CN118831181B

CN118831181B - A method for constructing a triple-mutant Alzheimer's disease mouse model and its application

Info

Publication number: CN118831181B
Application number: CN202411313775.4A
Authority: CN
Inventors: 冯睿; 韩冰舟; 来自颖; 孙士妍
Original assignee: Jiangsu Jicui Yaokang Biotechnology Co ltd
Current assignee: Jiangsu Jicui Yaokang Biotechnology Co ltd
Priority date: 2024-09-20
Filing date: 2024-09-20
Publication date: 2025-02-11
Anticipated expiration: 2044-09-20
Also published as: CN118831181A

Abstract

The present specification provides a method for constructing a triple-mutant Alzheimer's disease mouse model and its application, the construction method comprising: (1) preparing a transgenic fragment of the first exogenous gene hAPP, a transgenic fragment of the second exogenous gene hPSEN1, and a transgenic fragment of the third exogenous gene hMAPT; (2) inserting the three transgenic fragments into the genome of a mouse fertilized egg; (3) transplanting the fertilized egg into a pseudo-pregnant female mouse, and screening to obtain F0 generation mice in which all three exogenous genes are positive; (4) based on the F0 generation mice, obtaining an Alzheimer's disease mouse model 3-FAD homozygous positive mouse. The Alzheimer's disease mouse model provided in the present specification has a disease progression consistent with that of human patients, with blood markers appearing first, followed by pathological manifestations, and finally behavioral characteristics, providing a more accurate and effective approach for the study and drug treatment of Alzheimer's disease.

Description

Construction method and application of three-mutation Alzheimer disease mouse model

Technical Field

The specification relates to the technical field of genetic engineering, in particular to a construction method and application of a three-mutation Alzheimer's disease mouse model.

Background

Alzheimer's Disease (AD) is a common degenerative disease of the central nervous system in the elderly or in a pre-senile stage, a major type of dementia, accounting for 60-70%. It is typically manifested by early memory loss, cognitive dysfunction, followed by behavioral and psychotic symptoms such as anxiety, delusions, and the like. The main pathological features of AD include amyloid plaques in the brain, neurofibrillary tangles (NFTs), and neurodegeneration of the hippocampus and cortex. Beta amyloid (aβ) is produced by cleavage of Amyloid Precursor Protein (APP) by β and γ secretases, aβ42 is more toxic due to high aggregation, and amyloid plaques formed by accumulation thereof can cause neuronal damage and death through various mechanisms, exacerbating neurodegeneration. Tau protein is abnormally phosphorylated in AD, breaks away from microtubules and aggregates to form NFTs, disrupting neuronal structure and function, leading to severe cognitive impairment.

To study AD pathology and develop therapies, scientists have constructed various transgenic mouse models, introducing mutations in AD-related genes (such as APP, PS1, and Tau genes) to mimic pathological features. A common strategy is that mutant human APP genes mimic aβ accumulation, and mice carrying mutations in human APP and PS1 genes can develop amyloid plaques and cognitive impairment earlier. Double transgenic mice showed memory and synaptic transmission impairment at 6 months, an ideal model for studying early pathology, but failed to reproduce Tau protein aggregation well in human patients. Although models were compiled for three mutant genes, mice developed aβ pathology in 6 months and NFTs in 12 months, the behavior and pathological changes were not completely consistent with clinical patients and could only be used to study specific relationships. In addition, there are non-transgenic models that induce AD-like pathological changes by toxin injection or natural aging. However, to date, although a variety of AD-related mouse models have been constructed, none have been able to match the disease course changes in human patients.

It is therefore desirable to construct a mouse model that can match the changes in the course of a human Alzheimer's disease patient.

Disclosure of Invention

In order to provide a mouse model which can be matched with the disease course change of a human Alzheimer disease patient, a more accurate and effective way is provided for the research and administration treatment of Alzheimer disease, one or more embodiments of the specification provide a method for constructing a three-mutation Alzheimer disease mouse model, which comprises (1) preparing a transgenic fragment of a first exogenous gene hAPP, a transgenic fragment of a second exogenous gene hPSEN and a transgenic fragment of a third exogenous gene hMAPT, (2) inserting the three transgenic fragments into the genome of a fertilized egg of a mouse, (3) transplanting the fertilized egg into a pseudopregnant female mouse, screening to obtain F0 generation mice with positive identification of the three exogenous genes, and (4) obtaining a 3-FAD homozygous positive mouse of the Alzheimer disease mouse model based on the F0 generation mice;

Wherein the transgene fragment of the first exogenous gene hAPP carries a Swedish mutation, the transgene fragment of the second exogenous gene hPSEN carries a hPSEN M146V mutation, and the transgene fragment of the third exogenous gene hMAPT carries a hMAPT P L mutation.

One or more embodiments of the present disclosure provide an application of a three-mutant alzheimer's disease mouse model in researching a pathological mechanism of alzheimer's disease or screening a drug for treating alzheimer's disease, where a genome of the mouse model includes a transgenic fragment of a first exogenous gene hAPP, a transgenic fragment of a second exogenous gene hPSEN and a transgenic fragment of a third exogenous gene hMAPT, where the transgenic fragment of the first exogenous gene hAPP carries a Swedish mutation, the transgenic fragment of the second exogenous gene hPSEN carries a hPSEN M146V mutation, and the transgenic fragment of the third exogenous gene hMAPT carries a hMAPT P243L mutation.

The embodiment of the specification has at least the following beneficial effects of providing a three-mutation Alzheimer's disease mouse model which can be inherited stably and has the disease course completely consistent with that of a human patient, wherein the mouse model has the prospect of researching the pathology mechanism of Alzheimer's disease and screening medicaments for treating Alzheimer's disease when the development of the pathology course or stage of Alzheimer's disease is consistent with that of a clinical patient.

Drawings

The present specification will be further elucidated by way of example embodiments, which will be described in detail by means of the accompanying drawings. The embodiments are not limiting, in which like numerals represent like structures, wherein:

FIG. 1 is a graph of the detection of clinical early blood detection indicators of Alzheimer's disease in 3-FAD mouse blood according to some embodiments of the present disclosure;

FIG. 2 is a graph of the results of detection of Abeta deposition in the brain region of a 2 month old 3-FAD mouse, according to some embodiments of the present disclosure;

FIG. 3 is a graph of the results of an assay showing the presence of aggregation and plaque formation as the course of disease progresses in the brain region of 3-FAD mice, according to some embodiments of the present disclosure;

FIG. 4 is a graph of the results of detection of p-Tau181 in the brain region of a1 month old 3-FAD mouse according to some embodiments of the present disclosure;

FIG. 5 is a graph of the results of detection of p-Tau217 in the brain region of a2 month old 3-FAD mouse according to some embodiments of the present disclosure;

FIG. 6 is a graph of the results of detection of p-Tau (Thr 231) in the brain region of a2 month old 3-FAD mouse according to some embodiments of the present disclosure;

FIG. 7 is a graph of the results of detection of p-Tau (Ser 202, thr 205) in the brain region of a 3-FAD 3-month-old mouse according to some embodiments of the present disclosure;

FIG. 8 is a graph of the results of detection of p-Tau (Ser 396) in the brain region of a 3-FAD 3-month old mouse according to some embodiments of the present disclosure;

FIG. 9 is a graph of the results of detection of p-Tau181 as a function of disease in the brain region of a 3-FAD mouse according to some embodiments of the present disclosure;

FIG. 10 is a graph of the detection of p-Tau (Ser 202, thr 205) as a function of course in the brain region of a 3-FAD mouse according to some embodiments of the present disclosure;

FIG. 11 is a graph of experimental results of Morris water maze experiments performed on 3-FAD mice of 3 months of age according to some embodiments of the present disclosure;

FIG. 12 is a graph showing the results of testing blood of mice following administration according to some embodiments of the present disclosure;

fig. 13 is a graph of the results of detection of brain regions of mice following administration, as shown in some examples of the present specification.

Detailed Description

In order to more clearly illustrate the technical solutions of the embodiments of the present specification, the drawings that are required to be used in the description of the embodiments will be briefly described below. It is apparent that the drawings in the following description are only some examples or embodiments of the present specification, and it is possible for those of ordinary skill in the art to apply the present specification to other similar situations according to the drawings without inventive effort. Unless otherwise apparent from the context of the language or otherwise specified, like reference numerals in the figures refer to like structures or operations.

As used in this specification and the claims, the terms "a," "an," "the," and/or "the" are not specific to a singular, but may include a plurality, unless the context clearly dictates otherwise. In general, the terms "comprises" and "comprising" merely indicate that the steps and elements are explicitly identified, and they do not constitute an exclusive list, as other steps or elements may be included in a method or apparatus.

One or more embodiments of the present disclosure provide a three-mutant alzheimer's disease (3-FAD) mouse model, a construction method and an application thereof, in which three transgenic fragments of exogenous genes (including hPSEN M146V mutation, HAPP SWEDISH mutation and hMAPT P243L mutation) are randomly inserted into a mouse genome in a gene editing manner, and under the regulation of a promoter nerve-specific element, the three transgenic fragments of exogenous genes can be specifically expressed in a brain region of the mouse, thereby obtaining a three-mutant alzheimer's disease (3-FAD) mouse model which can be stably inherited and has a disease development process substantially consistent with that of a human patient.

The embodiment of the specification provides a method for constructing a three-mutation Alzheimer's disease (3-FAD) mouse model, which comprises the following steps:

(1) A transgenic fragment of the first exogenous gene hAPP, a transgenic fragment of the second exogenous gene hPSEN1, and a transgenic fragment of the third exogenous gene hMAPT were prepared.

The hAPP gene (human Amyloid Precursor Protein gene, a human amyloid precursor protein gene) is a gene encoding Amyloid Precursor Protein (APP), is located in the long arm (21q21.3) of chromosome 21 of human, and has important significance in the fields of biomedicine and neuroscience, and is especially related to Alzheimer's Disease (AD).

In some embodiments, the transgenic fragment of the first exogenous gene hAPP carries a Swedish mutation. The Swedish mutation (Swedish mutation) is a specific mutation of the hAPP gene, and is named "Swedish mutation" because it was originally found in several families in Swedish, and it is closely related to early-onset familial Alzheimer's Disease (Familial Alzheimer's Disease, FAD). Swedish mutations involve two nucleotide changes in the coding region of the hAPP gene, specifically at amino acids 670 and 671 on the encoded Amyloid Precursor Protein (APP).

In some embodiments, the transgenic fragment of the first exogenous gene hAPP comprises the nucleotide sequence set forth in SEQ ID NO. 1.

In some embodiments, the transgenic fragment of the first exogenous gene hAPP comprises the hAPP 5' utr and the mutant hAPP CDS sequence, which carries the Swedish mutation.

In some embodiments, the transgenic fragment of the first exogenous gene hAPP further comprises the nucleotide sequence shown as SEQ ID NO. 2.

HPSEN1 Gene (human Presenilin-1 gene, i.e., human presenilin 1 gene) is a gene encoding presenilin 1 protein (PRESENILIN-1, PS 1), presenilin 1 (PSEN 1) is a catalytic subunit constituting a gamma-secretase responsible for processing amyloid precursor protein (Amyloid Precursor Protein, APP) to produce Abeta. hPSEN1 Gene located on the long arm of human chromosome 14 (14q24.3), PSEN1 gene mutation is one of the major causative factors of early-onset Familial Alzheimer's Disease (FAD).

