] Primary Parkinson's disease (PD) is a neurodegenerative disease characterized by the deposition of α-synuclein (α-syn)-positive inclusion bodies in neurons and / or glial cells. Its main clinical features are resting tremor, bradykinesia, and rigidity. In later stages, motor symptoms such as postural instability may appear. PD has a high incidence rate, increasing with age. Early symptoms of PD are often insidious, making diagnosis difficult and lacking effective treatment. This severely damages the physical and mental health and quality of life of PD patients, imposing a heavy economic burden on society and families. If PD patients receive early and accurate diagnosis and timely treatment, the prognosis is better and survival can be prolonged. Therefore, research into the pathological mechanisms of PD is extremely urgent.

[0003] Currently, Parkinson's disease (PD) mouse models include induced animal models such as the MPTP model and striatal stereotactic injection of α-syn, and gene-modified animal models such as the LRRK2 transgenic model and the PINK1 gene knockout model. Each model has its own advantages and disadvantages. The MPTP model is quick and cost-effective to create, but its success rate is low, and it suffers from insensitivity in some mice and reversibility, which does not accurately reflect the progressive worsening of symptoms in actual clinical Parkinson's patients. This limits the study of the pathogenesis and early stages of PD. The striatal stereotactic injection of α-syn model has a high success rate, irreversible symptoms, and a good temporal simulation of the entire process of Parkinson's disease occurrence and development, which is beneficial for discovering and identifying new therapeutic targets. However, because it directly injects pathological proteins into the lesion site, it cannot study the impact of hazardous environments on the pathogenesis. Gene-modified models have a long creation cycle and can be used for basic research to obtain corresponding phenotypes, but they do not reflect the progressive loss of dopamine neurons and cannot accurately simulate the pathological process of PD. These shortcomings limit the study of the pathogenesis of PD; therefore, there is an urgent need to develop a more reliable PD animal model.

[0004] In summary, Parkinson's disease is a progressive neurodegenerative disease. Currently available animal models are difficult to simulate the progressive pathogenesis and pathological process, and most are acute reversible models, which limits the study of the pathogenesis of PD. Summary of the Invention

[0005] The purpose of this invention is to address the problem that existing animal models are difficult to simulate progressive pathogenesis and pathological processes, and are mostly acute and reversible models, which limits the study of PD pathogenesis. This invention provides a guideRNA targeting the mouse Gba gene and a method for constructing and applying a Parkinson's disease mouse model.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a guideRNA targeting the mouse Gba gene, the guideRNA is designed based on the 11th exon of the mouse Gba-202 transcript, and the nucleotide sequence of the guideRNA is shown in SEQ ID No.1.

[0007] This invention also provides the application of the above-mentioned guideRNA in the study of the pathogenesis of primary Parkinson's disease.

[0008] The present invention also provides a donor DNA vector, the sequence of which is shown in SEQ ID No. 2.

[0009] This invention also provides a method for constructing a Parkinson's mouse model, comprising the following steps:

[0010] S1. Cas9 mRNA, the guideRNA of claim 1 and the donor DNA vector of claim 2 are injected into mouse zygotes to obtain F0 generation mice. The F0 generation mice are identified by PCR and sequencing to screen out positive F0 generation mice.

[0011] S2. Positive F0 generation mice were bred with WT mice to obtain F1 generation mice. The F1 generation mice were genetically identified by PCR and sequencing. Positive F1 generation heterozygous mice were screened out, which are Gba gene point mutation mice.

[0012] S3. Gba gene point mutant mice were given 0.1 mL / g of fresh milk by gavage daily for 8 weeks.

[0013] S4. Every 4 weeks, mice were weighed and their behavioral phenotypes were assessed, including open field test, rotundus test, footprint test, balance beam test, and elevated cross maze test.

[0014] S5. After 8 weeks of feeding, based on the results of behavioral phenotype assessment and brain tissue pathological examination, the Parkinson's mouse model was determined to have been successfully constructed.

[0015] Preferably, the nucleotide sequences of the PCR amplification primers F and R in S1 and S2 are as shown in SEQ ID No. 3 and SEQ ID No. 4.

[0016] Preferably, in step S5, the pathological examination of mouse brain tissue involves immunohistochemistry and TH staining of mouse brain tissue.

[0017] This invention also provides the application of the above-mentioned construction method in the study of the pathogenesis of primary Parkinson's disease.

