KR100462180B1 - Transgenic animal with genes relating to Alzheimer's disease and the method of producing thereof - Google Patents

Abstract

The present invention relates to an animal transformed with an Alzheimer's disease etiology gene and a method of manufacturing the same, and more specifically, to an expression vector capable of simultaneously expressing an APP (amyloid precursor protein) and a PS1 (presenilin 1) mutant gene, Aβ ( Beta amyloid) expression vectors and animals transformed with expression vectors capable of simultaneously expressing APP, PS1 and beta cyclase (BACE) genes, and expression cassettes, recombinant plasmids, recombinant expression vectors for making such animal models. And the production method thereof.

Description

Transgenic animal with genes relating to Alzheimer's disease and the method of producing

The present invention relates to an animal transformed with an Alzheimer's disease etiology gene and a method of manufacturing the same, and more specifically, to an expression vector capable of simultaneously expressing an APP (amyloid precursor protein) and a PS1 (presenilin 1) mutant gene, Aβ ( Beta amyloid) expression vectors and animals transformed with expression vectors capable of simultaneously expressing APP, PS1 and beta cyclase (BACE) genes, and expression cassettes, recombinant plasmids, recombinant expression vectors for making such animal models. And the production method thereof.

Alzheimer's disease accounts for 50 to 70% of dementia resulting from gradual neuronal degeneration, resulting in familial Alzheimer's disease caused by genetic factors and sporadic incidence in a large number of patients. It is divided into Alzheimer's disease. Senile plaques and neurofibrillary tangles appear as pathological features in the brains of patients with Alzheimer's disease.

Among them, the senile plaque is formed by accumulation of proteins and dead cells on the outside of the cell. The main component is a peptide called β-amyloid (Aβ) composed of 42 or 43 amino acids. The cytoskeleton clumps abnormally and looks like a bundle of threads. Overly phosphorylated tau protein acts as the nucleus.

β-amyloid is produced through a proteolysis process from amyloid precursor protein (APP). The proprotein, APP, is a protein with a single transmembrane domain, expressed by several isotypes by alternating splicing, and known to go through two metabolic pathways in cells. One is the pathway in which p3 and sAPPα are produced by α-secretase and γ-secretase, and the other is the pathway in which β-amyloid and APPβ are produced by β-secretase and γ-secretase. Mutations have been found in this APP protein in patients with familial Alzheimer's disease. Mutations discovered so far include APP670 / 671 (Swedish), APP672 (Flemish), APP716 (Florida), and APP717 (London), and these mutations have been found to increase Aβ formation.

Another gene that represents mutations that cause familial Alzheimer's disease is presenilin 1 (PS1). PS1 is a protein with eight transmembrane domains and plays an important role in the development process. Recently, the possibility of γ-secretase itself or a major factor regulating it has been studied. More than 45 mutations that cause familial Alzheimer's disease have been reported throughout PS1, and these mutations have also been shown to increase A [beta] formation.

One of the major factors known recently for Alzheimer's disease is BACE (β-site APP cleaving enzyme), which produces β-amyloid by cleaving the β site of APP. This protease has long been known for its existence and its role in APP metabolism, but only recently (1999) has been reported by protein purification and gene cloning. Aspartyl protease (Aspartyl protease) as a kind of active conditions are known a lot, but the direct association with Alzheimer's disease is still under study. However, increased activity of the enzyme is expected to cause pathological lesions by increasing the amount of β-amyloid, an important factor in Alzheimer's disease, and related facts have been reported.

Therefore, it is important to identify the mechanism by which BACE, along with the mutations in APP and PS1, induce Alzheimer's disease.

In recent decades, several studies have attempted to establish transgenic mice for the study of the pathogenesis of Alzheimer's disease. Transgenic genes that were the main targets of these trials included genes such as tau and ApoE4, in addition to APP and PS1, which have been identified as the causative genes in familial Alzheimer's disease. However, the transgenic mice currently developed and used in the study often expressed only one gene of APP or PS1, which made it difficult to study related mechanisms between these two genes, which are considered to be the main cause genes, and also have characteristics of Alzheimer's disease. Amyloid spots that are thought to be formed only after 9-12 months have elapsed. In 1996, the Duff group successfully developed a double transgenic mouse obtained by crossing a single transgenic mouse with APP and PS1. In fact, in the case of double transgenic mice, the synergistic effect of the two genes (Synergistic Effect) can be seen to advance the spot formation period from 3 to 9 months. However, since most of these mice are obtained by crossing each transgenic mouse of APP and PS1, expression of each gene is regulated independently from each promoter, and the promoters used are not specifically expressed in neurons. There have been many difficulties in studying the pathogenesis of Alzheimer's disease in the nervous system.