In some embodiments, the transgenic fragment of the second exogenous gene hPSEN carries the hPSEN M146V mutation.

As used herein, "hPSEN M146V mutation" refers to the mutation of methionine M at position 146 to valine V in the hPSEN protein which is transcribed and translated after mutation of the hPSEN1 gene. Wherein, the 146 th methionine M is obtained by taking hPSEN protein sequence (GI: 15079861) or homologous sequence thereof as a reference.

In some embodiments, the transgenic fragment of the second exogenous gene hPSEN includes the sequence shown as SEQ ID NO. 3.

In some embodiments, the transgenic fragment of the second exogenous gene hPSEN includes a mutant hPSEN1 CDS sequence, the mutant hPSEN CDS sequence carrying the hPSEN M146V mutation.

MAPT gene codes microtubule-associated protein Tau, which is a microtubule-associated protein, and intracellular Tau protein is hyperphosphorylated and then reduced in solubility, and hyperphosphorylated Tau competes with tubulin for binding to normal Tau and other microtubule-associated proteins, thereby losing biological activity promoting microtubule assembly, resulting in microtubule depolymerization and impaired axon transport, resulting in neuronal degeneration and neuronal apoptosis, and finally resulting in AD. Mutations in the microtubule-associated protein Tau (MAPT) gene are a major contributor to other tauopathies neurodegenerative diseases such as AD, corticobasal syndrome, and Progressive Supranuclear Palsy (PSP) syndrome.

In some embodiments, the transgenic fragment of the third exogenous gene hMAPT carries the hMAPT P L mutation.

As used herein, the "hMAPT P L mutation" refers to the mutation of proline P to leucine L at position 243 in human microtubule-associated protein Tau (MAPT) obtained by mutating, transcribing and translating the hMAPT gene. Wherein, the proline P at 243 is obtained by taking hMAPT protein sequence (GI: 8400711) or homologous sequence thereof as reference.

In some embodiments, the transgenic fragment of third exogenous gene hMAPT includes mutant hMAPT CDS sequence, mutant hMAPT CDS sequence carrying the hMAPT P243L mutation.

In some embodiments, the transgenic fragment of third exogenous gene hMAPT includes a nucleotide sequence as set forth in SEQ ID NO. 5.

In some embodiments, the transgenic fragment of the first exogenous gene hPSEN, the transgenic fragment of the second exogenous gene hAPP, and the transgenic fragment of the third exogenous gene hMAPT each include a Thy1 promoter.

In some embodiments, the transgenic fragment of the first exogenous gene hAPP comprises elements operably linked and arranged in the order from 5' to 3' of the Thy1 promoter, hAPP 5' UTR, mutant hAPP CDS sequence (carrying a Swedish mutation), and the transgenic fragment of the first exogenous gene hAPP further comprises the sequence shown as SEQ ID NO: 2.

In some embodiments, the transgenic fragment of the second exogenous gene hPSEN1 includes elements operably linked and arranged from 5 'to 3' in the order of the Thy1 promoter, mutant hPSEN1 CDS sequence (carrying the hPSEN 1M 146V mutation), and the transgenic fragment of the second exogenous gene hPSEN1 also includes the sequence shown as SEQ ID NO. 4.

In some embodiments, the transgenic fragment of third exogenous gene hMAPT, comprising elements operably linked and arranged in the order from 5 'to 3' is the Thy1 promoter, mutant hMAPT CDS sequence (carrying the hMAPT P243L mutation), and the transgenic fragment of third exogenous gene hMAPT further comprises the sequence as set forth in SEQ ID NO: 6.

Transgenic fragments of exogenous genes can be obtained in a variety of ways. For example, the transgene fragment may be obtained directly by artificial in vitro synthesis. In some embodiments, the transgenic fragment can be obtained by constructing a plasmid for PCR amplification.

In some embodiments, preparing the transgenic fragment comprises:

a. PCR products of the Thy1 promoter, the first exogenous gene hAPP, the second exogenous gene hPSEN1, and the third exogenous gene hMAPT were obtained.

In some embodiments, primers are designed to obtain PCR products of the Thy1 promoter, the hAPP CDS, hPSEN CDS, hMAPT CDS fragments, respectively, by PCR.

B. And respectively connecting the Thy1 promoter with PCR products of the first exogenous gene hAPP, the second exogenous gene hPSEN and the third exogenous gene hMAPT and a framework vector to form a recombinant vector, and transforming the recombinant vector into competent cells.

In some embodiments, recombinant vectors formed by the Thy1 promoter and three fragments of the hAPP CDS, hPSEN CDS, hMAPT CDS, respectively, can be transformed into competent cells by heat shock and then cultured overnight in a 37 ℃ incubator.

As used herein, "recombinant vector" refers to a means for carrying, replicating, and expressing a fragment of a foreign gene. In some embodiments, the vectors of the recombinant vector include, but are not limited to, plasmid vectors, eukaryotic cell expression vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors.

C. Performing PCR identification and sequencing after transformation, and selecting clones with correct sequencing as a transgenic vector;

In some embodiments, the transformed monoclonal colonies are selected for PCR identification, clones identified as positive are transferred to test tubes, plasmids are extracted, restriction identification is performed, the correctly restriction clones are sent for measurement, and the correctly sequenced clones are selected as transgenic vectors of the exogenous genes.

D. The transgenic fragments are prepared based on the transgenic vectors.

In some embodiments, the constructed transgenic vector is used for preparing a transgenic fragment of the exogenous gene by an enzyme digestion mode

(2) Three transgenic fragments were inserted into the genome of the mouse fertilized eggs.

The "insertion" of the three transgenic fragments into the fertilized mouse egg refers to the addition of integration and/or integration substitutions into the mouse genome, either by random or site-directed insertion.

In some embodiments, the transgenic fragments are microinjected into fertilized eggs of mice, randomly targeted for insertion into the mouse genome.

(3) The fertilized ovum is transplanted into a pseudopregnant female mouse body, and F0 generation mice with exogenous genes identified as positive are obtained through screening.

In some embodiments, the identification is accomplished by a PCR reaction using primer pairs as shown in SEQ ID NO:7 and SEQ ID NO:8 and SEQ ID NO:9, respectively, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO: 17. Wherein SEQ ID NO. 7 and 8 and SEQ ID NO. 9 are used to identify the first exogenous gene hAPP, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 and 13 are used to identify the second exogenous gene hPSEN1, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17 are used to identify the third exogenous gene hMAPT.

(4) Based on the F0 generation mice, the mice model 3-FAD homozygous positive mice of Alzheimer's disease are obtained.

In some embodiments, F0 mice are backcrossed with wild type mice (WT) until F0N5 mice are obtained, and mice model 3-FAD homozygous positive mice for Alzheimer's disease are obtained from F0N5 mice selfing.

The embodiment of the specification provides an application of the three-mutation Alzheimer's disease mouse model obtained by the construction method in researching the pathological mechanism of Alzheimer's disease.

The embodiment of the specification provides an application of the three-mutation Alzheimer disease mouse model obtained by the construction method in screening medicines for treating Alzheimer disease.

The present embodiments also provide a mouse model of triple mutant Alzheimer's disease whose genome comprises a transgene fragment from a first exogenous gene hAPP, a transgene fragment from a second exogenous gene hPSEN1, and a transgene fragment from a third exogenous gene hMAPT. The "mouse model" as referred to herein corresponds to homozygote positive mice.

In some embodiments, the transgenic fragment of the first exogenous gene hAPP carries a Swedish mutation.

In some embodiments, the transgenic fragment of the first exogenous gene hAPP comprises the sequence shown as SEQ ID NO. 1.

In some embodiments, the transgenic fragment of third exogenous gene hMAPT includes a sequence as set forth in SEQ ID NO. 5.

In some embodiments, the transgenic fragment of the first exogenous gene hAPP, the transgenic fragment of the second exogenous gene hPSEN, and the transgenic fragment of the third exogenous gene hMAPT each comprise a Thy1 promoter.

Embodiments of the present disclosure provide a mouse model of tri-mutant alzheimer's disease, which in some embodiments is prepared by the aforementioned construction method.

The embodiment of the specification provides a three-mutation Alzheimer's disease mouse model, which can be used for researching the pathological mechanism of Alzheimer's disease or screening medicines for treating Alzheimer's disease.

The embodiments of the present specification also provide methods for studying the pathological mechanism of Alzheimer's disease using the above-described triple mutant Alzheimer's disease mouse model, and also provide methods for screening drugs for treating Alzheimer's disease using the above-described triple mutant Alzheimer's disease mouse model.

In some embodiments, the method of studying the pathological mechanism of alzheimer's disease comprises the steps of:

(1) Randomly inserting the transgenic fragments of the three exogenous genes into the genome of a mouse to prepare a mouse model of the three-mutant Alzheimer's disease;

(2) Adding a specific marker or hybridizing with other mice to perform research related to disease mechanism, periodically detecting biological characteristics of the mice, and collecting tissue blocks of tissue structures of the mice when the biological markers of the Alzheimer's disease appear;

(3) Assessing the expression of DNA, RNA or protein in the tissue mass collected in step (2);

(4) Identifying genes or proteins in the tissue mass that are associated with changes in the hierarchical organization of diseased cells, diseased processes, or biological characteristics.

The specific marker may be a virus or lentivirus, including adeno-associated virus (AAV), lentivirus (lentivirus), and the like.

In some embodiments, the method of screening for a drug for treating alzheimer's disease comprises the steps of:

(2) Administering a drug to be screened to the mouse model;

(3) Periodically detecting biological characteristics related to Alzheimer's disease in a mouse model, wherein the biological characteristics comprise but are not limited to blood, cerebrospinal fluid, pathology and behavior indexes, and the detection objects comprise but are not limited to proteins, DNA or RNA and the like;

(4) Screening out drugs for inhibiting biological characteristics related to Alzheimer's disease of the mouse model.

The embodiment of the specification has at least the following beneficial effects that the transgene fragments of the first exogenous gene (containing HAPP SWEDISH mutations), the second exogenous gene (containing hPSEN M146V mutations) and the third exogenous gene (containing hMAPT P243L mutations) are randomly inserted into the genome of a mouse in a gene editing mode, the transgene fragments containing hPSEN M146V mutations, HAPP SWEDISH mutations and hMAPT P243L mutations can be specifically expressed in the brain region of the mouse under the regulation of a nerve-specific element of the Thy1 promoter, so that a three-mutation Alzheimer's disease (3-FAD) mouse model which can be stably inherited and has the complete consistency of the pathogenesis of the human patient is obtained, the 3-FAD mouse accords with the human patient in the pathogenesis of the Alzheimer's disease, the blood markers appear firstly, then the pathological characterization appears, and finally the behavioural characteristics appear, and a more accurate and effective way is provided for the research and the administration treatment of the Alzheimer's disease.

The technical scheme of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. Some of the content in these embodiments may also be replaced or combined with corresponding content in other embodiments, thereby forming new embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagent companies. The quantitative tests in the following examples were all set up in triplicate and the results averaged. It should be understood that the following examples are for better explaining the present invention and are not intended to limit the present invention.

