Construction method and application of an "entero-brain axis" mouse model of Alzheimer's disease

Abstract

The application provides a construction method and application of a "gut-brain axis" Alzheimer's disease mouse model, and belongs to the technical field of biological medicines. A gene fragment containing a tetracycline-inducible expression system is inserted by using CRISPR / Cas9 technology, and Sox10MCS4 is combined with CFOS to ensure that the expression of the inserted gene fragment is limited in the enteric nervous system. In the above system, rtTA3G is expressed under the drive of a specific promoter, at this time, the conformation of rtTA3G cannot be combined with the pTRE3G promoter; when tetracycline is added, the conformation of rtTA3G is changed, so that rtTA3G is combined with the pTRE3G promoter to start the expression of MAPT, so as to realize the time and space induction expression. The model can conditionally induce the expression of a pathogenic protein with a fluorescent label in the background mouse, avoids the interference of the high background value of Tau protein, and dynamically analyzes the propagation path and the brain propagation mode of the aggregate, so as to provide a basis for exploring the pathogenesis of the gut-derived Alzheimer's disease, the neural circuit and the early diagnosis of Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a method for constructing an animal model, particularly a method for constructing and applying a "gut-brain axis" Alzheimer's disease mouse model. Background Technology

[0002] Mouse models are essential tools for studying disease pathogenesis and treatment strategies. Currently, the most common method for establishing mouse models is CRISPR-Cas9 technology.

[0003] Tau protein, encoded by the MAPT gene, is an important pathological protein in Alzheimer's disease (AD) and can aggregate to form neurofibrillary tangles. Tau protein aggregation is highly correlated with cognitive impairment. Recent studies have found Tau protein aggregation in the gut of AD patients, and some studies have shown that intestinal injection of patient-derived Tau protein can induce neurofibrillary tangles in the brain, suggesting that Alzheimer's disease may spread to the brain via the gut. However, a reliable mouse model of Tau protein-induced Alzheimer's disease along the gut-brain axis is currently lacking, hindering research on the impact of the peripheral system, particularly the gut system, on central cognitive function. Summary of the Invention

[0004] To address the shortcomings of existing models, the present invention aims to provide a method for constructing and applying a "gut-brain axis" Alzheimer's disease mouse model, so as to better study the Alzheimer's disease caused by the Tau protein "gut-brain axis".

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] This invention discloses a method for constructing a "gut-brain axis" Alzheimer's disease mouse model, comprising the following steps:

[0007] 1) By linking MAPT, P2A, RFP, Sox10MCS4, CFOS and rtTA3G to the pTRE3G expression vector, a donor vector was obtained;

[0008] 2) The Cas9 protein, the donor vector obtained in step 1), and gRNA were microinjected into the fertilized eggs of the donor mice, and then the fertilized eggs were transplanted into the recipient mice for pregnancy and reproduction.

[0009] 3) Mice with offspring carrying the exogenous MAPT gene in their genome were mated with wild-type mice. The offspring were then administered tetracycline by gavage until they showed Alzheimer's-like pathological changes. This was considered to be a "gut-brain axis" Alzheimer's disease mouse model.

[0010] Preferably, in step 1), MAPT, P2A, RFP, and polyA tails are connected to the pTRE3G vector to obtain pTRE3G-MAPT-P2A-RFP; CFOS and Sox10MCS4 are fused and connected to rtTA3G-polyA to obtain Sox10MCS4-CFOS-rtTA3G; and the obtained Sox10MCS4-CFOS-rtTA3G is then connected to pTRE3G-MAPT-P2A-RFP to obtain the donor vector.

[0011] Preferably, in step 2), the sequence of the gRNA is as shown in SEQ ID NO.15.

[0012] Preferably, in step 2), the volume ratio of Cas9 protein, donor vector, and gRNA is 1:8:2.

[0013] Preferably, in step 2), the donor mouse is a C57BL / 6JGpt female mouse.

[0014] Preferably, in step 2), the recipient mouse is an ICR female mouse.

[0015] Preferably, in step 3), the tetracycline is tetracycline hydrochloride at a concentration of 10 mg / mL.

[0016] Preferably, in step 3), the offspring mice obtained from mating are 2 months old.

[0017] Preferably, in step 3), the offspring mice obtained from mating are given tetracycline by gavage until the mice show Alzheimer's-like pathological changes and gradually worsen, which is considered to be obtaining a "gut-brain axis" Alzheimer's disease mouse model.

