WO2006004287A1

WO2006004287A1 - Transgenic mice inducing alzheimer's disease expressing mutant app

Info

Publication number: WO2006004287A1
Application number: PCT/KR2005/000923
Authority: WO
Inventors: Pyung Lim Han; Kang-Woo Lee; Sung-Don Yang; Jin-Sook Song
Original assignee: Neurotech Pharmaceuticals Co., Ltd.
Priority date: 2004-03-30
Filing date: 2005-03-30
Publication date: 2006-01-12
Also published as: KR20050096347A; KR100584914B1

Abstract

The present invention is related to a transgenic animal showing Alzheimer's disease pathology. More particularly, the present invention provides a transgenic vector comprising a mutant human beta amyloid precursor protein whith Swedish double mutation and Indiana mutation simultaneously, and a transgenic mouse showing Alzheimer's disease generated by microinjection of the above vector into a pronuclei of a fertilized oocyte followed by generating mice. The transgenic mouse of the present invention exhibited clnicical symptoms of Alzheimer's disease, such as decreases of cognitive ability and memory, and increases of anxiety. Therefore, the transgenic mouse of the present invention will be a useful animal model for a research of Alzheimer's disease. Particularly, because the transgenic mouse of the present invention showed more profound increase of anxiety than any other transgenic animal model for Alzheimer's disease known in the art, the transgenic mouse of the present invention can be used as an animal model for anxiety-related disorders.

Description

[DESCRIPTION]

[invention Title]

TRANSGENIC MICE INDUCING ALZHEIMER'S DISEASE EXPRESSING MUTANT APP

[Technical Field]

The present invention relates to a transgenic mouse with induced Alzheimer' s disease pathology, more precisely, a transgenic mouse that shows Alzheimer' s disease pathology induced by the insertion of a cDNA of mutant human amyloid beta precursor protein into chromosomal DNA.

[Background Art]

Alzheimer' s disease (AD) is characterized by biochemical and histological changes in the brain, showing amyloid deposition, nerve fiber concentration, and synaptic and neuronal loss. AD patients show multiple cognitive deficits including the retardation of memory, increased anxiety and hypersensitivity. Transgenic mice generated by inducing mutation of Alzheimer's disease related gene, APP (amyloid precursor protein, Games et al. , Nature, 1995, 373:523-527; Hsiao et al. , Science, 1996, 274:99-102; Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA., 1997, 94:13287-13292; Moechars et al. , J. Biol. Chem._r 1999, 274:6483-6492; Chishti et al. , J. Biol. Chem., 2001, 276:21562-21570), presenilin 1 (PSl, Duff et al., Nature, 1996, 383:710-713; Begley et al. , J. Neurochem. , 1999, 72:1030-1039; Barrow et al. , Neurobiol Bis, 2000, 7:119-126) or PS2 (Oyama et al., N. Neurochem. , 1998, 71;313-322), have been confirmed to mimic general characteristic symptoms of AD such as beta-amyloidosis, tauopathy, neuronal loss, gliosis and cognitive deficits, etc.