In order to improve this problem, the present invention is transformed by injecting an expression vector designed to simultaneously express the mutant gene of amyloid proprotein (APP) and presenilin 1 (PS1) and BACE known as the Alzheimer's disease pathogen gene It is an object of the present invention to provide an animal and a method for producing the same.

Another object of the present invention is to provide a transgenic animal in which the genes of amyloid proprotein (APP) and presenilin 1 (PS1) can be regulated under one promoter, and a method of preparing the same.

In another aspect, the present invention is to provide a transformed animal and a method for producing the same by injecting the Αβ expression vector known as the final product of the Alzheimer's disease etiology genes.

In addition, the present invention includes an expression cassette and the expression cassette, characterized in that it comprises an APP, PS1 mutant gene and a promoter specific for neurons capable of regulating the two mutant genes at once to prepare the transformed animal. Another object is to provide a recombinant expression vector.

In this case, the promoter used is preferably NSE (Neuron Specific Enolase), an internal ribosomal entry site between two genes so that the APP mutant gene and the PS1 mutant gene can be simultaneously expressed. Is preferably inserted.

The present invention also provides a recombinant plasmid in which Aβ ₄₂ (amyloid-beta 42), an APP mutant gene, a PS1 mutant gene, and a beta secretase gene (BACE) are inserted, and a recombinant expression vector prepared by combining these plasmids. Another purpose.

The ultimate object of the present invention is to express the APP mutant gene and the PS1 mutant gene under the control of one promoter and transform the animal, and the APP mutant and PS1 mutant protein and BACE protein can be specifically expressed in neurons at the same time. By providing a transgenic animal prepared by injecting a recombinant expression vector and an animal model transformed with an Aβ expression vector, it contributes to the study of the pathogenesis of Alzheimer's disease.

1 shows the simultaneous expression cassette of APP and PS1 mutant genes.

Figure 2 is a restriction digestion photograph of the NSE-APP-IRES-PS1 vector.

3 shows a vector comprising Aβ and an NSE promoter.

Figure 4 shows the cloning process of NSE-pcDNA3.0.

5 shows the cloning procedure of NSE intron 1-pcDNA3.0.

6 shows a clone of NSE1-APPswe-pcDNA3.0.

Figure 7 shows the cloning process of NSE1-PS1 M146L / L166P mutant-pcDNA3.0.

8 shows the cloning procedure of NSEI-BACE-pcDNA 3.1.

9 is an electrophoretic picture of an insert for microinjection into mice.

Lane 1: DNA Ladder (Intron)

Lane 2: NSEI-APPswe Sall-BssHII fragment

Lane 3: NSEI-PS1ML / LP Sall-BssHII fragment

Lane 4: NSEI-BACE Sall-BssHII Fragment

Figure 10 is an electrophoretic picture showing the genotype of the transformed mouse.

+,-: Control group, M: size marker

In order to achieve the above object, the present invention comprises an expression cassette, characterized in that it is possible to regulate the mutant genes of amyloid proprotein (APP) and presenilin 1 (PS1) at a time with one promoter specific for neurons To provide a recombinant expression vector. At this time, the promoter is preferably neuron specific enolase (NSE; Neuron Specific Enolase). The sequence of the NSE promoter was disclosed by Forss-Petter et al. (Transgenic mice expressing β-galactosidase in mature neurons under neuron-specific enolase promoter control, Neuron 5, 187-197 (1990)), the sequence of the promoter used in the present invention. This sequence is set forth in SEQ ID NO: 11 in the Sequence Listing.

In another aspect, the present invention provides an expression cassette characterized in that an internal ribosomal entry site (IRES) is inserted between the APP mutant gene and the PS1 mutant gene so as to simultaneously express the two genes.