[0018] Preferably, the "gut-brain axis" Alzheimer's disease mouse model can exhibit symptoms of intestinal dysfunction, motor dysfunction, cognitive impairment, and pathological protein deposition in the gut and brain.

[0019] The present invention also discloses the application of the mouse model obtained by the above-mentioned method for constructing the "gut-brain axis" Alzheimer's disease mouse model in exploring the pathogenesis and neural circuits of gut-derived Alzheimer's disease.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention provides a method for constructing a "gut-brain axis" Alzheimer's disease mouse model. First, using a donor vector as the target gene vector, a gene fragment containing a tetracycline-induced expression system is inserted into the H11 site of the mouse using CRISPR / Cas9 technology. The promoter sequence in the gene fragment is a CFOS promoter sequence, combined with a Sox10MCS4 enhancer sequence, ensuring that the expression of the inserted gene fragment is confined to the enteric nervous system, enabling the constructed system to achieve spatially induced expression. Taking the "Tet-on" system as a breakthrough, the transgenic fragment contains a tetracycline-induced expression system. Tetracycline initiates the temporal induction of the constructed system's expression. In the aforementioned tissues, rtTA3G is expressed under the drive of a specific promoter; at this time, the conformation of rtTA3G cannot bind to the pTRE3G promoter. When tetracycline is added, the conformation of rtTA3G is changed, allowing it to bind to the pTRE3G promoter, initiating MAPT expression, thereby achieving temporal and spatial induction of expression. Secondly, the entire modeling process utilizes tetracycline hydrochloride via gavage, inducing the expression of pathogenic proteins in the gut with minimal crossing of the blood-brain barrier. The behavioral phenotype of the mouse model is similar to that of typical Alzheimer's disease animals, representing a novel perspective for studying the transmission mechanism of Alzheimer's. Finally, because this animal model is derived from the mating of genetically modified mice, a large number of model mice can be obtained, ensuring reproducibility between experimental batches and the stability of experimental results. Histological and behavioral methods validated the constructed mouse model, revealing that MAPT mice not only exhibit neuronal lesions in multiple Alzheimer's-related brain regions but also demonstrate typical behavioral changes such as cognitive impairment. The model can conditionally induce the expression of fluorescently tagged pathogenic proteins in background mice, avoiding interference from high background Tau levels, while dynamically analyzing the propagation pathways of aggregates and the mode of transmission within the brain, better simulating the pathogenesis and behavioral changes of Alzheimer's disease. Similar to the typical animal behavior of Alzheimer's disease, this mouse model helps to study the transmission mechanism of Alzheimer's pathological proteins from a completely new perspective, providing a new model basis for exploring the pathogenesis and neural circuits of intestinal neurodegenerative diseases and for the early diagnosis of neurodegenerative diseases (including Alzheimer's disease). Attached Figure Description

[0022] Figure 1 This diagram illustrates the site-specific insertion of the pTRE3G-MAPT-P2A-RFP-Sox10MCS4-rtTA3G gene fragment into the H11 site of mice using CRISPR / Cas9 technology, followed by histological and behavioral validation. A represents the donor vector construction process, and B represents the histological and behavioral experimental procedures.

[0023] Figure 2 This is a plasmid diagram of the donor vector;

[0024] Figure 3 Examples of nucleic acid electrophoresis results for identifying positive overexpressing mouse genotypes using PCR; where A is the marker, B is the PCR result of the upstream primer of the inserted gene, C is the PCR result of the downstream primer of the inserted gene, and D is the PCR result of WT;

[0025] Figure 4 Image showing the results of immunofluorescence staining for aggregates of intestinal pathogenic proteins in MAPT mice, scale bar = 50 μm;

[0026] Figure 5 The image shows the results of immunofluorescence staining for protein aggregates in the brains of MAPT mice. In the image, A shows the results of double immunofluorescence staining of Tau protein and red fluorescent protein in the control group and 12M-MAPT group mice, and B shows the results of double immunofluorescence staining of AT8 and NeuN in the ACC brain region of the control group, 8M-MAPT group and 12M-MAPT group mice. The scale bar is 50 μm.