Examples

EXAMPLE 1 construction of Alzheimer's disease mouse (3-FAD mouse) model

The construction of the 3-FAD mouse model comprises the following steps:

(1) The preparation of the transgenic fragment of the first exogenous gene hAPP, the transgenic fragment of the second exogenous gene hPSEN1, and the transgenic fragment of the third exogenous gene hMAPT specifically includes:

a. Designing primers, and respectively obtaining PCR products of four fragments of a Thy1 promoter, an hAPP CDS, a hPSEN CDS and a hMAPT CDS by a PCR mode;

b. The Thy1 promoter is respectively connected with three fragments of hAPP CDS, hPSEN CDS and hMAPT CDS and a skeleton carrier in vitro, and is transformed into competent cells in a heat shock mode, and is cultured on a flat plate overnight;

c. Selecting a monoclonal colony on a flat plate for PCR identification, identifying a positive clone transfer test tube, extracting plasmids, carrying out enzyme digestion identification, carrying out enzyme digestion correct clone transfer test, and finally selecting clones with correct sequencing as three transgenic final vectors of Thy1-APP, thy1-PSEN1 and Thy 1-MAPT.

D. and preparing the constructed transgenic vector into a transgenic fragment of the exogenous gene by an enzyme digestion mode.

Specific information of the three exogenous genes is as follows:

The first exogenous gene comprises a Thy1 promoter, an hAPP 5' UTR, a mutant hAPP CDS sequence and a mutant hAPP CDS sequence carrying a Swedish mutation;

The second exogenous gene comprises a Thy1 promoter, a mutant hPSEN A CDS sequence and a mutant hPSEN A CDS sequence carrying a hPSEN M146V mutation;

The third exogenous gene comprises a Thy1 promoter, a mutant hMAPT CDS sequence and a mutant hMAPT CDS sequence carrying a hMAPT P243L mutation;

To keep the transcription and translation efficiency of hAPP, hPSN 1 and hMAPT stable, kozak (GCCGCCACC) sequences were added to the N-terminal of each of the three genes.

Wherein, the three exogenous gene sequences are specifically shown as follows:

The mutated hAPP CDS sequence of the transgenic fragment of the first exogenous gene is shown in SEQ ID NO. 1 (wherein the bold font is the mutation site sequence and the direction is 5'-3'）：atgctgcccggtttggcactgctcctgctggccgcctggacggctcgggcgctggaggtacccactgatggtaatgctggcctgctggctgaaccccagattgccatgttctgtggcagactgaacatgcacatgaatgtccagaatgggaagtgggattcagatccatcagggaccaaaacctgcattgataccaaggaaggcatcctgcagtattgccaagaagtctaccctgaactgcagatcaccaatgtggtagaagccaaccaaccagtgaccatccagaactggtgcaagcggggccgcaagcagtgcaagacccatccccactttgtgattccctaccgctgcttagttggtgagtttgtaagtgatgcccttctcgttcctgacaagtgcaaattcttacaccaggagaggatggatgtttgcgaaactcatcttcactggcacaccgtcgccaaagagacatgcagtgagaagagtaccaacttgcatgactacggcatgttgctgccctgcggaattgacaagttccgaggggtagagtttgtgtgttgcccactggctgaagaaagtgacaatgtggattctgctgatgcggaggaggatgactcggatgtctggtggggcggagcagacacagactatgcagatgggagtgaagacaaagtagtagaagtagcagaggaggaagaagtggctgaggtggaagaagaagaagccgatgatgacgaggacgatgaggatggtgatgaggtagaggaagaggctgaggaaccctacgaagaagccacagagagaaccaccagcattgccaccaccaccaccaccaccacagagtctgtggaagaggtggttcgagttcctacaacagcagccagtacccctgatgccgttgacaagtatctcgagacacctggggatgagaatgaacatgcccatttccagaaagccaaagagaggcttgaggccaagcaccgagagagaatgtcccaggtcatgagagaatgggaagaggcagaacgtcaagcaaagaacttgcctaaagctgataagaaggcagttatccagcatttccaggagaaagtggaatctttggaacaggaagcagccaacgagagacagcagctggtggagacacacatggccagagtggaagccatgctcaatgaccgccgccgcctggccctggagaactacatcaccgctctgcaggctgttcctcctcggcctcgtcacgtgttcaatatgctaaagaagtatgtccgcgcagaacagaaggacagacagcacaccctaaagcatttcgagcatgtgcgcatggtggatcccaagaaagccgctcagatccggtcccaggttatgacacacctccgtgtgatttatgagcgcatgaatcagtctctctccctgctctacaacgtgcctgcagtggccgaggagattcaggatgaagttgatgagctgcttcagaaagagcaaaactattcagatgacgtcttggccaacatgattagtgaaccaaggatcagttacggaaacgatgctctcatgccatctttgaccgaaacgaaaaccaccgtggagctccttcccgtgaatggagagttcagcctggacgatctccagccgtggcattcttttggggctgactctgtgccagccaacacagaaaacgaagttgagcctgttgatgcccgccctgctgccgaccgaggactgaccactcgaccaggttctgggttgacaaatatcaagacggaggagatctctgaagtgaatctggatgcagaattccgacatgactcaggatatgaagttcatcatcaaaaattggtgttctttgcagaagatgtgggttcaaacaaaggtgcaatcattggactcatggtgggcggtgttgtcatagcgacagtgatcgtcatcaccttggtgatgctgaagaagaaacagtacacatccattcatcatggtgtggtggaggttgacgccgctgtcaccccagaggagcgccacctgtccaagatgcagcagaacggctacgaaaatccaacctacaagttctttgagcagatgcagaactag（SEQ ID NO:1）.. It is noted that the purpose of the mutated sequence (bold representation) herein is to cause amino acid mutation at the corresponding position in the protein after transcriptional translation, so that the mutated sequence herein can be replaced with a sequence having the same transcriptional translation result (e.g., aacctt, aacctc and aaccta).