The APP mutant gene is used a gene in which amino acids 670 and 671 of APP 695 are mutated, and the PS1 mutant gene is used as a gene in which amino acid 146 is mutated.

In another aspect, the present invention provides a recombinant expression vector comprising Aβ ₄₂ (amyloid-beta 42), characterized in that the NSE as a promoter to prepare another Alzheimer's disease-related transformed animal.

The present invention also provides a method for preparing a vector comprising an NSE promoter and intron 1, in order to prepare a recombinant expression vector capable of simultaneously expressing an APP mutant gene, a PS1 mutant gene, and a BACE protein in neurons. The sequence of intron 1 was disclosed by Sakimura, K. et al. (Upstream and intron regulatory regions for expression of the rat neuron-specific enolase gene, Mol. Brain Res. 28, 19-28 (1995)) Set forth in SEQ ID NO: 12 of this Sequence Listing.

The present invention also provides a method for producing a recombinant plasmid in which an APP mutant is inserted and a recombinant plasmid in which a PS1 mutant gene is inserted into a vector including the NSE promoter and intron 1 prepared above. In this case, a double mutant gene in which amino acids 146 and 166 are mutated is used as the PS1 mutant gene. The present invention also provides a recombinant plasmid containing an NSE promoter and intron 1, into which a BACE gene is inserted. The amino acid sequence of BACE was disclosed by Vassar, R. et al. (Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE, Science 286 (5440), 735-741 (1999)) The amino acid sequence is described in No. 13.

Each of these recombinant plasmids is combined to provide a recombinant expression vector capable of simultaneously expressing an APP mutant gene, a PS1 mutant gene, and a BACE protein in neurons.

In addition, by using the three prepared plasmid prepared by insert (insert) for injection into the animal, and injected into the fertilized egg of the animal provides a method for producing a transformed animal model. At this time, the animal is preferably a mammal, and more preferably a mouse. More specifically, the present invention cloned the Swedish type mutant gene of APP, the M146L, L166P double mutant gene of PS-1, the cDNA of BACE to infiltrate transgenes into animals, and human APP for Aβ transformation. A method of cloning the Αβ moiety from cDNA by PCR was used. In addition, these constructs were injected into the embryos of animals to be transformed to produce new animal models.

The fertilized egg of the mouse transformed with the Alzheimer's disease etiology gene of the present invention prepared as described above was deposited at the Korea Biotechnology Research Institute Gene Bank of Korea, an international microbial deposit institution, on October 4, 2001, and the accession number is KCTC 10085BP .

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are described for the purpose of illustrating the present invention, but the scope of the present invention is not limited thereto.

Example 1 Preparation of Expression Cassette

A neuron-specific promoter known to be expressed in fully differentiated neurons to make an expression cassette capable of regulating the mutant genes of APP and PS1 at one time with a neuron-specific promoter Was used. As the APP gene, Swedish type cDNA, which is mutated from amino acids 670 and 671 of the isotype APP 695, which is mainly expressed in the brain, was used. The PS1 gene was cDNA mutated to amino acid 146. In order to allow these two genes to be regulated by a single promoter, an internal ribosomal entry site (IRS) was inserted between them to allow biscistronic, that is, both to be expressed simultaneously (see FIG. 1).

The expression cassette was cloned from two genes, cDNA and NSE promoter, based on the pIRESneo vector (Clontech). This was digested with various restriction enzymes to determine cloning (see FIG. 2) and confirmed through sequencing.

Example 2 Cloning of an Αβ Transgene

In order to make an animal model that is particularly toxic among amyloid beta, which is thought to be the cause of Alzheimer's disease, and overexpresses Aβ1-42, which is found to be a major component of senile plaques, the NSE promoter was subjected to PCR by analyzing the Aβ portion from human APP cDNA. It was cloned into the containing vector (refer FIG. 3).

Two-piece DNA obtained from two PCR reactions from human APP cDNA to include a signal sequence and start codons for positioning Aβ1-42 in the cell membrane in the cDNA portion of Aβ1-42 After ligation, it was ligation back to the mammalian protein expression vector. The sequence of the specific primer used at this time is as follows.