[0018] The beneficial effects of this invention are as follows:

[0019] (I) This invention provides an innovative and reliable method for constructing a Parkinson's disease mouse model by combining the PD risk gene Gba mutant mice with a high-dairy diet and a high-risk environment. This Parkinson's disease mouse model overcomes the limitations of existing PD animal models, such as long cycle, absence of α-syn positive inclusion bodies, lack of progressive loss of dopaminergic neurons, and acute reversibility. The Parkinson's disease mouse model in this invention can better simulate the occurrence and development of PD, conforms to the pathological progression of progressive PD, can monitor early changes in PD, and can dynamically quantify the motor impairment and pathological progression of PD, providing research basis for different stages of PD.

[0020] (ii) The model is simple and convenient to make, has a short cycle, and a high success rate of over 80%, which can well meet the needs of PD research. Attached Figure Description

[0021] Figure 1 This is a diagram illustrating the construction of the Gba gene point mutation mouse in Example 1;

[0022] Figure 2 This is an electrophoresis image used to verify the PCR amplification products in Example 1;

[0023] Figure 3 This is a peak shape diagram near the mutation site in the Gba gene point mutant mouse in Example 1;

[0024] Figure 4 The results of the mouse behavioral tests in Example 2;

[0025] Figure 5 The results of immunohistochemistry of the mouse brain in Example 2;

[0026] Figure 6 The results of TH staining in the mouse brain in Example 2. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0028] The mouse zygotes were zygotes from C57BL / 6J mice and were purchased from Nanmo Biotechnology.

[0029] Example 1: Construction of Gba gene point mutant mice

[0030] There are five transcripts of the Gba gene locus. Based on the Gba gene structure, the Gba-202 transcript was selected for analysis. This transcript has 12 exons, and protein translation begins at exon 3 and terminates at exon 12. The human GBAL444P corresponds to the mouse mutation L426P, which is located at exon 11 of the Gba-202 transcript. Referring to the Ensemnl database, the Gba-202 ID is ENSMUST00000167998.1. A guideRNA was designed based on exon 11 of the Gba-202 transcript. The nucleotide sequence of the guideRNA is shown in SEQ ID No. 1.

[0031] Using CRISPR / Cas9 gene editing technology, the exon 11 of the Gba-202 transcript was targeted. Guide RNA, Cas9 mRNA, and donor DNA vector were injected into the fertilized eggs of C57BL / 6J mice under a microscope. The Gba-202 exon 11 transcript was homologously recombinated with oligo donor DNA carrying the point mutation gene to obtain F0 generation mice. Positive F0 generation mice were screened from the offspring by PCR and sequencing. Positive F0 generation mice were bred with WT mice to obtain F1 generation mice. The F1 generation mice were genetically identified by PCR and sequencing, and positive F1 generation heterozygous mice were selected, which are the Gba gene point mutation mice.

[0032] The construction of the Gba gene point mutant mouse in this embodiment is shown in the attached figure. Figure 1 The donor DNA vector sequence is shown in SEQ ID No. 2; the nucleotide sequences of PCR amplification primers F and R are shown in SEQ ID No. 3 and SEQ ID No. 4, the PCR amplification fragment is shown in SEQ ID No. 5, and the electrophoresis diagram of the amplification product verification is attached. Figure 2 As shown in the attached figure, the peak shape near the mutation site in Gba gene point mutant mice is as follows. Figure 3 As shown.

[0033] Example 2: Construction of a Parkinson's Mouse Model

[0034] 1. Laboratory animals

[0035] Forty 6-8 week old SPF-grade Gba gene point mutant mice prepared in Example 1 were randomly selected, with half males and half females. They were randomly divided into two groups of 20 mice each, with half males and half females, serving as the control group and the high-milk diet group (model group). All animal experiments were approved by the Institutional Animal Care and Use Committee of Zhengzhou University.

[0036] 2. Establishment of a mouse model of Parkinson's disease

[0037] 2.1 Gavage

[0038] Mice in the high-milk diet group were given 0.1 mL / g of fresh milk by gavage before their first feeding each morning, while mice in the control group were given an equal volume of sterile PBS by gavage at the same time. The intervention lasted for 8 weeks. The weight of the mice was monitored weekly.

[0039] 2.2 Behavioral Evaluation

[0040] 2.2.1 Rotating bar experiment

[0041] After modeling, mice in both the model group and the control group underwent a rotarod test to assess overall motor function. The rotarod was operated in acceleration mode: accelerating from 6 rpm / min to 30 rpm / min within 240 seconds, with intervals of at least 15 minutes between each group. The time it took for the mouse to fall was recorded; if the mouse remained in the rotarod for more than 240 seconds, it was recorded as 240 seconds. This was performed once a week, with three groups per week, and the maximum value was recorded.