Among many AD models developed so far, specific mouse lines were proved to bear pathophysiological characteristics of AD, attracting researchers' attention. PDAPP mouse expresses human APP (695, 751 and 770) gene including Indiana mutant (V717F) under the control of platelet-derived growth factor-beta promoter. The mouse line showed such symptoms as extracellular amyloid plaque deposition, synaptic loss, increased gliosis and cognitive deficits at 6-15 months of age (Games et al., Nature, 1995, 373:523-527; Irizary et al., J. Neurosci. , 1997, 17:7053- 7059; Chen et al., Nature, 2000, 408:975-979) . Tg2576 mouse expresses human APP695 gene containing Swedish double mutation (K670N, M671L) under the regulation of hamster prion protein (PrP) promoter. The mouse line at 9-18 months showed elevated Abeta42, amyloid deposition, neuritic decrease, inflammatory changes and impaired memory retention. However, locomotor activity of the mouse line was either normal or insignificantly reduced. The anxiety was reduced in the Tg2576 mouse line at 17 months (Hsiao et al., Science, 1996, 274:99-102; Benzing et al., Neurobiol Aging, 1999, 20:581-589; King et al. , Behav Brain Res, 1999, 103:145-162; King et al . , Physiol Behav, 2002, 75:627-642; Kawarabayashi et al., J Neurosci, 2001, 15:372-381; Terai et al._r Neuroscience, 2001, 104:299-310; Lalonde et al., Brain Res, 2003, 977:38-45) . TgCRND8 mouse expresses APP695 gene bearing both Swedish mutation (K670N, M671L) and London mutation (V717I) under the regulation of hamster PrP promoter. Abeta plaque deposition and spatial perception deficit were detected in the mouse at three months (Janus et al., Nature, 2000, 408:979-982; Chishti et al., J Biol Chem, 2001, 276:21562- 21570) . APP23 mouse expresses human APP695 gene bearing Swedish mutation under the regulation of mouse thy-1 promoter. The mouse line showed Abeta deposition, synaptic loss and spatial perception deficit at 6 months (Sturchler-Pierrat et al., Proc Natl Acad Sci USA, 1997, 94:13287-13292; Sturchler-Pierrat et al., Ann NY Acad Sci, 2000, 920:134-139) but anxiety and locomotor activity of the mouse were not changed until at 16 months (Lalonde et al., Brain Res, 2002, 956:36-44) . Other transgenic mice expressing APP and PSl or tau have been developed as well, and these also showed plaque pathogenesis and taupathy at the same time (Borchelt et al., Neuron, 1997, 19:939-945; Holcomb et al. , Nature Med, 1998, 4:97-100; Dewachter et al., J Neurosci, 2000, 20:6452-6458; Lewis et al., Science, 2001, 293:1487-1491; Oddo et al. , J. Neurochem. , 2003, 71:313-322) . Most AD animal models have been generated so far by the inbred of those having the same genetic background. For example, PDAPP mouse was generated by the inbred of Swiss Webster and the hybrids of C57BL/6 and SJL; Tg2576 mouse was generated by the inbred of the hybrids of C57BL/6 and SJL; TgCRND8 mouse was generated by the inbred of the hybrids of C3H and C57BL/6; and APP23 mouse was generated by the inbred of the hybrids of C57BL/6 and DBA2. Unfortunately, all the developed AD animal models are not able to completely bear the whole AD symptoms, suggesting that AD characteristics are not detected only in individual mouse line. Therefore, it is required to develop a new transgenic mouse bearing the characteristics of AD-like brain disease.

Thus, the present inventors tried to establish animal models bearing clinical symptoms of AD, and finally prepared transgenic mice by the insertion of mutant human amyloid beta precursor protein into the mouse chromosome. And further, the inventors have completed the present invention by confirming that this newly created transgenic mouse clearly shows AD symptoms.

[Disclosure]

[Technical Problem]

The object of the present invention is to provide a transgenic vector that can be used to create transgenic mice showing AD pathology. ^■ Another object of the present invention is to create a genetically stable transgenic mouse carrying the above vector.

[Technical Solution] To achieve the above objects, the present invention provides a transgemic vector that contains a gene coding mutant human amyloid beta precursor protein (APP) bearing Swedish mutation and Indiana mutation simultaneously.

The present invention also provides a transgenic mouse in which AD is induced by injection of the vector into the nucleus of a fertilized egg of mice, followed by transferring injected eggs into the oviduct of foster mothers to generate mice.

[Description of Drawings] Fig. 1 is a set of schematic diagrams showing the cleavage maps of PDGF-APP(Sw, V717F)-pA (A) and PDGF- intron-APP (Sw, V717F)-pA (B), vectors for transformation constructed in the present invention.

Fig. 2 is a photograph of Southern blotting confirming the insertion of mutant APP751 gene into transgenic animals of the present invention ^ΛTg-APP/B6(- intron) ' and ^ΛTg-APP/B6 (+intron) ' . In the photograph, the arrow presents 2.3 kb APP751 fragment digested by Spe I.

Fig. 3 is a photograph of Northern blotting confirming the expression of mutant APP751 gene in transgenic animals of the present invention ^ΛTg-APP/Bβ(- intron) ' and ^ΛTg-APP/B6 (+intron) ' . In the photograph, the upper arrow presents internal APP transcript and the lower arrow presents mutant APP transcript of the present invention.