Aβ1-42-5 ': 5'-AGA AGC TTG GTA CCA CGC CTA TCG AT-3 '(SEQ ID NO: 1)

Aβ1-42-3 ': 5'- AGT CTA GAC TAC GC ATG ACA ACA CCG CC -3' (SEQ ID NO: 2)

The 5 'primer contains the HindIII restriction enzyme sequence for ligation, and the 3' primer contains the Xba I recognition sequence and the stop codon.

Example 3 Gene Expression Vector of Alzheimer's Disease Pathogenesis for Co-injection

1) NSE-pcDNA3.0 / pcDNA3.1 Cloning Step

Vectors were constructed that include promoters of neuronal specific enolase (NSE) known to specifically express in differentiated neurons. For this purpose, the CMV promoters of well-known mammalian expression vectors pcDNA3.0 and pcDNA3.1 / myc-his A (invitrogen) were replaced with NSE promoters.

To this end, the NSE promoter site was cut from the pNSECAT containing the rat NSE promoter mentioned by Forss-Petter et al. (Neuron, 1990) with restriction enzymes EcoRI and HindIII, transferred to a multi-cloning site, and cut back into EcoRV and HindIII. A fragment having a blunt end at the 5 'side was obtained. The CMV promoter regions of pcDNA 3.0 and pcDNA 3.1 vectors were cut out with NruI and HindIII, ligation of the NSE fragment cut with EcoRV and HindIII at this location, and transformed into DH5α to obtain a clone. This process is summarized in FIG.

2) NSE Intron 1-pcDNA3.0 / pcDNA3.1 Step

Intron via PCR from genomic DNA extracted from rat brain to clone the intron 1 site of NSE, which is known to play an important role in neurofibrillary specific enolase expression specifically in neurons 1 was obtained.

A sequence recognized by XhoI and HindIII was attached to the ends of the primers complementarily binding to the end of exon 1 and the beginning of exon 2 so that a restriction enzyme recognition site was present at the end of the amplified DNA fragment.

The sequence of each primer is as follows.

NSEI-F: 5'-TTC CCT CGA GTT GGC TGG ACA-3 '(SEQ ID NO: 3)

NSEI-R: 5'-ACA AAG CTT ATG GCT GGG ATC TCC TTG G-3 '(SEQ ID NO: 4)

The genomic DNA of rats to be used as a template for PCR reaction was extracted from the rat brain by genomic DNA extraction (Molecular cloning, 1984), and 1 μg template DNA and 1 unit Ex-Taq (TAKARA), 600 μM After addition of dNTP, each 200 nM primer, the synthesis was performed for 35 cycles with an appropriate amount of attachment buffer at 95 ° C. for 30 seconds, annealing at 53 ° C. for 1 minute, and 72 ° C. for 1 minute and 30 seconds. The synthesized product was electrophoresed on a 1% agarose gel, extracted from the gel, cut with XhoI and HindIII, and substituted with the NSE promoter extracted from the clone obtained in step 1 above, and replaced with xhoI, HindIII, pcDNA3.0 and pcDNA3.1. DNA fragments cut into This was also transformed into DH5α to obtain pcDNA 3.0, pcDNA3.1 vector including the NSE promoter and intron 1. This was called NSEI-pcDNA3.0 and NSEI-pcDNA3.1, respectively. This process is summarized in FIG.

3) NSEI-APPswe-pcDNA3.0 cloning step

2.2 kb of Swedish amyloid cDNA of human APP695, an isotype that is specifically expressed in the central nervous system of amyloid precursor protein, was digested with BamHI and XbaI restriction enzymes from pCB6 mammalian expression vector (Amy CY et al., JBC, 1994) DNA fragments were obtained. The fragment was linked to the positions of BamHI, XbaI of NSEI-pcDNA3.0 obtained from 1 and 2 above, and then transformed into DH5α to obtain a clone (see FIG. 6).

4) NSEI-PS-1 M146L / L166P Mutant-pcDNA3.0 Cloning Step

PCR was used to detect L166P, a novel mutation recently known to significantly increase amyloid-β production from M146L mutant cDNA, one of the mutations responsible for familial Alzheimer's disease of human presenilin-1 (PS-1). Site-directed mutations.

First, the NSEI-PS1ML-pcDNA3.0 plasmid was formed by connecting the two DNA fragments by cutting the NSEI-pcDNA3.0 vector prepared in Nos. 1 and 2 with BamHI and XbaI and the pIND vector containing the PS1M146L mutant. Got it.