[0042] 2.2.2 Footprint Experiment

[0043] Mice were coated with non-toxic green dye on their hind paws and non-toxic red dye on their forelegs. They were then guided through a 50cm long, 8cm wide paper track into a closed black box. Footprint analysis included six parameters: ① Stride distance, measuring the distance the mouse's forelimbs and hindlimbs moved forward, including left forelimb, left hindlimb, right forelimb, and right hindlimb. ② Base width, measuring the average distance between the left and right footprints of the mouse's forelimbs and hindlimbs, including anterior base width and posterior base width. For each parameter, three consecutive sets of clear footprints were selected for each measurement, and the average value was taken.

[0044] 2.2.3 Balance Beam

[0045] This experiment was used to assess the balance, muscle strength, and motor coordination of mice. The balance beam was square, measuring 1m long and 14mm wide. One side of the beam was placed inside a black box, and the mouse was placed on the other side. The time it took for the mouse to cross the beam into the black box and the number of slips within 60 seconds were recorded. The experiment consisted of a training period and a testing period, lasting 3 days. Training period: On day 1, the balance beam was used, and the mouse was guided to cross the square beam into the small box. Each mouse was trained 3 times, with a 2-hour interval between each training session. The same steps were repeated on day 2. Testing period: Day 3 was the testing phase. The mouse was guided to cross the beam to reach the small box, and the time to reach the box and the number of slips were recorded. If the mouse failed to reach the box, the time was recorded as 60 seconds.

[0046] 2.2.4 Open Field Experiment

[0047] Open field tests were conducted on mice in the model group and control group to observe their spontaneous exploratory movement activity and anxiety behavior. Each mouse underwent one test, placed facing a wall in one of the four corner squares, and allowed to freely explore the environment for 5 minutes. Parameters such as the mouse's activity time in the central area, total distance traveled, activity time in the surrounding areas, and rest time were recorded. After each mouse's test, the testing area was wiped with a damp cloth and then dried with a dry cloth.

[0048] 2.2.5 Elevated Cross Maze

[0049] Place the experimental animals in the central area of ​​the maze, with their heads facing the open arms, ensuring that each animal remains in the same position thereafter. Simultaneously, activate a video monitor to record the number of times each animal enters the open and closed arms within 5 minutes, as well as the time taken to enter each arm. During the experiment, the experimenter must maintain a distance of 1 meter from the maze.

[0050] The results of the behavioral tests on the swivel, footprints, open field, elevated platform, and balance beam are attached. Figure 4 As shown, compared with the control group, mice in the high-dairy diet group (model group) showed decreased motor function and reduced spontaneous activity and exploration ability, which worsened over time.

[0051] 2.3 Pathological examination

[0052] Brain tissues from mice in the high-dairy diet group were subjected to histochemical staining and TH staining, respectively.

[0053] 2.3.1 Immunohistochemistry

[0054] ① Dewaxing: Xylene I for 20 min; Xylene II for 15 min. ② Hydration: 100% ethanol I for 15 min; 100% ethanol II for 15 min; 95% ethanol for 5 min; 85% ethanol for 5 min; 75% ethanol for 5 min; rinse with PBS for 3 min. ③ Blocking endogenous catalase: Add 3% H2O2 and deionized water, incubate at room temperature for 5 min; rinse with PBS for 3 min. ④ High-temperature and high-pressure antigen retrieval: Place the tissue in sodium citrate buffer, place in an autoclave, start timing from steam rise, autoclave at high temperature and pressure for 5 min; cool and rinse with PBS for 3 min. ⑤ Enzymatic digestion antigen retrieval: Add trypsin diluted 1:2 to the tissue and incubate at room temperature for 1 min; rinse with PBS for 3 min. ⑥ Blocking: Block with goat serum blocking solution at room temperature for 20 min. ⑦ Adding primary antibody: Pα-syn antibody diluted 1:800 and incubated at 4℃ for 24 h, then rinse with PBS 3 times, 5 min each time. ⑧ Add secondary antibody: Dilute goat anti-mouse secondary antibody at a ratio of 1:300 and add it to the tissue, incubate slightly, and incubate at room temperature for 30 min; wash three times with PBS, 5 min each time. ⑨ DAB staining: Prepare the DAB solution according to the specified ratio in the dark, incubate at room temperature for 5 min, and rinse with tap water for 10 min. ⑩ Counterstaining: Hematoxylin in the dark for 80 s; differentiate with hydrochloric acid differentiation solution for 20 s; rinse with tap water for 5 min. Dehydration: 75% ethanol 1 min; 85% ethanol 1 min; 95% ethanol 1 min; 100% ethanol II 10 min; 100% ethanol I 10 min; xylene II 10 min; xylene I 1 min. Mounting: Mount with neutral resin. Observe under a microscope after mounting. Immunohistochemical results are as follows. Figure 5 As shown, this indicates the presence of α-syn positive inclusion bodies in the mouse brain.