Fig. 4 is a photograph of Western blotting confirming the production of APP751 protein in Tg-APP/Bβ transgenic mice of the present invention. The upper panel presents the result analyzed by using monoclonal APP751 antibody and the lower panel presents the result analyzed by using polyclonal APP751 antibody. The short arrow indicates the location of internal APP protein and the long arrow indicates the location of mutant APP751 protein.

Fig. 5 is a photograph of Western blotting showing the possibility of the production of human APP Abeta protein (6E10) in Tg-APP/Bβ transgenic mice of the present invention and the comparison of the expression levels of human APP Abeta protein between homo- and hetero- transgenic mice.

Fig. 6 is a photograph (left) of Western blotting showing the expressions of betaCTF and αCTF proteins in

Tg-APP/B6 transgenic mice of the present invention and a graph (right) showing the results above presented in relative expression levels.

Fig. 7 is a photograph of immunohistochemical analysis showing the expression of mutant APP751 protein in CX and CAl, CA3 and DG regions of HP of the brain of Tg-APP/Bβ transgenic mice of the present invention.

Fig. 8 is a photograph of immunohistochemical analysis showing that calbindin expression was reduced in

CAl, CA3 and DG regions of HP of the brain of Tg-APP/Bβ transgenic mice of the present invention, compared to the wild type controls, while parvalbumin and calretinin expressions were not changed.

Fig. 9 is a photograph of immunohistochemical analysis showing that c-fos expression was reduced in CAl,

CA3 and DG regions of HP of the brain of Tg-APP/B6 transgenic mice of the present invention, compared to the wild type controls.

Fig. 10 shows the results of open filed test and rota-rod test investigating the locomotor activity of Tg- APP/B6 transgenic mice of the present invention (homo- and hetero-) . In the graph, * indicates a difference at the p<0.05 level in each group (student's t-test) .

Fig. 11 shows the results of Morris water maze test and passive avoidance test investigating cognitive deficits of Tg-APP/Bβ transgenic mice of the present invention (hetero) at 7 - 14 months. In the graph, * indicates a difference at the p<0.05 level in each group (student's t-test) .

Fig. 12 shows the results of elevated plus maze test

(graph and photograph of locomotor image) investigating the increased anxiety of Tg-APP/B6 transgenic mice of the present invention (homo and hetero) at 7 - 13 months. In the graph, * indicates a difference at the p<0.05 level in each group (student's t-test) . In the photograph of locomotor image, CA means closed arm and OA means open arm.

Fig. 13 shows the results of microarray assay and RT-PCR investigating any changed expression of gene in Tg- APP/Bβ transgenic mice of the present invention.

[Best Mode]

Hereinafter, the present invention is described in detail.

The present invention provides a transgemic vector that contains a gene coding mutant human amyloid beta precursor protein (APP) bearing Swedish mutation and Indiana mutation simultaneously.

The mutant human amyloid beta precursor protein

(APP) is preferably selected from a group consisting of

APP751(Sw, V717F) in which lysine (K), the 651^st amino acid of APP751 represented by SEQ. ID. No 2, is replaced with asparagine (N) , methionine (M) , the 652^nd amino acid, is replaced with leucine (L) , and valine (V) , the 698^th amino acid, is replaced with phenylalanine (F), APP695(Sw,

V717F) in which lysine (K) , the 595^th amino acid of APP 695 represented by SEQ. ID. No 3, is replaced with asparagine (N) , methionine (M) , the 596^th amino acid, is replaced with leucine (L) and valine (V) , the 642^nd amino acid, is replaced with phenylalanine (F) and APP770(Sw,

V717F) in which lysine (K), the 670^th amino acid of APP770 represented by SEQ. ID. No 4, is replaced with asparagine

(N) , methionine (M) , the 671^st amino acid, is replaced with leucine (L) and valine (V) , the 717^th amino acid, is replaced with phenylalanine (F) . In the above, the replacement of the 670^th amino acid lysine (K) of APP770 represented by SEQ. ID. No 4 with asparagine (N) and of the 671^st amino acid methionine (M) with leucine (L) is called "Swedish mutation' . The SEQ. ID. No 2 and No 3 are isoforms generated by selective splicing of APP gene, having similar functions though they have different number of amino acid residues. Thus, mutations of the SEQ. ID.

No 2 and No 3 corresponding to Swedish mutation of the SEQ.