In order to convert amino acid 166 of PS1M146L cDNA of this plasmid from leucine to proline, PCR was performed using primers in which TTA encoding leucine was substituted with one base with CTA encoding proline. Double mutants were made by PCR again the product.

The primer sequences used are as shown in the sequence listing herein.

PS1LP-1 5'-CTC GGA TCC ACT AGT CCA GT-3 '(SEQ ID NO: 5)

PS1LP-2 5'-CAT GCC TGG CCT ATT ATA TCA-3 '(SEQ ID NO: 6)

PS1LP-3 5'-TGA TAT AAT AGG CCA GGC ATG-3 '(SEQ ID NO: 7)

PS1LP-4 5'-GAG GGA CAG TCA TCT AGG GCC-3 '(SEQ ID NO: 8)

First, the results were obtained using the primers SEQ ID NO: 5, 6 and 7, 8, and then PCR with the resultant and primers 5 and 8 to obtain a 1.3kb DNA fragment. This was cut with restriction enzymes XhoI and BsrGI, and NSEI-pcDNA3.0 containing PS1 M146L cDNA was linked to the fragment cut with the same restriction enzyme to obtain a vector containing a double mutant PS1 gene. This process is summarized in FIG.

5) NSEI-BACE-pcDNA 3.1 Cloning Step

During the metabolism of APP, cDNA of human BACE known as β-secretase, which plays an important role in the formation of amyloid-β, was obtained by RT-PCR from human cell line SY5Y and produced in 1 and 2 times. Cloned into NSEI-pcDNA3.1.

Whole RNA was isolated from guandium thiocyanate from SY5Y, a human neuroblastoma cell line, and reverse transcription was carried out using M-MLV protranscriptase and oligo dT primers. Was performed. The cDNA thus produced was PCR with a specific primer capable of amplifying the entire cDNA of BACE as a template. The sequence of primers used is as follows.

BACE-5: 5'-GGC GAA TTC GTG CCG ATG TAG CGG GCT CCG-3 '(SEQ ID NO: 9)

BACE-3: 5'-GGC CTC GAG CTG GAA CCC ACC TTG CCA GCC-3 '(SEQ ID NO: 10)

At the ends of each primer, EcoRI and XhoI recognized nucleotide sequences, respectively, and the resulting PCR product was digested with EcoRI and XhoI and cloned into NSEI-pcDNA3.1 / myc-his A.

This vector was used to enable labeling of myc and his proteins at the carboxyl terminus of BACE. This process is summarized in Figure 5.

6) Insert preparation step for microinjection

1% agarose gel after cleavage using Sal I and BssH II among the enzymes present in the vector backbone to make linear DNA from which plasmid DNA made in steps 3, 4, and 5 was removed as much as possible. Was electrophoresed (see FIG. 9) to elute only the insert site. The isolated insert DNA was extracted two or more times with a phenol / chloroform solvent, and then purified by passing through an elutip-D column (Schleicher & Schuell, Germany), and finally, 10 mM Tris-Cl (pH 8.0). After dialysis under a /0.1 mM EDTA solution, the concentrations were adjusted so that each insert DNA was 10 ng / μl in the same solution, and they were mixed in equal amounts to inject three insert DNAs together into mouse embryos.

7) microinjection step

a) Preparation of mouse fertilized eggs

To induce polyovulation, 5 IU of PMSG (Sigma, USA) was intraperitoneally injected into female mice of C57BL / 6 strain, and 48 hours later, hCG (Sigma, USA) was injected in the same manner, followed by SJL male mice. And induce ovulation and fertilization. The next day, the presence of the vagina was checked, and only individuals with the vagina were selected and sacrificed by cervical rupture. The fallopian tubes were extracted to collect the cumulus mass surrounding the fertilized egg. The recovered cumulus mass was treated with 300 unit / ml hyaluronidase (Sigma, USA) solution for 2-3 minutes to remove cumulus cells. Subsequently, centrifugation was performed at 15,000 rpm for 3 minutes to move the pigment granules in the cytoplasm so that the nuclear membrane was easily observed, and then transferred to a culture solution (M16 + 0.4% BSA) previously prepared in a 37 ° C. CO2 incubator and temporarily cultured.