The sequence of the transgenic fragment of the first exogenous gene Thy1-hAPP is shown in SEQ ID NO. 2 (italic Thy1 promoter sequence, underlined Kozak sequence, capital letter 5' UTR sequence, bolded hAPP CDS sequence with mutation, bolded and underlined mutation site sequence, orientation 5'-3'）：aattcagagaccgggaaccaaactagcctttaaaaaacataagtacaggagccagcaagatggctcagtgggtaaaggtgcctaccagcaagcctgacagcctgagttcagtccccacgaactacgtggtaggagaggaccaaccaactctggaaatctgttctgcaaacacatgctcacacacacacacacaaatagtataaacaattttaaatttcatttaaaaataatttgtaaacaaaatcattagcacaggttttagaaagagcctcttggtgacatcaagttgatgctgtagatggggtatcattcctgaggacccaaaaccgggtctcagcctttccccattctgagagttctctcttttctcagccactagctgaagagtagagtggctcagcactgggctcttgagttcccaagtcctacaactggtcagcctgactactaaccagccatgaagaaacaaggagtggatgggctgagtctgctgggatgggagtggagttagtaagtggccatggatgtaatgaccccagcaatgctggctagaaggcatgcctcctttccttgtctggagacggaacgggagggatcatcttgtactcacagaagggagaacattctagctggttgggccaaaatgtgcaagttcacctggaggtggtggtgcatgcttttaactccagtactcaggaggcagggccaggtggatctctgtgagttcaagaccagcctgcactatggagagagttttgggacagccagagttacacagaaaaatcctggtggaaaatctgaaagaaagagagaaagaaagaaagaaagaaaggaagaaagaaagaaagagtggcaggcaggcaggcaggaggaaggaaggaaggaaggaaggaaggaaggaaggaaggaaggaaaataggtgcgacttcaagatccggagttacaagcagaatgcactgtttccctaacagggccaagtgttttgagtaactgaaggtgggcatgatgcctgggaagcagaaacaagccaggcagatgcaccccttgccttgcttccgaagggctgcagtagcatggaaaacatggaaaacaaccaatccattccctttgctgatataacaggctccaaagccaaaacctgtcactggaggctcaagagcagatctccagccaagaggcaaaggaatgggggaagctggagggcctccctctggttatccaggcttctgaaggttcaagcaaagaaagggttacaaccttaaaaggagagcgtcccggggtatgggtagaagactgctccaccccgacccccagggtccctaaccgtcttttccctgggcgagtcagcccaatcacaggactgagagtgcctctttagtagcagcaagccacttcggacacccaaatggaacacctccagtcagccctcgccgaccaccccaccccctccatccttttccctcagcctccgattggctgaatctagagtccctccctgctcccccctctctccccacccctggtgaaaactgcgggcttcagcgctgggtgcagcaactggaggcgttggcgcaccaggaggaggctgcagctaggggagtccaggtgagagcaggccgacgggagggacccgcacatgcaaggaccgccgcagggcgaggatgcaagccttccccagctacagttttgggaaaggataccagggcgctcctatatgggggcgcgggaactggggaaagaaggtgctcccaggtcgaggtgggagaggaaggcagtgcggggtcacgggctttctccctgctaacggacgctttcgaagagtgggtgccggaggagaaccatgaggaaggacatcaaggacagcctttggtccccaagctcaaatcgctttagtggtgcgaatagagggaggaggtgggtggcaaactggagggagtccccagcgggtgacctcgtggctggctgggtgcggggcaccgcaggtaagaaaaccgcaatgttgcgggaggggactgggtggcaggcgcgggggaggggaaagctagaaaggatgcgagggagcggaggggggagggagcgggagaatctcaactggtagaggaagattaaaatgaggaaatagcatcagggtggggttagccaagccgggcctcagggaaagggcgcaaagtttgtctgggtgtgggcttaggtgggctgggtatgagattcggggcgccgaaaacactgctgcgcctctgccaaatcacgctacccctgtatctagttctgccaggcttctccagccccagccccaattcttttctctagtgttcccccttccctcccctgaatctcaagcccacactccctcctccataacccactgttatcaaatctaagtcatttgccacccaacaaccatcaggaggcggaagcagacgggaggagtttgagatcaacttgggctacatcacgagttccaggctcaccaaggcttcttaaggagaccttgtctctaaaattaattaattaattaattaatagtcccctttctctgccacagaaccttgggatctggctcctggtcgcagctccccccaccccaggctgacattcactgccatagcccatccggaaatcctagtctatttccccatggatcttgaactgcagagagaatggcagagtggcccgccctgtgcaaaggatgttcctagcctaggtggagctcgcgaactcgcagactgtgcctctcttgggcaaggacaggctagacagcctgccggtgtgttgagctagggcactgtggggaaggcagagaacctgtgcagggcagcaatgaacacaggaccagaaaactgcagccctaggaacactcaagagctggccatttgcaagcatctctggcctccgtgcttctcactcatgtcccatgtcttatacaggcctctgtggcacctcgcttgcctgatctcatccctagccgttaagctttctgcatgacttatcacttggggcataatgctggatacctaccattttcttagaccccatcaaaatcctatttgagtgtacggttcggagaacctcatttatccggtaaatgtcttttactctgctctcagggagctgaggcaggacatcctgagatacattgggagaggagatacagtttcaataaaataataggttgggtggaggtacatgcctataatgccaccactcaggaaatggtggcagcttcgtgagtttgaggccaacccaagaaacatagtgaaaccctgtcagtaaataagtaagcaagtatttgagtatctactatatgctagggctgacctggacattaggggtcatcttctgaacaaactagtgcttgagggaggtatttggggtttttgtttgtttaatggatctgaatgagttccagagactggctacacagcgatatgactgagcttaacacccctaaagcatacagtcagaccaattagacaataaaaggtatgtatagcttaccaaataaaaaaattgtattttcaagagagtgtctgtctgtgtagccctggctgttcttgaactcactctgtagaccaggctggcctggaaatccatctgcctgcctctgcctctctgcctctctgcctctctgcctctctctctgcctctctctgcctctctctgcccctctctgcccctctctgcccctctctgccgccctctgccttttgccctctgccctctgttctctggcctctgccctctgccctctggcctctggcctctgcctctgcctcttgagtgctggaatcaaaggtgtgagctctgtaggtcttaagttccagaagaaagtaatgaagtcacccagcagggaggtgctcagggacagcacagacacacacccaggacataggctcccacttccttggctttctctgagtggcaaaggaccttaggcagtgtcactccctaagagaaggggataaagagaggggctgaggtattcatcatgtgctccgtggatctcaagccctcaaggtaaatggggacccacctgtcctaccagctggctgacctgtagctttccccaccacagaatccaagtcggaactcttggcacctagaggatcgccgccaccaGTTTCCTCGGCAGCGGTAGGCGAGAGCACGCGGAGGAGCGTGCGCGGGGGCCCCGGGAGACGGCGGCGGTGGCGGCGCGGGCAGAGCAAGGACGCGGCGGATCCCACTCGCACAGCAGCGCACTCGGTGCCCCGCGCAGGGTCGCGatgctgcccggtttggcactgctcctgctggccgcctggacggctcgggcgctggaggtacccactgatggtaatgctggcctgctggctgaaccccagattgccatgttctgtggcagactgaacatgcacatgaatgtccagaatgggaagtgggattcagatccatcagggaccaaaacctgcattgataccaaggaaggcatcctgcagtattgccaagaagtctaccctgaactgcagatcaccaatgtggtagaagccaaccaaccagtgaccatccagaactggtgcaagcggggccgcaagcagtgcaagacccatccccactttgtgattccctaccgctgcttagttggtgagtttgtaagtgatgcccttctcgttcctgacaagtgcaaattcttacaccaggagaggatggatgtttgcgaaactcatcttcactggcacaccgtcgccaaagagacatgcagtgagaagagtaccaacttgcatgactacggcatgttgctgccctgcggaattgacaagttccgaggggtagagtttgtgtgttgcccactggctgaagaaagtgacaatgtggattctgctgatgcggaggaggatgactcggatgtctggtggggcggagcagacacagactatgcagatgggagtgaagacaaagtagtagaagtagcagaggaggaagaagtggctgaggtggaagaagaagaagccgatgatgacgaggacgatgaggatggtgatgaggtagaggaagaggctgaggaaccctacgaagaagccacagagagaaccaccagcattgccaccaccaccaccaccaccacagagtctgtggaagaggtggttcgagttcctacaacagcagccagtacccctgatgccgttgacaagtatctcgagacacctggggatgagaatgaacatgcccatttccagaaagccaaagagaggcttgaggccaagcaccgagagagaatgtcccaggtcatgagagaatgggaagaggcagaacgtcaagcaaagaacttgcctaaagctgataagaaggcagttatccagcatttccaggagaaagtggaatctttggaacaggaagcagccaacgagagacagcagctggtggagacacacatggccagagtggaagccatgctcaatgaccgccgccgcctggccctggagaactacatcaccgctctgcaggctgttcctcctcggcctcgtcacgtgttcaatatgctaaagaagtatgtccgcgcagaacagaaggacagacagcacaccctaaagcatttcgagcatgtgcgcatggtggatcccaagaaagccgctcagatccggtcccaggttatgacacacctccgtgtgatttatgagcgcatgaatcagtctctctccctgctctacaacgtgcctgcagtggccgaggagattcaggatgaagttgatgagctgcttcagaaagagcaaaactattcagatgacgtcttggccaacatgattagtgaaccaaggatcagttacggaaacgatgctctcatgccatctttgaccgaaacgaaaaccaccgtggagctccttcccgtgaatggagagttcagcctggacgatctccagccgtggcattcttttggggctgactctgtgccagccaacacagaaaacgaagttgagcctgttgatgcccgccctgctgccgaccgaggactgaccactcgaccaggttctgggttgacaaatatcaagacggaggagatctctgaagtgaatctggatgcagaattccgacatgactcaggatatgaagttcatcatcaaaaattggtgttctttgcagaagatgtgggttcaaacaaaggtgcaatcattggactcatggtgggcggtgttgtcatagcgacagtgatcgtcatcaccttggtgatgctgaagaagaaacagtacacatccattcatcatggtgtggtggaggttgacgccgctgtcaccccagaggagcgccacctgtccaagatgcagcagaacggctacgaaaatccaacctacaagttctttgagcagatgcagaactagtcgaggtccttcctctgcagaggtcttgcttctcccggtcagctgactccctccccaagtccttcaaatatctcagaacatggggagaaacggggaccttgtccctcctaaggaaccccagtgctgcatgccatcatcccccccaccctcgcccccacccccgccacttctccctccatgcataccactagctgtcattttgtactctgtatttattccagggctgcttctgattatttagtttgttctttccctggagacctgttagaacataagggcgtatggtgggtaggggaggcaggatatcagtccctggggcgagttcctccctgccaaccaagccagatgcctgaaagagatatggatgagggaagttggactgtgcctgtacctggtacagtcatactctgttgaaagaatcatcggggaggggggggggctcaagaggggagagctctgctgagcctttgtggaccatccaatgaggatgagggcttagattctaccaggtcattctcagccaccacacacaagcgctctgccatcactgaagaagccccctagggctcttgggccagggcacactcagtaaagatgcaggttcagtcagggaatgatggggaaaggggtaggaggtgggggagggatcaccccctcctctaaaacacgagcctgctgtctccaaaggcctctgcctgtagtgagggtggcagaagaagacaaggagccagaactctgactccaggatctaagtccgtgcaggaaggggatcctagaaccatctggttggacccagcttaccaagggagagcctttattcttctttcccttgcccctctgtgccagcccctcttgctgtccctgatcccccagacagcgagagtcttgcaacctgcctcttccaagacctcctaatctcaggggcaggcggtggagtgagatccggcgtgcacactttttggaagatagctttcccaaggatcctctcccccactggcagctctgcctgtcccatcaccatgtataataccaccactgctacagcatctcaccgaggaaagaaaactgcacaataaaaccaagcctctggagtgtgtcctggtgtctgtctcttctgtgtcctggcgtctgtctcttctgtgttcttccaaggtcagaaacaaaaaccacacacttcaacctggatggctcggctgagcacttctgtgtgcagaaggtccaaccagactctggggtaccccggccctccctattcccttgcctcctgtctcccgctttttatagctccctatgctgggcttctctggagagtgaaatctttgcccaaatcaatgcgcattctctctgctgagtcatctggcgacagcagttgagttcacccgccaacacatgggcccagctatgtagccgaaccctggctctggaagtgccagggactttgtgcataagtatgtaccatgcccttttttcacagtcctagctctgcagaagtgcagcctgaaggcctgtctgctgagaggacatgccctggagccctgaaacaggcacagtgggaggaggaacggaggatgacaggcatcaggccctcagtccaaaagcaaccacttgagaatgggctggagtacgaaacatggggtcccgtccctggatccctcctcaaagagtaataagtaaaatataaacaggtaccccaggccgttctgggtttgggttgtaatgggatccatttgcagagaactattgagacagcccagccgtactgtgacaggcaatgtgggggaggaggttgaatcacttggtatttagcatgaatagaataattccctgaacatttttcttaaacatccatatctaaattaccaccactcgctcccagtcttcctgcctttgcgccagcctcctgtctggccatgcctgaagaaggctggagaagccacccacctcaggccatgacactgccagccacttggcaggtgcagccaaacctgagctgtcccagaaagggacattctcaagacccaggcaccctgatcagcactgacttggagctacaagtgtcatgccagaaaagtctctaagaaaaccttttcagggaaaagggggtgactcaacaccgggcaagtttgggaagccccacccttcgagtgatggaagagcagataggaagcctcagaagagagacaccggcacccaggtaacgttcctcatgtggtctctgtcacactaggtgctcttccctggacatctccgtgaccacactctcagttcttagggagatgcgggtgctctctgaggctatctcagagttgcagattctgaggcctagagtgactacagtcagcctaggaagccacagaggactgtggaccaggagggcagaagaggagaagggaagaaaaaccatcagataggacttgcaatgaaactaacccaagacaatcataatgcagacaggaatgttaaaggcgttcagcagctggccatgacacccatctgtccctctggccaagtcagcaagcctggaagacctgggactcctgcccatatgtcctaagctccccacccacccactcgttcactgtccttattctctctctaccttcagccacttagtttcctaccttaagtcctagaattgatcctggcgtaatagcgaagaggcccgcaccgat（SEQ ID NO:2）.

The mutated hPSEN CDS sequence of the transgenic fragment of the second exogenous gene is shown in SEQ ID NO.3 (the bolded font is mutation site sequence, the direction 5'-3'）：atgacagagttacctgcaccgttgtcctacttccagaatgcacagatgtctgaggacaaccacctgagcaatactgtacgtagccagaatgacaatagagaacggcaggagcacaacgacagacggagccttggccaccctgagccattatctaatggacgaccccagggtaactcccggcaggtggtggagcaagatgaggaagaagatgaggagctgacattgaaatatggcgccaagcatgtgatcatgctctttgtccctgtgactctctgcatggtggtggtcgtggctaccattaagtcagtcagcttttatacccggaaggatgggcagctaatctataccccattcacagaagataccgagactgtgggccagagagccctgcactcaattctgaatgctgccatcatgatcagtgtcattgttgtcgtgactatcctcctggtggttctgtataaatacaggtgctataaggtcatccatgcctggcttattatatcatctctattgttgctgttctttttttcattcatttacttgggggaagtgtttaaaacctataacgttgctgtggactacattactgttgcactcctgatctggaattttggtgtggtgggaatgatttccattcactggaaaggtccacttcgactccagcaggcatatctcattatgattagtgccctcatggccctggtgtttatcaagtacctccctgaatggactgcgtggctcatcttggctgtgatttcagtatatgatttagtggctgttttgtgtccgaaaggtccacttcgtatgctggttgaaacagctcaggagagaaatgaaacgctttttccagctctcatttactcctcaacaatggtgtggttggtgaatatggcagaaggagacccggaagctcaaaggagagtatccaaaaattccaagtataatgcagaaagcacagaaagggagtcacaagacactgttgcagagaatgatgatggcgggttcagtgaggaatgggaagcccagagggacagtcatctagggcctcatcgctctacacctgagtcacgagctgctgtccaggaactttccagcagtatcctcgctggtgaagacccagaggaaaggggagtaaaacttggattgggagatttcattttctacagtgttctggttggtaaagcctcagcaacagccagtggagactggaacacaaccatagcctgtttcgtagccatattaattggtttgtgccttacattattactccttgccattttcaagaaagcattgccagctcttccaatctccatcacctttgggcttgttttctactttgccacagattatcttgtacagccttttatggaccaattagcattccatcaattttatatctag（SEQ ID NO:3）. is noted that the purpose of the mutated sequence is to make the protein after transcription and translation have amino acid mutation at the corresponding position, so that the mutated sequence can be replaced by sequences (such as gtt, gtc and gta) with the same transcription and translation result.