[0027] Figure 6 The graph shows the results of the new object recognition and water maze tests to evaluate the cognitive behavior of MAPT mice. In the graph, A is the new object recognition test of mice, and the vertical axis represents the new object recognition index (RI = (time spent by the mouse on the new object - time spent by the mouse on the old object) / (time spent by the mouse on the new object + time spent by the mouse on the old object) × 100%). B is the test of the spatial memory behavior of mice in the water maze, and the vertical axis represents the comparison of the time spent by the mice in the target quadrant on the fifth day with the control group. Detailed Implementation

[0028] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0029] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0030] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0031] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0032] This invention provides a method for constructing a "gut-brain axis" Alzheimer's disease mouse model, mainly including three aspects. For example... Figure 1 The following are the main points: 1. Construction and identification of the MAPT mouse model; 2. Analysis of the relevant phenotypes of pathogenic protein aggregates in the intestines and brains of the identified MAPT mice using histological methods; 3. Verification of the Alzheimer's disease behavioral phenotypes of the identified MAPT mice using behavioral experimental methods.

[0033] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0034] The following examples use conventional instruments and equipment in the art, and the raw materials and reagents used (e.g., organic solvents, inorganic solvents, kinases, substrates, antibodies, buffer solutions, reaction solutions, etc.) are all commercially available products, or can be prepared or formulated by known methods or reagent instructions. Experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. All raw materials used in the following examples are conventional commercial products with specifications in the art, unless otherwise stated. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to weight parts, and "ratio" refers to weight proportion.

[0035] I. Acquisition and Identification of Conditional Overexpression Gene Knockout Mice

[0036] 1. Construct the donor carrier

[0037] 1) Obtaining the human MAPT gene

[0038] Search for the human MAPT gene at https: / / www.ncbi.nlm.nih.gov / gene / 4173. Transcript MGI (ENSG00000186868).

[0039] 2) Amplification

[0040] Using human MAPT gene cDNA as a template, primers MAPT-F and MAPT-R were designed, with sequences shown in Table 1 as SEQ ID NO.1 and SEQ ID NO.2. The MAPT fragment was amplified by PCR to obtain MAPT cDNA.

[0041] Using the P2A plasmid (sequence shown in SEQ ID NO.3 in Table 1) as a template, primers P2A-F and P2A-R were designed, with sequences shown in SEQ ID NO.4 and SEQ ID NO.5 in Table 1. The P2A fragment was amplified by PCR to obtain P2A.

[0042] Using RFP (sequence shown in SEQ ID NO.6 in Table 1) as a template, primers RFP-F and RFP-R were designed, with sequences shown in SEQ ID NO.7 and SEQ ID NO.8 in Table 1. The RFP fragment was amplified by PCR, and a polyA tail sequence was added to the end of the long primer to obtain RFP-polyA.

[0043] 3) Connect MAPT, P2A and RFP-polyA

[0044] Using the pTRE3G conventional plasmid as a template, primers pTRE3G-F and pTRE3G-R were designed, with sequences shown in Table 1 as SEQ ID NO.9 and SEQ ID NO.10. The pTRE3G fragment was amplified by PCR.

[0045] Using an infusion kit (In-Fusion HD Cloning kits, TAKARA, Clontech 639648), the MAPT cDNA, P2A, and RFP-polyA fragments obtained in step 2) were sequentially ligated into the tetracycline-dependent inducible expression vector pTRE3G, yielding pTRE3G-MAPT-P2A-RFP. Sequencing was performed to verify the sequence and ensure the absence of any base mutations.

[0046] 4) Connect rtTA3G, CFOS and Sox10MCS4

[0047] Using conventional plasmid backbones of rtTA3G, human CFOS promoter and Sox10MCS4 enhancer as templates, each fragment was cloned by PCR. The human CFOS promoter and Sox10MCS4 enhancer were fused and ligated to rtTA3G to obtain Sox10MCS4-CFOS-rtTA3G.

[0048] The sequences of the infusion primers rtTA3G-F and rtTA3G-R are shown in Table 1 as SEQ ID NO.11 and SEQ ID NO.12.

[0049] 5) Connect MAPT-RFP-rtTA3G-hCFOS-Sox10MCS4 final

[0050] The Sox10MCS4-CFOS-rtTA3G gene fragment obtained in step 4) was ligated into the pTRE3G-MAPT-P2A-RFP vector obtained in step 3), resulting in the donor vector. The plasmid map is shown below. Figure 2 As shown.