b) Microinjection of insert DNA

DNA purified using a micromanipulator (Leica, Germany) was injected into the nucleus of the mouse fertilized egg. Observing the fertilized egg under an inverted microscope equipped with a differential interference contrast (DIC) system, DNA was injected into the nucleus with a microinjector to confirm that the nuclear membrane expanded.

c) introduction of fertilized eggs into surrogate mother mice

Embryos injected with DNA were incubated for 1 to 2 hours in a CO 2 incubator to select only those embryos with good condition. About 30 fertilized eggs per surrogate mother were introduced into the oviduct of a surrogate mother of ICR-type surrogate mice that induced fertility status by males undergoing vasculature ligation.

d) Identification of transgenic (gene transplant) mice

To determine the transgenic mice in the born offspring, at 3 to 4 weeks of age, a product was removed from the ear and digested with the DNA, and the insertion of the inserted DNA was confirmed by PCR. In order to decompose the tissue, the detached tissue was dissolved in 20 µl of lysate (50 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM EDTA.1% SDS solution in a 20 mg / ml proteinase K ratio of 1:19). Furnace) was decomposed for 2 hours in a 55 ℃ thermostat. After confirming that the tissue was dissolved, 180 μl of water was added and heat was added for 5 minutes in boiling water to inactivate proteinase. PCR was performed using 1 μl of this solution. PCR was repeated (or simultaneously) using each of the primers used to construct each vector to identify transgenic mice with each insert introduced.

e) genotyping to confirm exogenous insertion of the injected mouse

Mice obtained by injecting the DNA into embryos obtained from B6 / SJL hybrid mice were selected by PCR with primers specific thereto (see FIG. 10).

The pathogenesis of APP and PS1, which are known as the leading cause of Alzheimer's disease, causes Alzheimer's disease is still unclear. However, various studies have shown that APP and PS1 contribute to the formation of spots by excess accumulation of Aβ. Previous studies have shown that mutant APP acts better as a substrate for β- and γ-secretase, and PS1 acts as an important regulator for the action of key factor γ-secretase. Or the possibility of acting as γ-secretase itself is emerging.

Therefore, it is possible to assume that the action of both APP and PS1 genes is important for the natural development of Alzheimer's disease, and that animal models for the study of this mechanism should be made to examine the action of both genes. Can be. The animal model created in this study allows these two genes to be generated at the same time, allowing them to examine the actions of both genes at the same time, and in particular by specifically expressing them in neurons, the nervous system that is a problem in Alzheimer's disease. It seems to be suitable for the study of Alzheimer's disease etiology in that it can focus only on changes in Essence.

In addition, the present invention is expected to be a good model for the study of the action as β-secretase, which has not yet been well-defined in Alzheimer's disease due to the introduction of the recently cloned BACE. Since the gene is configured to maximize the production of β-amyloid, it is possible to establish a model in which the onset time and progression are advanced by increasing its production amount. It is expected to be expressed in the cell and to observe the actual onset of Alzheimer's disease.

These animal models compensate for problems that may arise in animal models that overexpress one gene currently used in several studies, models obtained by crossing two lines, or models using non-neuronal cell specific promoters. This will make it easier and faster to interpret the findings. This will be of great help in the study of the pathogenesis of Alzheimer's disease, which is not yet clearly defined.

In addition to basic research, such as the pathogenesis of Alzheimer's disease, it may be possible to use it as a better laboratory animal to test the efficacy of new drugs.