The sequence of the transgenic fragment of the second exogenous gene Thy1-hPSEN1 is shown as SEQ ID NO. 4 (italic Thy1 promoter sequence, underlined Kozak sequence, bolded hPSEN CDS sequence with mutation, bolded and underlined mutation site sequence, orientation 5'-3'）：aattcagagaccgggaaccaaactagcctttaaaaaacataagtacaggagccagcaagatggctcagtgggtaaaggtgcctaccagcaagcctgacagcctgagttcagtccccacgaactacgtggtaggagaggaccaaccaactctggaaatctgttctgcaaacacatgctcacacacacacacacaaatagtataaacaattttaaatttcatttaaaaataatttgtaaacaaaatcattagcacaggttttagaaagagcctcttggtgacatcaagttgatgctgtagatggggtatcattcctgaggacccaaaaccgggtctcagcctttccccattctgagagttctctcttttctcagccactagctgaagagtagagtggctcagcactgggctcttgagttcccaagtcctacaactggtcagcctgactactaaccagccatgaagaaacaaggagtggatgggctgagtctgctgggatgggagtggagttagtaagtggccatggatgtaatgaccccagcaatgctggctagaaggcatgcctcctttccttgtctggagacggaacgggagggatcatcttgtactcacagaagggagaacattctagctggttgggccaaaatgtgcaagttcacctggaggtggtggtgcatgcttttaactccagtactcaggaggcagggccaggtggatctctgtgagttcaagaccagcctgcactatggagagagttttgggacagccagagttacacagaaaaatcctggtggaaaatctgaaagaaagagagaaagaaagaaagaaagaaaggaagaaagaaagaaagagtggcaggcaggcaggcaggaggaaggaaggaaggaaggaaggaaggaaggaaggaaggaaggaaaataggtgcgacttcaagatccggagttacaagcagaatgcactgtttccctaacagggccaagtgttttgagtaactgaaggtgggcatgatgcctgggaagcagaaacaagccaggcagatgcaccccttgccttgcttccgaagggctgcagtagcatggaaaacatggaaaacaaccaatccattccctttgctgatataacaggctccaaagccaaaacctgtcactggaggctcaagagcagatctccagccaagaggcaaaggaatgggggaagctggagggcctccctctggttatccaggcttctgaaggttcaagcaaagaaagggttacaaccttaaaaggagagcgtcccggggtatgggtagaagactgctccaccccgacccccagggtccctaaccgtcttttccctgggcgagtcagcccaatcacaggactgagagtgcctctttagtagcagcaagccacttcggacacccaaatggaacacctccagtcagccctcgccgaccaccccaccccctccatccttttccctcagcctccgattggctgaatctagagtccctccctgctcccccctctctccccacccctggtgaaaactgcgggcttcagcgctgggtgcagcaactggaggcgttggcgcaccaggaggaggctgcagctaggggagtccaggtgagagcaggccgacgggagggacccgcacatgcaaggaccgccgcagggcgaggatgcaagccttccccagctacagttttgggaaaggataccagggcgctcctatatgggggcgcgggaactggggaaagaaggtgctcccaggtcgaggtgggagaggaaggcagtgcggggtcacgggctttctccctgctaacggacgctttcgaagagtgggtgccggaggagaaccatgaggaaggacatcaaggacagcctttggtccccaagctcaaatcgctttagtggtgcgaatagagggaggaggtgggtggcaaactggagggagtccccagcgggtgacctcgtggctggctgggtgcggggcaccgcaggtaagaaaaccgcaatgttgcgggaggggactgggtggcaggcgcgggggaggggaaagctagaaaggatgcgagggagcggaggggggagggagcgggagaatctcaactggtagaggaagattaaaatgaggaaatagcatcagggtggggttagccaagccgggcctcagggaaagggcgcaaagtttgtctgggtgtgggcttaggtgggctgggtatgagattcggggcgccgaaaacactgctgcgcctctgccaaatcacgctacccctgtatctagttctgccaggcttctccagccccagccccaattcttttctctagtgttcccccttccctcccctgaatctcaagcccacactccctcctccataacccactgttatcaaatctaagtcatttgccacccaacaaccatcaggaggcggaagcagacgggaggagtttgagatcaacttgggctacatcacgagttccaggctcaccaaggcttcttaaggagaccttgtctctaaaattaattaattaattaattaatagtcccctttctctgccacagaaccttgggatctggctcctggtcgcagctccccccaccccaggctgacattcactgccatagcccatccggaaatcctagtctatttccccatggatcttgaactgcagagagaatggcagagtggcccgccctgtgcaaaggatgttcctagcctaggtggagctcgcgaactcgcagactgtgcctctcttgggcaaggacaggctagacagcctgccggtgtgttgagctagggcactgtggggaaggcagagaacctgtgcagggcagcaatgaacacaggaccagaaaactgcagccctaggaacactcaagagctggccatttgcaagcatctctggcctccgtgcttctcactcatgtcccatgtcttatacaggcctctgtggcacctcgcttgcctgatctcatccctagccgttaagctttctgcatgacttatcacttggggcataatgctggatacctaccattttcttagaccccatcaaaatcctatttgagtgtacggttcggagaacctcatttatccggtaaatgtcttttactctgctctcagggagctgaggcaggacatcctgagatacattgggagaggagatacagtttcaataaaataataggttgggtggaggtacatgcctataatgccaccactcaggaaatggtggcagcttcgtgagtttgaggccaacccaagaaacatagtgaaaccctgtcagtaaataagtaagcaagtatttgagtatctactatatgctagggctgacctggacattaggggtcatcttctgaacaaactagtgcttgagggaggtatttggggtttttgtttgtttaatggatctgaatgagttccagagactggctacacagcgatatgactgagcttaacacccctaaagcatacagtcagaccaattagacaataaaaggtatgtatagcttaccaaataaaaaaattgtattttcaagagagtgtctgtctgtgtagccctggctgttcttgaactcactctgtagaccaggctggcctggaaatccatctgcctgcctctgcctctctgcctctctgcctctctgcctctctctctgcctctctctgcctctctctgcccctctctgcccctctctgcccctctctgccgccctctgccttttgccctctgccctctgttctctggcctctgccctctgccctctggcctctggcctctgcctctgcctcttgagtgctggaatcaaaggtgtgagctctgtaggtcttaagttccagaagaaagtaatgaagtcacccagcagggaggtgctcagggacagcacagacacacacccaggacataggctcccacttccttggctttctctgagtggcaaaggaccttaggcagtgtcactccctaagagaaggggataaagagaggggctgaggtattcatcatgtgctccgtggatctcaagccctcaaggtaaatggggacccacctgtcctaccagctggctgacctgtagctttccccaccacagaatccaagtcggaactcttggcacctagaggatcgccgccaccatgacagagttacctgcaccgttgtcctacttccagaatgcacagatgtctgaggacaaccacctgagcaatactgtacgtagccagaatgacaatagagaacggcaggagcacaacgacagacggagccttggccaccctgagccattatctaatggacgaccccagggtaactcccggcaggtggtggagcaagatgaggaagaagatgaggagctgacattgaaatatggcgccaagcatgtgatcatgctctttgtccctgtgactctctgcatggtggtggtcgtggctaccattaagtcagtcagcttttatacccggaaggatgggcagctaatctataccccattcacagaagataccgagactgtgggccagagagccctgcactcaattctgaatgctgccatcatgatcagtgtcattgttgtcgtgactatcctcctggtggttctgtataaatacaggtgctataaggtcatccatgcctggcttattatatcatctctattgttgctgttctttttttcattcatttacttgggggaagtgtttaaaacctataacgttgctgtggactacattactgttgcactcctgatctggaattttggtgtggtgggaatgatttccattcactggaaaggtccacttcgactccagcaggcatatctcattatgattagtgccctcatggccctggtgtttatcaagtacctccctgaatggactgcgtggctcatcttggctgtgatttcagtatatgatttagtggctgttttgtgtccgaaaggtccacttcgtatgctggttgaaacagctcaggagagaaatgaaacgctttttccagctctcatttactcctcaacaatggtgtggttggtgaatatggcagaaggagacccggaagctcaaaggagagtatccaaaaattccaagtataatgcagaaagcacagaaagggagtcacaagacactgttgcagagaatgatgatggcgggttcagtgaggaatgggaagcccagagggacagtcatctagggcctcatcgctctacacctgagtcacgagctgctgtccaggaactttccagcagtatcctcgctggtgaagacccagaggaaaggggagtaaaacttggattgggagatttcattttctacagtgttctggttggtaaagcctcagcaacagccagtggagactggaacacaaccatagcctgtttcgtagccatattaattggtttgtgccttacattattactccttgccattttcaagaaagcattgccagctcttccaatctccatcacctttgggcttgttttctactttgccacagattatcttgtacagccttttatggaccaattagcattccatcaattttatatctagtcgaggtccttcctctgcagaggtcttgcttctcccggtcagctgactccctccccaagtccttcaaatatctcagaacatggggagaaacggggaccttgtccctcctaaggaaccccagtgctgcatgccatcatcccccccaccctcgcccccacccccgccacttctccctccatgcataccactagctgtcattttgtactctgtatttattccagggctgcttctgattatttagtttgttctttccctggagacctgttagaacataagggcgtatggtgggtaggggaggcaggatatcagtccctggggcgagttcctccctgccaaccaagccagatgcctgaaagagatatggatgagggaagttggactgtgcctgtacctggtacagtcatactctgttgaaagaatcatcggggaggggggggggctcaagaggggagagctctgctgagcctttgtggaccatccaatgaggatgagggcttagattctaccaggtcattctcagccaccacacacaagcgctctgccatcactgaagaagccccctagggctcttgggccagggcacactcagtaaagatgcaggttcagtcagggaatgatggggaaaggggtaggaggtgggggagggatcaccccctcctctaaaacacgagcctgctgtctccaaaggcctctgcctgtagtgagggtggcagaagaagacaaggagccagaactctgactccaggatctaagtccgtgcaggaaggggatcctagaaccatctggttggacccagcttaccaagggagagcctttattcttctttcccttgcccctctgtgccagcccctcttgctgtccctgatcccccagacagcgagagtcttgcaacctgcctcttccaagacctcctaatctcaggggcaggcggtggagtgagatccggcgtgcacactttttggaagatagctttcccaaggatcctctcccccactggcagctctgcctgtcccatcaccatgtataataccaccactgctacagcatctcaccgaggaaagaaaactgcacaataaaaccaagcctctggagtgtgtcctggtgtctgtctcttctgtgtcctggcgtctgtctcttctgtgttcttccaaggtcagaaacaaaaaccacacacttcaacctggatggctcggctgagcacttctgtgtgcagaaggtccaaccagactctggggtaccccggccctccctattcccttgcctcctgtctcccgctttttatagctccctatgctgggcttctctggagagtgaaatctttgcccaaatcaatgcgcattctctctgctgagtcatctggcgacagcagttgagttcacccgccaacacatgggcccagctatgtagccgaaccctggctctggaagtgccagggactttgtgcataagtatgtaccatgcccttttttcacagtcctagctctgcagaagtgcagcctgaaggcctgtctgctgagaggacatgccctggagccctgaaacaggcacagtgggaggaggaacggaggatgacaggcatcaggccctcagtccaaaagcaaccacttgagaatgggctggagtacgaaacatggggtcccgtccctggatccctcctcaaagagtaataagtaaaatataaacaggtaccccaggccgttctgggtttgggttgtaatgggatccatttgcagagaactattgagacagcccagccgtactgtgacaggcaatgtgggggaggaggttgaatcacttggtatttagcatgaatagaataattccctgaacatttttcttaaacatccatatctaaattaccaccactcgctcccagtcttcctgcctttgcgccagcctcctgtctggccatgcctgaagaaggctggagaagccacccacctcaggccatgacactgccagccacttggcaggtgcagccaaacctgagctgtcccagaaagggacattctcaagacccaggcaccctgatcagcactgacttggagctacaagtgtcatgccagaaaagtctctaagaaaaccttttcagggaaaagggggtgactcaacaccgggcaagtttgggaagccccacccttcgagtgatggaagagcagataggaagcctcagaagagagacaccggcacccaggtaacgttcctcatgtggtctctgtcacactaggtgctcttccctggacatctccgtgaccacactctcagttcttagggagatgcgggtgctctctgaggctatctcagagttgcagattctgaggcctagagtgactacagtcagcctaggaagccacagaggactgtggaccaggagggcagaagaggagaagggaagaaaaaccatcagataggacttgcaatgaaactaacccaagacaatcataatgcagacaggaatgttaaaggcgttcagcagctggccatgacacccatctgtccctctggccaagtcagcaagcctggaagacctgggactcctgcccatatgtcctaagctccccacccacccactcgttcactgtccttattctctctctaccttcagccacttagtttcctaccttaagtcctagaattgatcctggcgtaatagcgaagaggcccgcaccgat（SEQ ID NO:4）.