[0055] 2.3.2 TH staining

[0056] ① Wash frozen sections with PBS (pH 7.4) for 5 min × 3 times. ② Immerse sections in freshly prepared 0.3% hydrogen peroxide for 5–10 min to inactivate endogenous enzymes, then wash with distilled water for 5 min × 3 times. ③ Block with normal goat serum. ④ Add primary antibody TH monoclonal antibody at a 1:1000 ratio. Incubate overnight at 4°C. The control group used normal goat serum instead of the primary antibody, and the result was negative. ⑤ Add secondary antibody biotinylated goat anti-rabbit serum, incubate at 37°C for 1 h, then wash with PBS for 5 min × 3 times. ⑥ Add SA / HRP, incubate at 37°C for 1 h, then wash with PBS for 5 min × 3 times. ⑦ Develop with DAB (diaminobenzidine), controlling the color development under a microscope for 3–10 min at room temperature. Stop the development when the color development is satisfactory. Remove the slides, dehydrate, clear, mount with neutral resin, and observe under a microscope. TH staining results are as follows. Figure 6 As shown, the model mice have a reduction in dopaminergic neurons.

[0057] Pathological examination revealed the presence of α-syn positive inclusion bodies in the mouse brain; TH staining showed a reduction in dopaminergic neurons in the model mice.

[0058] In summary, this invention provides a method for constructing a Parkinson's disease mouse model through the combined effects of Gba mutant mice (PD risk gene) and a high-dairy diet environment. The Parkinson's model mice constructed by this invention exhibit significant motor function decline and a decrease in spontaneous activity and exploratory abilities, with behavioral differences worsening over time. Furthermore, the pathological changes in the brain, including the appearance of α-synucleosis and a reduction in the number of dopaminergic neurons, all indicate that the method of this invention successfully constructs a Parkinson's disease mouse model that can well simulate the occurrence and development of PD and conforms to the progressive pathological progression of PD. This model can be widely used in the study of the pathogenesis of Parkinson's disease.

[0059] The specification and drawings of this invention are intended to be illustrative rather than restrictive. Based on this invention, those skilled in the art can make substitutions and modifications to some of the technical features without creative effort, and all such modifications are within the scope of protection of this invention.

Claims

1. A guideRNA targeting the mouse Gba gene, characterized in that, A guideRNA was designed based on the 11th exon of the mouse Gba-202 transcript, and the nucleotide sequence of the guideRNA is shown in SEQ ID No.

1.

2. A donor DNA vector, characterized in that, The donor DNA vector sequence is shown in SEQ ID No.

2.

3. A method for constructing a Parkinson's disease mouse model, characterized in that, Includes the following steps: S1. Cas9 mRNA, the guideRNA of claim 1 and the donor DNA vector of claim 2 are injected into mouse zygotes to obtain F0 generation mice. The F0 generation mice are identified by PCR and sequencing to screen out positive F0 generation mice. S2. Positive F0 generation mice were bred with WT mice to obtain F1 generation mice. The F1 generation mice were genetically identified by PCR and sequencing. Positive F1 generation heterozygous mice were screened out, which are Gba gene point mutation mice. S3. Gba gene point mutant mice were given 0.1 mL / g of fresh milk by gavage daily for 8 weeks. S4. Every 4 weeks, mice were weighed and their behavioral phenotypes were assessed, including open field test, rotundus test, footprint test, balance beam test, and elevated cross maze test. S5. After 8 weeks of feeding, based on the results of behavioral phenotype assessment and brain tissue pathological examination, the Parkinson's mouse model was determined to have been successfully constructed.

4. The construction method according to claim 3, characterized in that, The nucleotide sequences of the PCR amplification primers F and R in S1 and S2 are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively.

5. The construction method according to claim 3, characterized in that, In S5, the pathological examination of mouse brain tissue involves immunohistochemistry and TH staining of mouse brain tissue.

6. The application of the guideRNA as described in claim 1 in the study of the pathogenesis of primary Parkinson's disease.

7. The application of the construction method as described in any one of claims 3 to 5 in the study of the pathogenesis of primary Parkinson's disease.

Images