[0051] Table 1 Sequence List

[0052]

[0053]

[0054] 6) Primers donor-F and donor-R were designed based on the base sequence, as shown in SEQ ID NO.13 and SEQ ID NO.14 in Table 1. Using the donor vector synthesized in step 5) as a template, PCR was performed according to the PCR reaction system and conditions in Table 2. The PCR product was purified and recovered by agarose gel electrophoresis, and its concentration was determined to obtain donor DNA for subsequent microinjection.

[0055] Table 2 Reaction system and reaction conditions

[0056]

[0057] 2. Construction of gRNA

[0058] Using the CRISPR Design tool from MIT (http: / / crispr.mit.edu / ), a pair of 20 bp oligonucleotide sequences targeting the target DNA were designed based on the score to prepare gRNA, as shown in SEQ ID NO.15 in Table 1. The synthesized two single-stranded oligonucleotide gRNA sequences were annealed (95°C, then allowed to cool to room temperature for 5 minutes) to form double-stranded DNA, and a gRNA expression vector was constructed. The gRNA expression vector was linearized, purified by phenol-chloroform extraction, and used as a template for in vitro transcription. gRNA was then synthesized in vitro using the MEGAshortscript Kit (Thermo Fisher, AM1354).

[0059] 3. In vitro Cas9 protease cleavage activity detection

[0060] Following the instructions of the Cas9 protein cleavage activity kit (purchased from Beijing Yingmaoshengye, PC1400), the reaction system shown in Table 3 was prepared, and the reaction conditions were 37℃ for 15 min, in order to detect the cleavage activity of Cas9 protein.

[0061] Table 3 Reaction System

[0062]

[0063] 4. Pronuclear microinjection of transgenic vectors

[0064] The Cas9 protein, purified donor vector, and gRNA were injected into the pronucleus of mouse zygotes using microinjection, allowing them to integrate with the mouse genome. Strain identification was then performed to establish a mouse model carrying the exogenous MAPT gene in its genome. The first generation of transgenic positive mice were designated as founder mice. Each founder mouse underwent PCR identification. The specific steps are as follows:

[0065] 1) Prepare the microinjection components according to Table 4, then centrifuge at 4°C for 10 min and take out 15 μL for subsequent microinjection experiments.

[0066] Table 4 Injection Concentration and Volume

[0067] Cas9 protein 100g / μL lμL gRNA-F 20ng / μL lμL gRNA-R 20ng / μL lμL Donor DNA 50 ng / μL 8μL <![CDATA[Add RNase-free H2O to]]> 20μL

[0068] 2) After HCG induction, 6-week-old C57BL / 6JGpt female mice (donor mice) were mated with male mice in the same cage. Female mice with vaginal plugs were euthanized, and the oviducts were harvested. Fertilized eggs were isolated and cultured in vitro. Using a microinjector, 15 μL of the microinjection mixture prepared in step 1) was injected into the pronucleus of the donor mouse's fertilized eggs, allowing it to integrate with the donor mouse's genome. As the cells divide, each cell will carry this fragment.

[0069] 3) The fertilized eggs obtained in step 2) were transferred into the uterus of 7-9 week old ICR female mice (recipient mice). The birth date, number of mice, and sex of the F0 generation mice were recorded and marked with ear tags. After the F0 generation mice with the correct genotype reached sexual maturity, they were mated with wild-type C57 mice to produce offspring mice, namely founder mice.

[0070] 5. Gene identification of conditional overexpression knockout mice

[0071] 1) DNA sample preparation: Cut 1-2 mm of F1 generation founder mouse toes, number the toes, place them in EP tubes, add 180 μL of digestion solution (0.1 mol Tris-HCl pH=8.5; 5 mmol EDTA; 0.2% SDS; 0.2 M NaCl), add 10 μL of proteinase K, and incubate overnight at 56℃.

[0072] Meanwhile, WT mice were used as a control, and all other experimental conditions were the same.

[0073] 2) After incubating the solution overnight in a water bath in step 1), centrifuge at 12000 rpm for 10 min at 21°C. After the liquid separates into layers, aspirate the supernatant into another EP tube, being careful not to aspirate any impurities during transfer. Then, add 1 mL of anhydrous ethanol / isopropanol to the EP tube containing the supernatant, centrifuge at 12000 rpm for 10 min at 21°C, discard the supernatant, and a white transparent precipitate will form at the bottom of the tube. Add 1 mL of 75% ethanol (preferably freshly prepared, ultrapure water) to the tube containing the white transparent precipitate to wash the DNA, then discard the 75% ethanol and invert the EP tube to air dry. Finally, add 50–100 μL of dH2O (ultrapure water) to the dried EP tube and incubate at 37°C for 15 min in a water bath to dissolve the DNA.