The mutant hMAPT CDS of the third exogenous gene transgene fragment is shown in SEQ ID NO. 5 (wherein the bold font is the mutation site sequence and the direction is 5'-3'）：atggctgagccccgccaggagttcgaagtgatggaagatcacgctgggacgtacgggttgggggacaggaaagatcaggggggctacaccatgcaccaagaccaagagggtgacacggacgctggcctgaaagctgaagaagcaggcattggagacacccccagcctggaagacgaagctgctggtcacgtgacccaagctcgcatggtcagtaaaagcaaagacgggactggaagcgatgacaaaaaagccaagggggctgatggtaaaacgaagatcgccacaccgcggggagcagcccctccaggccagaagggccaggccaacgccaccaggattccagcaaaaaccccgcccgctccaaagacaccacccagctctggtgaacctccaaaatcaggggatcgcagcggctacagcagccccggctccccaggcactcccggcagccgctcccgcaccccgtcccttccaaccccacccacccgggagcccaagaaggtggcagtggtccgtactccacccaagtcgccgtcttccgccaagagccgcctgcagacagcccccgtgcccatgccagacctgaagaatgtcaagtccaagatcggctccactgagaacctgaagcaccagccgggaggcgggaaggtgcagataattaataagaagctggatcttagcaacgtccagtccaagtgtggctcaaaggataatatcaaacacgtcctgggaggcggcagtgtgcaaatagtctacaaaccagttgacctgagcaaggtgacctccaagtgtggctcattaggcaacatccatcataaaccaggaggtggccaggtggaagtaaaatctgagaagcttgacttcaaggacagagtccagtcgaagattgggtccctggacaatatcacccacgtccctggcggaggaaataaaaagattgaaacccacaagctgaccttccgcgagaacgccaaagccaagacagaccacggggcggagatcgtgtacaagtcgccagtggtgtctggggacacgtctccacggcatctcagcaatgtctcctccaccggcagcatcgacatggtagactcgccccagctcgccacgctagctgacgaggtgtctgcctccctggccaagcagggtttgtga（SEQ ID NO:5）.. It is noted that the purpose of the mutation sequence (bold) is to make the protein undergo amino acid mutation at the corresponding position after transcription and translation, so that the mutation sequence can be replaced by a sequence (such as ctt, ctc and cta) having the same transcription and translation result.

The sequence of the transgenic fragment of the third exogenous gene Thy1-hMAPT is shown as SEQ ID NO. 6 (italic Thy1 promoter sequence, underlined Kozak sequence, bolded hMAPT CDS sequence with mutation, bolded and underlined mutation site sequence, orientation 5'-3'）：aattcagagaccgggaaccaaactagcctttaaaaaacataagtacaggagccagcaagatggctcagtgggtaaaggtgcctaccagcaagcctgacagcctgagttcagtccccacgaactacgtggtaggagaggaccaaccaactctggaaatctgttctgcaaacacatgctcacacacacacacacaaatagtataaacaattttaaatttcatttaaaaataatttgtaaacaaaatcattagcacaggttttagaaagagcctcttggtgacatcaagttgatgctgtagatggggtatcattcctgaggacccaaaaccgggtctcagcctttccccattctgagagttctctcttttctcagccactagctgaagagtagagtggctcagcactgggctcttgagttcccaagtcctacaactggtcagcctgactactaaccagccatgaagaaacaaggagtggatgggctgagtctgctgggatgggagtggagttagtaagtggccatggatgtaatgaccccagcaatgctggctagaaggcatgcctcctttccttgtctggagacggaacgggagggatcatcttgtactcacagaagggagaacattctagctggttgggccaaaatgtgcaagttcacctggaggtggtggtgcatgcttttaactccagtactcaggaggcagggccaggtggatctctgtgagttcaagaccagcctgcactatggagagagttttgggacagccagagttacacagaaaaatcctggtggaaaatctgaaagaaagagagaaagaaagaaagaaagaaaggaagaaagaaagaaagagtggcaggcaggcaggcaggaggaaggaaggaaggaaggaaggaaggaaggaaggaaggaaggaaaataggtgcgacttcaagatccggagttacaagcagaatgcactgtttccctaacagggccaagtgttttgagtaactgaaggtgggcatgatgcctgggaagcagaaacaagccaggcagatgcaccccttgccttgcttccgaagggctgcagtagcatggaaaacatggaaaacaaccaatccattccctttgctgatataacaggctccaaagccaaaacctgtcactggaggctcaagagcagatctccagccaagaggcaaaggaatgggggaagctggagggcctccctctggttatccaggcttctgaaggttcaagcaaagaaagggttacaaccttaaaaggagagcgtcccggggtatgggtagaagactgctccaccccgacccccagggtccctaaccgtcttttccctgggcgagtcagcccaatcacaggactgagagtgcctctttagtagcagcaagccacttcggacacccaaatggaacacctccagtcagccctcgccgaccaccccaccccctccatccttttccctcagcctccgattggctgaatctagagtccctccctgctcccccctctctccccacccctggtgaaaactgcgggcttcagcgctgggtgcagcaactggaggcgttggcgcaccaggaggaggctgcagctaggggagtccaggtgagagcaggccgacgggagggacccgcacatgcaaggaccgccgcagggcgaggatgcaagccttccccagctacagttttgggaaaggataccagggcgctcctatatgggggcgcgggaactggggaaagaaggtgctcccaggtcgaggtgggagaggaaggcagtgcggggtcacgggctttctccctgctaacggacgctttcgaagagtgggtgccggaggagaaccatgaggaaggacatcaaggacagcctttggtccccaagctcaaatcgctttagtggtgcgaatagagggaggaggtgggtggcaaactggagggagtccccagcgggtgacctcgtggctggctgggtgcggggcaccgcaggtaagaaaaccgcaatgttgcgggaggggactgggtggcaggcgcgggggaggggaaagctagaaaggatgcgagggagcggaggggggagggagcgggagaatctcaactggtagaggaagattaaaatgaggaaatagcatcagggtggggttagccaagccgggcctcagggaaagggcgcaaagtttgtctgggtgtgggcttaggtgggctgggtatgagattcggggcgccgaaaacactgctgcgcctctgccaaatcacgctacccctgtatctagttctgccaggcttctccagccccagccccaattcttttctctagtgttcccccttccctcccctgaatctcaagcccacactccctcctccataacccactgttatcaaatctaagtcatttgccacccaacaaccatcaggaggcggaagcagacgggaggagtttgagatcaacttgggctacatcacgagttccaggctcaccaaggcttcttaaggagaccttgtctctaaaattaattaattaattaattaatagtcccctttctctgccacagaaccttgggatctggctcctggtcgcagctccccccaccccaggctgacattcactgccatagcccatccggaaatcctagtctatttccccatggatcttgaactgcagagagaatggcagagtggcccgccctgtgcaaaggatgttcctagcctaggtggagctcgcgaactcgcagactgtgcctctcttgggcaaggacaggctagacagcctgccggtgtgttgagctagggcactgtggggaaggcagagaacctgtgcagggcagcaatgaacacaggaccagaaaactgcagccctaggaacactcaagagctggccatttgcaagcatctctggcctccgtgcttctcactcatgtcccatgtcttatacaggcctctgtggcacctcgcttgcctgatctcatccctagccgttaagctttctgcatgacttatcacttggggcataatgctggatacctaccattttcttagaccccatcaaaatcctatttgagtgtacggttcggagaacctcatttatccggtaaatgtcttttactctgctctcagggagctgaggcaggacatcctgagatacattgggagaggagatacagtttcaataaaataataggttgggtggaggtacatgcctataatgccaccactcaggaaatggtggcagcttcgtgagtttgaggccaacccaagaaacatagtgaaaccctgtcagtaaataagtaagcaagtatttgagtatctactatatgctagggctgacctggacattaggggtcatcttctgaacaaactagtgcttgagggaggtatttggggtttttgtttgtttaatggatctgaatgagttccagagactggctacacagcgatatgactgagcttaacacccctaaagcatacagtcagaccaattagacaataaaaggtatgtatagcttaccaaataaaaaaattgtattttcaagagagtgtctgtctgtgtagccctggctgttcttgaactcactctgtagaccaggctggcctggaaatccatctgcctgcctctgcctctctgcctctctgcctctctgcctctctctctgcctctctctgcctctctctgcccctctctgcccctctctgcccctctctgccgccctctgccttttgccctctgccctctgttctctggcctctgccctctgccctctggcctctggcctctgcctctgcctcttgagtgctggaatcaaaggtgtgagctctgtaggtcttaagttccagaagaaagtaatgaagtcacccagcagggaggtgctcagggacagcacagacacacacccaggacataggctcccacttccttggctttctctgagtggcaaaggaccttaggcagtgtcactccctaagagaaggggataaagagaggggctgaggtattcatcatgtgctccgtggatctcaagccctcaaggtaaatggggacccacctgtcctaccagctggctgacctgtagctttccccaccacagaatccaagtcggaactcttggcacctagaggatcgccgccaccatggctgagccccgccaggagttcgaagtgatggaagatcacgctgggacgtacgggttgggggacaggaaagatcaggggggctacaccatgcaccaagaccaagagggtgacacggacgctggcctgaaagctgaagaagcaggcattggagacacccccagcctggaagacgaagctgctggtcacgtgacccaagctcgcatggtcagtaaaagcaaagacgggactggaagcgatgacaaaaaagccaagggggctgatggtaaaacgaagatcgccacaccgcggggagcagcccctccaggccagaagggccaggccaacgccaccaggattccagcaaaaaccccgcccgctccaaagacaccacccagctctggtgaacctccaaaatcaggggatcgcagcggctacagcagccccggctccccaggcactcccggcagccgctcccgcaccccgtcccttccaaccccacccacccgggagcccaagaaggtggcagtggtccgtactccacccaagtcgccgtcttccgccaagagccgcctgcagacagcccccgtgcccatgccagacctgaagaatgtcaagtccaagatcggctccactgagaacctgaagcaccagccgggaggcgggaaggtgcagataattaataagaagctggatcttagcaacgtccagtccaagtgtggctcaaaggataatatcaaacacgtcctgggaggcggcagtgtgcaaatagtctacaaaccagttgacctgagcaaggtgacctccaagtgtggctcattaggcaacatccatcataaaccaggaggtggccaggtggaagtaaaatctgagaagcttgacttcaaggacagagtccagtcgaagattgggtccctggacaatatcacccacgtccctggcggaggaaataaaaagattgaaacccacaagctgaccttccgcgagaacgccaaagccaagacagaccacggggcggagatcgtgtacaagtcgccagtggtgtctggggacacgtctccacggcatctcagcaatgtctcctccaccggcagcatcgacatggtagactcgccccagctcgccacgctagctgacgaggtgtctgcctccctggccaagcagggtttgtgatcgaggtccttcctctgcagaggtcttgcttctcccggtcagctgactccctccccaagtccttcaaatatctcagaacatggggagaaacggggaccttgtccctcctaaggaaccccagtgctgcatgccatcatcccccccaccctcgcccccacccccgccacttctccctccatgcataccactagctgtcattttgtactctgtatttattccagggctgcttctgattatttagtttgttctttccctggagacctgttagaacataagggcgtatggtgggtaggggaggcaggatatcagtccctggggcgagttcctccctgccaaccaagccagatgcctgaaagagatatggatgagggaagttggactgtgcctgtacctggtacagtcatactctgttgaaagaatcatcggggaggggggggggctcaagaggggagagctctgctgagcctttgtggaccatccaatgaggatgagggcttagattctaccaggtcattctcagccaccacacacaagcgctctgccatcactgaagaagccccctagggctcttgggccagggcacactcagtaaagatgcaggttcagtcagggaatgatggggaaaggggtaggaggtgggggagggatcaccccctcctctaaaacacgagcctgctgtctccaaaggcctctgcctgtagtgagggtggcagaagaagacaaggagccagaactctgactccaggatctaagtccgtgcaggaaggggatcctagaaccatctggttggacccagcttaccaagggagagcctttattcttctttcccttgcccctctgtgccagcccctcttgctgtccctgatcccccagacagcgagagtcttgcaacctgcctcttccaagacctcctaatctcaggggcaggcggtggagtgagatccggcgtgcacactttttggaagatagctttcccaaggatcctctcccccactggcagctctgcctgtcccatcaccatgtataataccaccactgctacagcatctcaccgaggaaagaaaactgcacaataaaaccaagcctctggagtgtgtcctggtgtctgtctcttctgtgtcctggcgtctgtctcttctgtgttcttccaaggtcagaaacaaaaaccacacacttcaacctggatggctcggctgagcacttctgtgtgcagaaggtccaaccagactctggggtaccccggccctccctattcccttgcctcctgtctcccgctttttatagctccctatgctgggcttctctggagagtgaaatctttgcccaaatcaatgcgcattctctctgctgagtcatctggcgacagcagttgagttcacccgccaacacatgggcccagctatgtagccgaaccctggctctggaagtgccagggactttgtgcataagtatgtaccatgcccttttttcacagtcctagctctgcagaagtgcagcctgaaggcctgtctgctgagaggacatgccctggagccctgaaacaggcacagtgggaggaggaacggaggatgacaggcatcaggccctcagtccaaaagcaaccacttgagaatgggctggagtacgaaacatggggtcccgtccctggatccctcctcaaagagtaataagtaaaatataaacaggtaccccaggccgttctgggtttgggttgtaatgggatccatttgcagagaactattgagacagcccagccgtactgtgacaggcaatgtgggggaggaggttgaatcacttggtatttagcatgaatagaataattccctgaacatttttcttaaacatccatatctaaattaccaccactcgctcccagtcttcctgcctttgcgccagcctcctgtctggccatgcctgaagaaggctggagaagccacccacctcaggccatgacactgccagccacttggcaggtgcagccaaacctgagctgtcccagaaagggacattctcaagacccaggcaccctgatcagcactgacttggagctacaagtgtcatgccagaaaagtctctaagaaaaccttttcagggaaaagggggtgactcaacaccgggcaagtttgggaagccccacccttcgagtgatggaagagcagataggaagcctcagaagagagacaccggcacccaggtaacgttcctcatgtggtctctgtcacactaggtgctcttccctggacatctccgtgaccacactctcagttcttagggagatgcgggtgctctctgaggctatctcagagttgcagattctgaggcctagagtgactacagtcagcctaggaagccacagaggactgtggaccaggagggcagaagaggagaagggaagaaaaaccatcagataggacttgcaatgaaactaacccaagacaatcataatgcagacaggaatgttaaaggcgttcagcagctggccatgacacccatctgtccctctggccaagtcagcaagcctggaagacctgggactcctgcccatatgtcctaagctccccacccacccactcgttcactgtccttattctctctctaccttcagccacttagtttcctaccttaagtcctagaattgatcctggcgtaatagcgaagaggcccgcaccgat（SEQ ID NO:6）.