ID. No 4 are also regarded as Swedish mutation. In the meantime, the replacement of the 717^th amino acid valine

(V) of APP770 represented by SEQ. ID. No 4 with phenylalanine (F) is called ^Indiana mutation' or VlIlF mutation. Likewise, the mutations of SEQ. ID. No 2 and No 3 corresponding to the Indiana mutation are regarded as Indiana mutation. In the preferred embodiment of the present invention, a gene coding mutant APP protein above was prepared, which was then named ^ΛλhAPP(Sw, V717F)". In order to construct the transgemic vector for AD, in the preferred embodiment of the present invention, APP751(Sw, VlIlF) represented by SEQ. ID. No 5 was used as mutant human amyloid beta precursor protein (APP) . It is preferred for the transgemic vector of the present invention to additionally include promoter and polyadenylation region. At this time, the promoter is not limited to a specific one but human PDGF-beta promoter is preferred (GenBank Accession No: Y00389) . The polyadenylation region is not limited to a specific region either but SV40 pA is preferably used. It is more preferred for a vector for transformation to induce AD of the present invention to additionally include Kozak sequence in the upstream of starting codon of a gene coding mutant human amyloid beta precursor protein bearing Swedish mutation and Indiana mutation together, to elevate translation efficiency.

In the preferred embodiment of the present invention, the transgemic vector includes human PDGF-beta promoter, a gene coding mutant human amyloid beta precursor protein, represented by SEQ. ID. No 5, and SV40 pA in that order, and presented as a cleavage map PDGF-APP(Sw, V717F) -polyA.

The present inventors constructed the transgemic vector to contain PDGF-beta promoter gene, Kozak sequence, mutant gene (hAPP(Swe, VlIlF) ) coding an amino acid sequence represented by SEQ. ID. No 5 and SV40 polyadenylation gene, and named the vector as "PDGF-APP(Sw, V717F) -polyA" (see Fig. 1) .

Further, a transgemic vector of the present invention preferably includes intron between a promoter and a gene coding human amyloid beta precursor protein. At this time, the intron is not limited to a specific one but the intron B originated from human beta-globin gene is preferred. In the preferred embodiment of the present invention, a transgemic vector preferably includes human PDGF-beta promoter, intron B of human beta-globin gene represented by SEQ. ID. No 6, a gene coding mutant human amyloid beta precursor protein represented by SEQ. ID. No 5 and SV40 pA, in that order, and is preferably presented as a cleavage map PDGF-APP(Sw, V717F) -polyA.

The introduction of the intron B gene of human beta- globin gene is to increase expression efficiency of mutant gene and transcription stability. So, in the present invention, a transgemic vector was constructed to include PDGF-beta promoter gene, intron B gene of human beta- globin gene, Kozac sequence, mutant gene coding an amino acid sequence represented by SEQ. ID. No 5 (hAPP751 (Swe, VlIlF) ) and SV40 polyadenylation gene, which was named "PDGF-intron-APP(Sw, V717F) -polyA" (see Fig. 1). The present invention also provides a genetically stable transgenic mouse carrying the above vector of the invention. PDGF-APP (Sw,V717F)-polyA or PDGF-intron-APP (Sw, V717F) -polyA vector for transformation is preferably introduced into a mouse chromosome to produce a transgenic mouse of the present invention, and PDGF-intron-APP(Sw, V717F)-polyA is more preferred. In the preferred embodiment of the present invention, PDGF-APP(Sw,V717F) - polyA or PDGF-intron-APP (Sw, V717F)-polyA vector for transformation was microinjected into the pronuclei of fertilized eggs prepared from inbred C75BL/6 mice, and the injected eggs were transplanted in surrogate mice. Comparison in expressions of APP(Sw, V717F) mutant gene among the second generation produced from the surrogate mice and also offsprings produced by inbred was made. As a result, the expression of the mutant gene was much higher in transgenic mice transformed with PDGF-intron- APP(Sw, V717F) -polyA vector than in other mice transformed with the other vector. The result indicates that it is preferred to transform a mouse by the introduction of PDGF-intron-APP(Sw, V717F)-polyA vector to increase the expression efficiency of APP(Sw, VlIlF) mutant gene of the present invention. In the present invention, a transgenic mouse prepared by introducing PDGF-intron-APP (Sw, V717F)-polyA vector into nucleus of a fertilized egg was named "Tg-

APP/B6". After confirming that APP mutant gene was successfully inserted into a mouse and so APP protein was expressed to the wanted level therein, the present inventors deposited the transgenic mouse of the invention at Korean Collection for Type Cultures (KCTC) of Korea

Research Institute of Bioscience and Biotechnology (KRIBB) on March 10, 2004 (Accession No: KCTC 10608BP) .