[0074] 3) Mix 20 μL of the mixture according to Table 5 and add it to a PCR tube. The primers used for the PCR reaction are shown in Table 6 as SEQ ID NO.16~SEQ ID NO.21:

[0075] Table 5 PCR reaction system

[0076]

[0077]

[0078] Table 6 PCR Primers

[0079]

[0080] 4) Two-step PCR amplification: pre-denaturation 95℃, 5 mins; amplification 98℃, 30 s; annealing 65℃, 30 s, repeat 20 cycles; extension 72℃, 4 min; terminate and store at 4℃.

[0081] 5) Electrophoretic separation and observation of PCR products: PCR products were separated using 1% agarose gel electrophoresis, and the size and position of the amplified bands were indicated by a DNA marker. Images were captured using a gel imaging system to obtain genotype identification results, such as... Figure 3As shown, the target bands were 1300–1400 bp (inserted gene band) and 400–500 bp (WT). Based on the PCR strategy diagram, PCR and sequencing confirmed that mice #58, #63, #69, and #72 were positive mice.

[0082] II. Construction of a "Gut-Brain Axis" Alzheimer's Disease Mouse Model

[0083] MAPT gene expression was induced in founder mice to obtain a gut-brain axis Alzheimer's disease animal model. The specific steps are as follows:

[0084] Two-month-old founder mice were administered tetracycline hydrochloride at a concentration of 10 mg / mL via gavage for eight weeks. After gavage was stopped, the mice were fed for more than two months to induce the aggregation of intestinal expressed proteins to form pathogenic aggregates and to cause intracerebral dissemination, thus obtaining the "gut-brain axis" Alzheimer's disease mouse model, namely, the MAPT mouse.

[0085] III. Histological methods for identifying the aggregation of pathogenic proteins in the intestine and brain of Alzheimer's disease mice along the gut-brain axis

[0086] Eight weeks after gavage in 2-month-old MAPT mice (4 months old), pathogenic protein aggregates appeared in the intestines and gradually spread to the brain. Immunofluorescence staining was performed on intestinal and brain tissues of MAPT mice that were gavage for 8 weeks and then fed until 8 months of age, and MAPT mice that were gavage for 8 weeks and then fed until 12 months of age, to determine the aggregation of pathogenic proteins in the enteric plexus and their dissemination in the brain. Details are as follows:

[0087] Two-month-old MAPT mice that underwent gavage for 8 weeks served as the control group. MAPT mice that underwent gavage for 8 weeks and were subsequently fed to four months of age served as the 8M-MAPT group. MAPT mice that underwent gavage for 8 weeks and were subsequently fed to twelve months of age served as the 12M-MAPT group. Mice in each group were fixed by perfusion with paraformaldehyde. Brain tissue and the entire intestine were removed. Intestinal contents were cleared. The jejunum was taken 1 cm below the stomach, the ileum 1 cm above the cecum, and the colon 1 cm below the cecum. The removed brain and intestinal tissues were placed in 4% PFA and soaked overnight at 4°C for post-fixation. The solution was then replaced with 30% sucrose solution for dehydration at 4°C for at least 48 hours. Once the tissues had completely settled, frozen sections were prepared to obtain brain tissue with a thickness of 20 μm and transverse intestinal sections.