(2) Microinjection of the three transgenic fragments into fertilized eggs of mice, random targeting insertion into mouse genome;

(3) Transplanting fertilized eggs into pseudopregnant female mice, and identifying and screening to obtain F0 generation mice with positive identification of three exogenous genes;

The primer pair sequences used for PCR identification were as follows:

Identifying a first exogenous gene hAPP primer pair 1: AAGTAATGAAGTCACCAGCAGG (SEQ ID NO: 7) and CGTTTCTCCCCATGTTCTGAGA (SEQ ID NO: 8), and primer pair 2: GGGTTGACAATATATATAGAGAGGAG (SEQ ID NO: 9) and CGTTTCTCCCCATGTTCTGAGA (SEQ ID NO: 8);

Primer pairs for identifying the second exogenous gene hPSEN1, primer pair 1, AAGTAATGAAGTCACCAGCAGG (SEQ ID NO: 10) and CGTACAGTATTGCTCAGGTGGTTG (SEQ ID NO: 11), primer pair 2, AGGAACTTTCCAGCAGAGTACTC (SEQ ID NO: 12) and CGTTTCTCCCCATGTTCTGAGA (SEQ ID NO: 13);

Primer pairs for identifying third exogenous gene hMAPT, primer pair 1: AAGTAATGAAGTCACCAGCAGG (SEQ ID NO: 14) and TGCCTGCTTCTTCAGCTTTCAG (SEQ ID NO: 15), primer pair 2: CAAGTGTGGCTCATTAGGCAC (SEQ ID NO: 16) and CGTTTCTCCCCATGTTCTGAGA (SEQ ID NO: 17);

Primer pair GTGCTGCTCACGCTGACCTTTAG (SEQ ID NO: 18) and CAGAGTGGAATACTGTTGCACC (SEQ ID NO: 19) of the reference gene.

(4) Backcrossing F0 generation mice with wild mice (WT) until F0N5 generation mice are obtained, and selfing F0N5 generation mice to obtain the mice model 3-FAD homozygous positive mice of Alzheimer's disease.

Example 2 detection of blood biomarkers

The detection method of the automatic ultrasensitive immunoassay-single molecule array is adopted to detect the clinical early blood detection index of the Alzheimer's disease in the plasma of the mice, the detected objects comprise 3-FAD (1M) mice with the age of 1 month, 3-FAD (2M) mice with the age of 2 months and C57BL/6J control mice (WT) with the age of 6 months as control groups, and the experimental results are shown in figure 1. In FIG. 1, (a), (b) and (c) show the results of blood (plasma) tests (unit: pg/mL) of Abeta 42/40, p-Tau181 and p-Tau217, respectively.

As can be seen from FIG. 1, first, three biomarkers of Abeta 42/40, p-Tau181 and p-Tau217 were detected simultaneously in the blood of 1M 3-FAD mice. However, no model capable of detecting these three biomarkers simultaneously from blood has been found on the market, and no detection is possible at 1 month of age.

Second, the up-regulation of the aβ42/40 ratio, specifically, the aβ42 was found at 1 month of age compared to the control group in which no aβ 42,3-FAD mice were detected, and the aβ42/40 ratio was increased over time in 2 months of age, but did not show a significant increase with the progression of the disease process, which was consistent with the clinical progression of alzheimer's disease.

Finally, the concentrations of p-Tau181, p-Tau217 gradually increased over time, which behavior was also consistent with the detection index in clinical patients.

From this, it can be concluded that 3-FAD mice can successfully detect Abeta 42/40, p-Tau181, p-Tau217 in plasma simultaneously, and that the presented disease index is consistent with the criteria for early detection of clinical blood.

Example 3 pathological brain region detection

Alzheimer's disease-marked neuropathological changes are diffuse neuritic plaques marked by extracellular β -amyloid deposition (Abeta deposition) and neurofibrillary tangles (formed by the intracellular aggregation of hyperphosphorylated Tau protein). Wherein aβ aggregation may extend through the entire stage of development of alzheimer's disease.

The detection step is that 6 3-FAD mice are taken and euthanized at the age of 2 months. And then, performing perfusion, taking out the brain, fixing and embedding, and detecting the expression of the Abeta plaque through immunohistochemical staining to obtain an Immunohistochemical (IHC) result. IHC results were obtained by treating control (WT) 2 month-old C57BL/6J2 mice and 4 month-old, 6 month-old 3-FAD mice in the same manner.

From the results, it can be seen that on the one hand, as shown in FIG. 2, abeta was detected in Cortex (Cortex, ctx), hippocampal lower tray (Subiculum, sub) of the 2-month-old 3-FAD mice and exhibited a speckle state, compared to the mice in the control group, which did not exhibit Abeta deposition. And as shown in fig. 3, in the 3-FAD mice of 1 month old, deposition of aβ was not revealed, whereas in the brains of the 3-FAD mice of 2 months old, deposition of aβ was revealed, while aβ42 could be detected in combination with the aforementioned 3-FAD mice of 1 month old in blood, which is consistent with actual clinical manifestations. In addition, as the course of disease progressed, aβ deposition appeared to aggregate and plaque in the cortex and hippocampal lower trays of 3-FAD mice until aβ plaque deposition occurred at 6 months of age, whereas control group at 6 months of age did not. The 3-FAD mice are consistent with the clinical representation of Alzheimer's disease in the pathological representation of the gradual deposition and expression of Abeta plaques.

On the other hand, various hyperphosphorylated Tau proteins were detected at different stages of age in 3-FAD mice. As shown in FIG. 4, p-Tau181 was detected in the cortex (Ctx), hippocampus (Hip) and cerebellum (Cere) of the 3-FAD mice of 1 month old, as shown in FIG. 5, p-Tau 217 was detected in the cortex (Ctx), hippocampus (Hip) and cerebellum (Cere) of the 3-FAD mice of 2 months old, as shown in FIG. 6, p-Tau (Thr 231) was detected in the cortex (Ctx), hippocampus (Hip) and cerebellum (Cere) of the 3-FAD (3M) mice of 3 months old, as shown in FIG. 7, p-Tau (Ser 202, thr 205) was detected in the cortex (Ctx), hippocampus (Hip) and cerebellum (Cere) of the 3-FAD mice of 3 months old, as shown in FIG. 8. Wherein, as shown in FIG. 9, p-Tau181 is phosphorylated with time progress and gradually forms tangled morphology, and as shown in FIG. 10, p-Tau (Ser 202, thr 205) appears after Abeta deposition is detected and is exacerbated with disease progress.