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acctggggga gctggccaaa ctgcgggacc tgcccaagct gagggccttg gtgctgctgg 660 acaacccctg tgccgatgag actgactacc gccaggaggc cctggtgca g atggcacacc 720 tagagcgcct agacaaagag tactatgagg acgaggaccg ggcagaagct gaggagatcc 780 gacagaggct gaaggaggaa caggagcaag aactcgaccc ggaccaagac atggaaccgt 840 acctcccgcc aacttagtgg ctcctctagc ctgcagggac agtaaaggtg atggcaggaa 900 ggcagccccc ggaggcaaag gctgggcacg cgggaggaga ggccagagtc agaggctgcg 960 ggtatctcag atatgaagga aagatgagag aggctcagga agaggtaaga aaagacacaa 1020 gagaccagag aagggagaag aattagagag ggaggcagag gaccgctgtc tctacagaca 1080 tagctggtag agactgggag gaagggatga accctgagcg catgaaggga aggaggtggc 1140 tggtggtata tggaggatgt agctggggcc agggaaaaga tcctgcactg gggatctgaa 1200 gctggggaga acaggacacg gggtggagag gcgaaaggag ggcagagtga agcagagaga 1260 ctgaggcctg gggatgtggg cattccggta gggcacacag ttcacttgtc ttctcttttt 1320 ccaggaggcc aaagatgctg acgtcaagaa ctcataatac cccagtgggg accaccgcat 1380 tcatagccct gttacaagaa gtgggagatg ttcctttttg tcccagactg gaaatccatt 1440 acatcccgag gctcaggttc tgtggtggtc atctctgtgt ggcttgtt ct gtgggcctac 1500 ctaaagtcct aagcacagct ctcaagcaga tccgaggcga ctaagatgct agtaggggtt 1560 gtctggagag aagagccgag gaggtgggct gtgatggatc agttcagctt tcaaataaaa 1620 aggcgttttt atattctgtg tcgagttcgt gaacccctgt ggtgggcttc tccatctgtc 1680 tgggttagta cctgccacta tactggaata aggagacgcc tgcttccctc gag 1733 <210> 12 <211> 513 <212> DNA <213> Artificial Sequence <220 > <223> INTRON <400> 12 gtgaggcccg tatcgggccg catcccctta ctctgtcctt gccgcgtcca ctccccgtta 60 tcctgggctt agcgcccccg ggtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 120 tgtgtgtgtg tgtgtgtgtg tgtgtgacgc tgcgtacctg tcacctgcct tccctggcac 180 cctccatccc cggccttcgc ggacagctcc cggctttctg gctgtcacct cctcctggtt 240 taggtaccag cttttctctc tctctctctc tctctctctc tctctctctc tctctctctc 300 tctctctctc cctgcacccg cttctctaac tccacggttc caccctcgcc tgcccctctc 360 cccatgcctg tattcgtcca gggtccggcc gatttgtttc ttaagttcga tgtctccgca 420 ggcggcgcgt gttttcctgg cacgt tcatt tttgctttgc gtttgcgaag ataccctctc 480 atccttcctt ggctgcttca gacttgctct aga 513 <210> 13 <211> 501 <212> PRT <213> Artificial Sequence <220> <223> BACE <400> 13 Met Ala Gln Ala Leu Pro Trp Leu Leu Leu Trp Met Gly Ala Gly Val 1 5 10 15 Leu Pro Ala His Gly Thr Gln His Gly Ile Arg Leu Pro Leu Arg Ser 20 25 30 Gly Leu Gly Gly Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp 35 40 45 Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val 50 55 60 Asp Asn Leu Arg Gly Lys Ser Gly Gln Gly Tyr Tyr Val Glu Met Thr 65 70 75 80 Val Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val Asp Thr Gly Ser 85 90 95 Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr 100 105 110 Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val 115 120 125 Tyr Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp 130 135 140 Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg Ala Asn Ile 145 150 155 160 Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 165 170 175 Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Pro Asp Asp 180 185 190 Ser Leu Glu Pro Phe Phe Asp Ser Leu Val Lys Gln Thr His Val Pro 195 200 205 Asn Leu Phe Ser Leu Gln Leu Cys Gly Ala Gly Phe Pro Leu Asn Gln 210 215 220 Ser Glu Val Leu Ala Ser Val Gly Gly Ser Met Ile Ile Gly Gly Ile 225 230 235 240 Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro Ile Arg Arg 245 250 255 Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu Ile Asn Gly Gln 260 265 270 Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp Lys Ser Ile Val 275 280 285 Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys Lys Val Phe Glu Ala 290 295 300 Ala Val Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu Lys Phe Pro Asp 305 310 315 320 Gly Phe Trp Leu Gly Glu Gln Leu Val Cys Trp Gln Ala Gly Thr Thr 325 330 335 Pro Trp Asn Ile Phe Pro Val Ile Ser Leu Tyr Leu Met Gly Glu Val 340 345 350 Thr Asn Gln Ser Phe Arg Ile Thr Ile Leu Pro Gln Gln Tyr Leu Arg 355 360 365 Pro Val Glu Asp Val Ala Thr Ser Gln Asp Asp Cys Tyr Lys Phe Ala 370 375 380 Ile Ser Gln Ser Ser Thr Gly Thr Val Met Gly Ala Val Ile Met Glu 385 390 395 400 Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala 405 410 415 Val Ser Ala Cys His Val His Asp Glu Phe Arg Thr Ala Ala Val Glu 420 425 430 Gly Pro Phe Val Thr Leu Asp Met Glu Asp Cys Gly Tyr Asn Ile Pro 435 440 445 Gln Thr Asp Glu Ser Thr Leu Met Thr Ile Ala Tyr Val Met Ala Ala 450 455 460 Ile Cys Ala Leu Phe Met Leu Pro Leu Cys Leu Met Val Cys Gln Trp 465 470 475 480 Arg Cys Leu Arg Cys Leu Arg Gln Gln His Asp Asp Phe Ala Asp Asp 485 490 495 Ile Ser Leu Leu Lys 500