[0088] Immunofluorescence double staining was performed on mouse intestinal sections using AT8 (a marker of Tau protein aggregates) and PGP9.5 (an intestinal neuron cell marker). Immunofluorescence double staining was performed on mouse brain tissue sections using Tau protein and red fluorescent protein. Immunofluorescence double staining was performed on mouse ACC brain region sections using AT8 and NeuN (a neuron cell marker). The specific steps are as follows: Before staining, to prevent the sections from falling off the slide, fix the sections with 4% PFA at room temperature for 30 min, and wash with PBS 3 × 10 min; incubate the sections in a humidified chamber with blocking buffer (3% BSA + 0.3% Triton) at room temperature for 1 hour; add the primary antibody prepared in the blocking buffer (tyrosine hydroxylase, TH, polyclonal antibody purchased from Santa Cruz Biotechnology, USA; phosphorylated Tau protein, AT8, polyclonal antibody purchased from Thermo Fisher Scientific, USA; protein gene product 9.5, PGP 9.5, polyclonal antibody purchased from GeneTex, USA; red fluorescent protein, RFP, polyclonal antibody purchased from GeneTex, USA; Tau N368 monoclonal antibody synthesized by Wuhan Jinkairui Biotechnology Co., Ltd.), and incubate overnight in a humidified chamber at room temperature; the next day, wash with PBS 3 × 10 min, and add secondary antibody (Alexa). 594-labeled donkey anti-mouse polyclonal secondary antibody, Alexa Donkey anti-rabbit polyclonal secondary antibody labeled with 647 was purchased from Cell Signaling, USA. The cells were incubated at room temperature for 2 h and washed with PBS 3 × 10 min. The nuclei were stained with DAPI (4',6-diamidino-2-phenylindole, DAPI) purchased from Sigma, Germany for 5 min. The cells were then mounted with 50% glycerol. The cells were observed and images were acquired using a laser confocal microscope.

[0089] The staining results of aggregates of intestinal pathogenic proteins are as follows: Figure 4 As shown in the figure, compared with the control group mice, Tau N368 aggregates appeared in the intestinal neurons of the 12M-MAPT group mice, indicating that the "gut-brain axis" Alzheimer's disease mouse model was successfully constructed.

[0090] The staining results of protein aggregates in the brain are as follows Figure 5 As shown, aggregates of Tau N368 protein were found in the brains of MAPT mice, and the Tau protein in the brain originated from the gut. Furthermore, comparisons with the control group, 8M-MAPT group, and 12M-MAPT group revealed that the number of Tau N368 protein aggregates gradually increased with age.

[0091] IV. Behavioral methods to verify the behavioral phenotype of Alzheimer's disease in mice with the gut-brain axis.

[0092] Based on commonly used behavioral assessment methods for Alzheimer's disease models, the basic motor function and cognitive level of MAPT mice were evaluated using novel object recognition and water maze tests. The specific steps are as follows:

[0093] Four-month-old MAPT mice without gavage were used as the control group (4 months old); six-month-old MAPT mice without gavage were used as the control group (6 months old); eight-month-old MAPT mice without gavage were used as the control group (8 months old); two-month-old MAPT mice that were gavageed for 8 weeks and then fed to four-month-old mice were used as the 4M-MAPT group; two-month-old MAPT mice that were gavageed for 8 weeks and then fed to six-month-old mice were used as the 6M-MAPT group; and two-month-old MAPT mice that were gavageed for 8 weeks and then fed to eight-month-old mice were used as the 8M-MAPT group. Behavioral experiments were conducted on mice in each group. The steps are as follows:

[0094] Morris Water Maze: Before the water maze experiment, prepare a 120cm diameter pool and a 10cm diameter platform. Fill the pool with water until it covers the platform, and add a whitening agent to the water. The experiment lasted for 5 days. Days 1-4 were for navigation and positioning. The pool was divided into four quadrants. The platform was placed in the fourth quadrant, and the mice were allowed to swim for 1 minute. If the mice successfully found the platform within 1 minute, the experiment ended. If the mice did not find the platform, they were guided to the platform location and memorized it for 5-7 seconds. Each mouse was placed into each of the four quadrants once, and the data was recorded. This was repeated for 4 days. On the last day, the spatial exploration experiment was conducted. The platform was removed, and each mouse was placed into each of the four quadrants once, and the mice were allowed to swim for 1 minute. The data was recorded and analyzed using software.

[0095] New Object Recognition Experiment: This experiment was conducted over two days. Two hours before the test or training, the mice were placed in the testing room to acclimatize to the environment. Day 1: The mice were placed in the testing area for 10 minutes to acclimatize. Training began on Day 2. Two identical objects, A and B, were placed at opposite corners of one side wall. The mice were placed with their backs to the objects, and the distance from the tip of their noses to the objects was the same. After 10 minutes, the recording device was immediately turned on to record the mice's contact with the objects, including the number of times their noses or mouths touched the objects and the time spent exploring within 2-3 cm of the objects (paws resting on the objects, sniffing the objects, licking the objects, etc., all constitute exploration; simply posing or climbing on the objects without moving does not count as exploration of a new object). After 10 minutes, the mice were immediately returned to their original enclosures and allowed to rest for 6 hours before the test (during which time the mice remained in the testing room). After the mice rested for 6 hours, the test was started. At this time, object B in the field was replaced with object C (different from object AB). The mice were still facing away from the two objects, with their noses at the same distance from the two objects. The test was recorded with video equipment, and the behavior of the mice was analyzed using the SMART system. Finally, the new object recognition index (RI = (new object - old object) / (new object + old object) × 100%) was calculated.