The transgenic mice with induced AD of the present invention (Tg-APP/Bβ) showed characteristic symptoms of AD such as motor coordination deficit, memory and cognitive deficits and increased anxiety, etc. In the preferred embodiment of the present invention, open field test, rota-rod test, Morris water maze test, elevated plus maze test and passive avoidance test were performed to investigate whether or not the transgenic mice of the invention could show clinical AD symptoms . In the open field test, locomotor activity of transgenic mice of the present invention was similar to that of wild type controls (see Fig. 10A) , whereas, in the rota-rod test, locomotor coordination and locomotor learning of transgenic mice of the present invention were reduced, compared to the wild type controls (see Fig. 10B) . In the Morris water maze test, transgenic mice of the present invention showed cognitive deficits, leading to the impairment of memory retention, compared to the wild type controls (see Fig. HA) . In the elevated plus maze test, transgenic mice of the present invention showed increased anxiety (see Fig. 12B) and in the passive avoidance test, the mice showed impaired memory retention (see Fig. 12C) . The above results indicate that the transgenic mice of the present invention show clinical AD symptoms such as memory and cognitive deficits and increased anxiety.

Microarray assay was also performed to investigate the possible reason for the increased anxiety shown above. As a result, expressions of TNF-α, T-LAK cell-originated protein kinase, hematopoietic stem cell specific Lyn substrate 1, staniocalcin, calmodulin 4, cytochrome c, type 17 procollagen alpha 1, prominin 1 and transthyretin were increased. RT-PCR results resembled the above (see Fig. 13), supporting the current reports. Precisely, the injection of recombinant human TNF-α into the cerebroventricle of a mouse caused anxiety-like symptoms (Connor et al. , Neuroscience, 1998, 84:923-933), in elevated plus maze test, and T-LAK cell-originated protein kinase was proved to be involved in TNF receptor activity (Abe et al. , J Leukoc Biol, 1995, 57:462-468). Elevated blood plasma rennin activity also had something to do with increased anxiety (Perini et al., J. Hypertens., 1994, 12:601-607) .

The increased anxiety is the most problemable symptom of AD (Folstein and Bylsma, 1999, In Alzheimer Disease (Eds by Terry et al. , 2^nd. Lippincott Williams & Wilkins, Philadelphia) . Thus, the transgenic mouse of the present invention showing increased anxiety will be very useful as an AD model and further for the study on APP protein and age-dependently increased anxiety. In particular, the transgenic mouse of the present invention showed increased anxiety more severly than any other conventional AD animal models, making the transgenic mouse useful candidate for the study on increased anxiety, one of characteristic pathological symptoms of AD, and in broad, for the study on general anxiety.

[Mode for Invention] Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

<Example 1> Preparation of human amyloid beta precursor protein cDNA and APP mutant gene

<!-!> Preparation of human amyloid beta precursor protein cDNA

The cDNA coding human amyloid beta precursor protein (referred ^ΛAPP' hereinafter) was prepared by PCR using Marathon-Ready cDNA library (Clontech, Palo Alto, CA, USA) constructed from human brain. The cDNA could not be amplified at once because the size of its open reading frame (ORF) was about 2.3 kb, taking APP770 as standard. Thus, the first half of the cDNA was amplified by using primer sets of app-lf primer represented by SEQ. ID. No 6 (5' -gcaagggtcgcgatqctgcccqgtttg-3' , the underlined part presented Nru I restriction enzyme recognition site) and app-2r primer represented by SEQ. ID. No 9 (5'- gacattctctctcggtgcttggcc-3' ) , and the resulting products were digested with Nru I and Xho I. The products were inserted into Sma I and Xho I restriction enzyme recognition sites of pBluescript II KS vector (Stratagene, USA) . The second half of the cDNA was amplified by using primer sets of app-2f primer represented by SEQ. ID. No 8 (5' -cctacaacagcagccagtacccctg-3' ) and app-lr primer represented by SEQ. ID. No 7 (5' -qqgqqactaqttctqcatctqctc- 3' , the underlined part presented Spe I restriction enzyme recognition site) , followed by digesting with Spe I and Xho I. Then, the resulting products were inserted into Spe I and Xho I restriction enzyme recognition sites of pBluescript II KS vector. The DNA fragments produced by digesting pBluescript II KS vector bearing the first half of the cDNA with BamH I and Xho I and the other DNA fragments produced by digesting pBluescript II KS vector bearing the second half of the cDNA with Xba I and Xho I were fused together with pBluescript II KS vector predigested with Xba ¹I and BamH I, leading to the preparation of a vector construct carrying the full length