Claims (21)

a) an APP mutant gene encoding amyloid proprotein (APP) mutated amino acids 670 and 671, b) a PS1 mutant gene encoding presenilin 1 (PS1) mutated amino acids 146, c) both A neuronal specific enolase (NSE) promoter having a sequence of SEQ ID NO: 11 capable of regulating a mutant gene at once, and d) an internal ribosomal entry site (IRS) inserted between the APP mutant and PS1 mutant genes And an expression cassette for producing an animal transformed with an Alzheimer's disease etiology gene, having a cleavage map of FIG. 1. delete delete delete delete The method of claim 1, The PS1 mutant gene is an expression cassette, characterized in that the double mutant gene encoding a double mutant PS1 is further mutated amino acid 166. A recombinant expression vector comprising the expression cassette of claim 1. delete a) an NSE promoter having the sequence of SEQ ID NO: 11, b) an Intron 1 comprising the sequence of SEQ ID NO: 12, and c) an APP mutant gene encoding an amyloid proprotein (APP) mutated by amino acids 670 and 671; Recombinant plasmid for producing an animal transformed with Alzheimer's disease etiology gene, comprising the cleavage map of FIG. 6. a) an NSE promoter having the sequence of SEQ ID NO: 11, b) an Intron 1 comprising the sequence of SEQ ID NO: 12, and c) a PS1 mutant gene encoding PS1 wherein amino acids 146 and 166 are mutated and A recombinant plasmid for producing an animal transformed with an Alzheimer's disease etiology gene with a cleavage map. delete a) an NSE promoter having the sequence of SEQ ID NO: 11, b) an Intron 1 comprising the sequence of SEQ ID NO: 12, and c) a gene encoding BACE (β-site APP cleaving enzyme) having the amino acid sequence of SEQ ID NO: 13 Recombinant plasmid for producing an animal transformed with Alzheimer's disease etiology gene, comprising the cleavage map of FIG. 8. delete delete delete delete Constructing a vector comprising an NSE promoter and an NSE intron 1; Preparing three plasmids by inserting the human APP mutant gene, the PS1 mutant gene, and the BACE gene into the vector and then cloning them; Eluting only an insert, which is a gene region to be inserted into an animal, from the three plasmids; Injecting the eluted inserts into the embryo of the animal to be transformed; Method for producing animals other than humans transformed with the Alzheimer's disease etiology gene comprising a. An APP mutant gene prepared by the method of claim 17 and encoding an amyloid proprotein (APP) mutated at amino acids 670 and 671, a PS1 mutant gene encoding PS1 mutated at amino acids 146 and 166, and a sequence Animals other than humans transformed with Alzheimer's disease etiology gene, transformed with a gene encoding BACE having the amino acid sequence of No. 13. 19. The animal of claim 18, wherein the animal is a mammal other than a human. 19. The animal of claim 18, wherein the animal is a mouse (except Accession Number KCTC 10085BP), characterized in that the animal is a mouse. An expression vector for specifically expressing a neuron-specific gene comprising a NSE promoter having a sequence of SEQ ID NO: 11 and an NSE intron 1 comprising a sequence of SEQ ID NO: 12 and having a cleavage map of FIG. 5.