From this, it can be concluded from FIGS. 2 to 10 that in the brain region of the 3-FAD mice, p-Tau181, p-Tau217, p-Tau (Thr 231), p-Tau (Ser 202, thr 205) and p-Tau (Ser 396) occur in chronological order, and that in the brain of the 3-FAD mice, p-Tau181 occurs before Abeta deposition, p-Tau (Ser 202, thr 205) occurs after Abeta deposition and is exacerbated as the course of the disease progresses. The development of the pathological process or stage is consistent with the situation of clinical patients, and has the prospect of researching the pathological mechanism of the Alzheimer's disease and screening medicaments for treating the Alzheimer's disease.

Example 4 behavioural verification

Loss of visual space memory is a characteristic performance characteristic of the progression of Alzheimer's disease. The Morris water maze experiment is an experimental method widely applied in the field of neuroscience, and is mainly used for evaluating the spatial learning and memory abilities and the sense of direction of experimental animals. The experiment is based on the animal's instinctive response to the water environment, i.e. the spatial memory capacity of the animal is assessed by observing the time and path it takes to find a platform hidden under water in the water maze.

Morris water maze experiments were performed on 3-FAD mice 3 months old, and C57BL/6J mice 3 months old were used as a control group (WT). Specifically, in a circular pool as shown in fig. 11 (a), the pool is divided into four quadrants (Q1, Q2, Q3, and Q4), and the water temperature of the pool is maintained at 23±1 ℃. A hidden fixed platform (with the diameter of 10 cm) is placed on Q2 to be about 1cm higher than the water surface on the first day, 2 times of visual training are carried out, and on the 2 th to 5 th days, the hidden fixed platform is placed under the Q2 to be immersed for 1cm below the water surface, and 4 times of training are carried out every day. The platform was removed from the maze on day 6, leaving the mice free to swim for 60 seconds. The time and distance each mouse passed over the active area (plateau) and the time and distance explored in the target quadrant (the quadrant in which the plateau was located), as well as the number of entries, were monitored and recorded.

As a result, as shown in FIGS. 11 (b) and (c), there was no significant difference in both the swimming distance (unit: M) and the swimming speed (unit: mm/s) between the 3-FAD mice and the control group, indicating that the 3M 3-FAD mice were consistent in locomotor ability with the control group, i.e., the locomotor function (Normal locomotor function) was not impaired. As shown in fig. 11 (d) and (e), the residence time of the 3-FAD mice was significantly shortened and the number of times of entry was significantly reduced compared to the control group, as seen from the number of times and residence time of entering the second quadrant, which is the quadrant in which the platform was located, indicating that the 3-FAD mice failed to memorize the position of the platform, i.e., the memory ability was lowered, in combination with the case where the exercise function was not impaired. Meanwhile, compared with the control group, the time from the time of putting the 3-FAD mice into the pool to the time of reaching the platform is obviously longer than that of the control group, as shown in (f) in fig. 11, and the number of times of shuttling near the platform is obviously smaller than that of the control group, as shown in (g) in fig. 11, and the memory capacity of the 3-FAD mice is also reduced by combining the situation that the exercise function is not damaged.

From this, it can be concluded that the motor function (Normal locomotor function) of 3-FAD mice of 3 months of age was not impaired, but exhibited a phenotype of reduced spatial memory consistent with clinical symptoms. And compared with the pathological representation that the Abeta deposition starts to appear at the age of 2 months, the behavioral representation is that the Abeta deposition starts to appear at the age of 3 months, and accords with the clinical pathogenesis of Alzheimer's disease.

In conclusion, the 3-FAD mouse can effectively simulate a mouse model of a human Alzheimer disease patient from clinical diagnosis markers, pathology to behavior change, and has important significance for deep exploration of pathogenesis of Alzheimer disease, discovery of new therapeutic targets and promotion of drug development.

EXAMPLE 5 drug screening Using 3-FAD mouse model

3-FAD mice were treated with drug Lecanemab, specifically, 4 3-FAD mice of 1 month old were given by intraperitoneal injection at a dose of 12 mpk per dose, once a week for 4 weeks, and the dose was recorded as experimental group (3-FAD+ Lecanemab). Two control groups were simultaneously set, one control group being 3 non-dosed 6 month old C57BL/6J mice (WT) and two control groups being 4 non-dosed 3-FAD mice (3-FAD). The results of measuring the amounts of p-Tau181 and p-Tau217 in the blood of the mice after 4 weeks are shown in FIG. 12, wherein (a) and (b) represent the results of measuring the amounts of p-Tau181 and p-Tau217 in the blood, respectively. As can be seen from FIG. 12, the contents of p-Tau181 and p-Tau217 in the blood of 3-FAD mice were significantly increased compared to C57BL/6J mice, but the contents of p-Tau181 and p-Tau217 in 3-FAD mice (3-FAD+ Lecanemab) after administration were decreased compared to 3-FAD mice (3-FAD) without administration, and the trend of the decrease of both blood markers was consistent with that of clinical AD patients, and was more evident compared to p-Tau181 and p-Tau217, and also consistent with that of clinical treatment patients detected by Lecanemab treatment.

The application also detects brain sections of a control group and an experimental group after 4 weeks of administration, and obtains IHC results shown in figure 13, compared with C57BL/6J mice, the 3-FAD mice (3-FAD) without administration have obvious Abeta deposition, but the 3-FAD mice (3-FAD+ Lecanemab) after administration have slow Abeta deposition, and plaque areas are smaller, and the detection results are consistent with those of clinical treatment patients after Lecanemab treatment, so that the 3-FAD mice of the application can be used for drug screening of Alzheimer's disease.

While the basic concepts have been described above, it will be apparent to those skilled in the art that the foregoing detailed disclosure is by way of example only and is not intended to be limiting. Although not explicitly described herein, various modifications, improvements, and adaptations to the present disclosure may occur to one skilled in the art. Such modifications, improvements, and modifications are intended to be suggested within this specification, and therefore, such modifications, improvements, and modifications are intended to be included within the spirit and scope of the exemplary embodiments of the present invention.

Meanwhile, the specification uses specific words to describe the embodiments of the specification. Reference to "one embodiment," "an embodiment," and/or "some embodiments" means that a particular feature, structure, or characteristic is associated with at least one embodiment of the present description. Thus, it should be emphasized and should be appreciated that two or more references to "an embodiment" or "one embodiment" or "an alternative embodiment" in various positions in this specification are not necessarily referring to the same embodiment. Furthermore, certain features, structures, or characteristics of one or more embodiments of the present description may be combined as suitable.

Likewise, it should be noted that in order to simplify the presentation disclosed in this specification and thereby aid in understanding one or more inventive embodiments, various features are sometimes grouped together in a single embodiment, figure, or description thereof. This method of disclosure does not imply that the subject matter of the present description requires more features than are set forth in the claims. Indeed, less than all of the features of a single embodiment disclosed above.

In some embodiments, numbers describing the components, number of attributes are used, it being understood that such numbers being used in the description of embodiments are modified in some examples by the modifier "about," approximately, "or" substantially. Unless otherwise indicated, "about," "approximately," or "substantially" indicate that the number allows for a 20% variation. Accordingly, in some embodiments, numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and employ a method for preserving the general number of digits. Although the numerical ranges and parameters set forth herein are approximations that may be employed in some embodiments to confirm the breadth of the range, in particular embodiments, the setting of such numerical values is as precise as possible.

Each patent, patent application publication, and other material, such as articles, books, specifications, publications, documents, etc., referred to in this specification is incorporated herein by reference in its entirety. Except for application history documents that are inconsistent or conflicting with the content of this specification, documents that are currently or later attached to this specification in which the broadest scope of the claims to this specification is limited are also. It is noted that, if the description, definition, and/or use of a term in an attached material in this specification does not conform to or conflict with what is described in this specification, the description, definition, and/or use of the term in this specification controls.

Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments of this specification. Other variations are possible within the scope of this description. Thus, by way of example, and not limitation, alternative configurations of embodiments of the present specification may be considered as consistent with the teachings of the present specification. Accordingly, the embodiments of the present specification are not limited to only the embodiments explicitly described and depicted in the present specification.

Claims

1. A triple-mutant Alzheimer's disease mouse model for use in studying the pathological mechanism of Alzheimer's disease or screening drugs for treating Alzheimer's disease, characterized in that the genome of the mouse model includes a transgenic fragment of a first exogenous gene hAPP, a transgenic fragment of a second exogenous gene hPSEN1, and a transgenic fragment of a third exogenous gene hMAPT;

Among them, the transgenic fragment of the first exogenous gene hAPP carries the Swedish mutation; the transgenic fragment of the second exogenous gene hPSEN1 carries the hPSEN1 M146V mutation; the transgenic fragment of the third exogenous gene hMAPT carries the hMAPT P243L mutation;

The CDS sequence of the transgenic fragment of the first exogenous gene hAPP is shown in SEQ ID NO: 1, the CDS sequence of the transgenic fragment of the second exogenous gene hPSEN1 is shown in SEQ ID NO: 3, and the CDS sequence of the transgenic fragment of the third exogenous gene hMAPT is shown in SEQ ID NO: 5;

The transgenic fragment of the first exogenous gene hAPP, the transgenic fragment of the second exogenous gene hPSEN1 and the transgenic fragment of the third exogenous gene hMAPT are inserted into the genome of a mouse fertilized egg; the fertilized egg is transplanted into a pseudo-pregnant female mouse, and F0 generation mice in which all three exogenous genes are positive are screened and obtained; the F0 generation mice are backcrossed with wild mice until F0N5 generation mice are obtained; and the Alzheimer's disease mouse model 3-FAD homozygous positive mice are obtained by selfing the F0N5 generation mice.

2. A method for constructing a triple-mutant Alzheimer's disease mouse model, the method comprising:

(1) preparing a transgenic fragment of the first exogenous gene hAPP, a transgenic fragment of the second exogenous gene hPSEN1, and a transgenic fragment of the third exogenous gene hMAPT;

(2) inserting the three transgenic fragments into the genome of mouse fertilized eggs;

(3) transplanting the fertilized eggs into pseudo-pregnant female mice, and screening to obtain F0 generation mice in which all three exogenous genes were positive;

(4) Based on the F0 generation mice, obtaining 3-FAD homozygous positive mice of the Alzheimer's disease mouse model;

The step (4) comprises backcrossing the F0 generation mice with wild mice until F0N5 generation mice are obtained; and self-crossing the F0N5 generation mice to obtain the Alzheimer's disease mouse model 3-FAD homozygous positive mice.

3. The construction method according to claim 2, characterized in that the preparation of the transgenic fragment in step (1) comprises:

a. Obtaining PCR products of the Thy1 promoter and the first exogenous gene hAPP, the second exogenous gene hPSEN1 and the third exogenous gene hMAPT;

b. connecting the Thy1 promoter to the PCR products of the first exogenous gene hAPP, the second exogenous gene hPSEN1 and the third exogenous gene hMAPT and the backbone vector to form a recombinant vector, and transforming the recombinant vector into competent cells;

c. After transformation, PCR identification and sequencing are performed, and the clones with correct sequencing are selected as transgenic vectors;

d. preparing the transgenic fragment based on the transgenic vector.

4. The construction method according to claim 2, characterized in that step (2) comprises:

The transgenic fragment is microinjected into the fertilized egg of the mouse and randomly inserted into the genome of the fertilized egg of the mouse.

5. The construction method according to claim 2, characterized in that the identification in step (3) is performed by a PCR reaction, and the primer pairs used in the PCR reaction are respectively as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9; SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13; SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17.