[0096] Statistical methods: Two-way ANOVA was used for statistical analysis.

[0097] The results are as follows Figure 6 To evaluate the cognitive function of mice, the Morris water maze and new object recognition experiments were selected. The MAPT experimental group mice showed a decrease in spatial memory ability. Figure 6 In the A-means, MAPT mice showed a significantly lower proportion of time spent at new objects compared to the control group in the new object recognition experiment (P<0.05), and this decrease was significant with increasing age of the mice (P<0.01). Figure 6 The results showed that after four days of training in the water maze experiment, on day 5, after the platform was removed, the MAPT group mice spent significantly less time in the target quadrant compared to the control group. Furthermore, significant differences were observed at 4 and 6 months of age (P<0.05), indicating significant cognitive decline in the MAPT mice. While late-stage Alzheimer's patients also experience cognitive decline, most Alzheimer's animal models cannot simulate this cognitive impairment. However, this model also evaluated the cognitive function of mice.

[0098] In summary, the MAPT mouse model conforms to current pathological research findings on Alzheimer's disease and also reflects the characteristics of the "gut-brain axis" transmission of the disease. It can provide a novel animal model for the pathogenesis and drug screening research of Alzheimer's disease, and can dynamically analyze the transmission pathways of aggregates and the transmission modes in the brain. This model provides a new model basis for exploring the pathogenesis and neural circuits of gut-derived Alzheimer's disease and for the early diagnosis of Alzheimer's disease.

[0099] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for constructing a "gut-brain axis" Alzheimer's disease mouse model, characterized in that, Includes the following steps: 1) MAPT, P2A, RFP, and polyA tails are ligated to the pTRE3G vector to obtain pTRE3G-MAPT-P2A-RFP; the CFOS promoter is fused with the Sox10MCS4 enhancer and ligated to rtTA3G-polyA to obtain Sox10MCS4-CFOS-rtTA3G; the obtained Sox10MCS4-CFOS-rtTA3G is then ligated to pTRE3G-MAPT-P2A-RFP to obtain the donor vector; 2) The Cas9 protein, the donor vector obtained in step 1), and gRNA were microinjected into the fertilized eggs of the donor mice, and then the fertilized eggs were transplanted into the recipient mice for pregnancy and reproduction. 3) Mice with offspring carrying the exogenous MAPT gene in their genome were mated with wild-type mice. The offspring were then administered tetracycline by gavage until they developed Alzheimer's-like pathological changes. This was considered to be a mouse model of Alzheimer's disease along the gut-brain axis.

2. The method for constructing a "gut-brain axis" Alzheimer's disease mouse model according to claim 1, characterized in that, In step 2), the sequence of the gRNA is shown in SEQ ID NO.

15.

3. The method for constructing a "gut-brain axis" Alzheimer's disease mouse model according to claim 1, characterized in that, In step 2), the volume ratio of Cas9 protein, donor vector, and gRNA is 1:8:

2.

4. The method for constructing a "gut-brain axis" Alzheimer's disease mouse model according to claim 1, characterized in that, In step 2), the donor mouse is a female C57BL / 6JGpt mouse.

5. The method for constructing a "gut-brain axis" Alzheimer's disease mouse model according to claim 1, characterized in that, In step 2), the recipient mouse is an ICR female mouse.

6. The method for constructing a "gut-brain axis" Alzheimer's disease mouse model according to claim 1, characterized in that, In step 3), the tetracycline is tetracycline hydrochloride at a concentration of 10 mg / mL.

7. The method for constructing a "gut-brain axis" Alzheimer's disease mouse model according to claim 1, characterized in that, In step 3), the offspring mice obtained from mating are 2 months old.

8. A method for constructing a "gut-brain axis" Alzheimer's disease mouse model according to any one of claims 1 to 7, characterized in that, The "gut-brain axis" Alzheimer's disease mouse model exhibits symptoms such as intestinal dysfunction, motor dysfunction, cognitive impairment, and pathological protein deposition in the gut and brain.

Images