^" of APP cDNA. In the meantime, three different isoforms were produced, according to the numbers of amino acid residues of encoded protein, from the same gene of human beta amyloid by selective splicing. So, according to the numbers of amino acid residues, APP cDNA carries three different isoforms, that is, APP770, APP751 and APP695. The DNA sequences of the cloned cDNA were analyzed. As a result, among those three isoforms, the cloned cDNA was confirmed to be APP751 cDNA represented by SEQ. ID. No 1 coding APP751 (represented by SEQ. ID. No 2) .

<l-2> Preparation of APP751 mutant gene "Swedish double mutation (in APP770 isoform, the 670^th amino acid lysine is replaced with asparagine, the 671^st amino acid methionine is replaced with leucine, K670N, M671L)" and "V717F mutation (in APP770 isoform, the 717^th amino acid valine is replaced with phenylalanine) " were induced by PCR in APP751 cDNA. More precisely, in order to induce "Swedish mutation", first, PCR was performed by using pBluescript II KS vector, in which the second half of APP751 cDNA prepared in the above <Example 1-1> was cloned, as a template with primer sets of app-swe primer (5 ' -aggagatctctgaagtgaatctggatgcaa-3 ' ) represented by SEQ. ID. No 10 and app-717-r primer (5V- caaggtgatgaagatcactgtcgc-3 ' ) represented by SEQ. ID. No 11 with the 32 cycles of denaturation at 95^°C for 1 minute, primer annealing at 57 ^°C for 40 seconds and extension at 72^°C for 1 minute. Then, in order to induce "V717F mutation", PCR was performed with the primer sets of app717-f primer (5 ' -gcgacagtgatcttcatcaccttg-3 ' ) represented by SEQ. ID. No 12 and app-lr primer represented by SEQ. ID. No 7 under the same conditions as described above. The PCR products from the two PCR above had "Vl7IF mutation". So, the two PCR products were separated, slowly cooled down, extended with Klenow enzyme and then fused into one fragment. The fragment was digested with BgI II and Spe I by taking advantage of BgI II restriction enzyme recognition site of app-swe primer and Spe I restriction enzyme recognition site of app-lr primer, which were inserted into pBluescript II KS vector which was also digested with BgI II and Spe I ahead of time and the second half of APP cDNA was inserted in, leading to the preparation of the mutant second half APP751 cDNA. In the meantime, the mutated DNA fragment prepared above was used to replace the corresponding region of pBluescript II KS vector where the full length of APP751 cDNA was inserted, resulting in APP751 mutant cDNA represented by SEQ. ID. No 4, and then named "hAPP(Swe, VlIlF)". The nucleotide sequence of the hAPP(Swe, V717F) mutant gene was confirmed by DNA sequencing.

<Example 2> Construction of an expression cassette containing APP751 mutant gene for transgenic animal

In order to creat an AD animal model, an expression cassette for transformation containing APP751 mutant cDNA prepared in the <Example l-2> was constructed. Particularly, pGK-neo-PA vector (Lee et al._f J. Neurosci. , 2002, 15:7931-7940) was amplified using primer sets of SV40pA-f primer represented by SEQ. ID. No 13 (5'- tccccgcgqtccagacatgataagatacattga-3' , the underlined part presented Sac II restriction enzyme recognition site) and SV40pA-r primer represented by SEQ. ID. No 14 (5'- qttcqaqctcataatcaqccataccacatttq~3' , the underlined part presented Sac I restriction enzyme recognition site) , resulting in 247 bp sized SV40-pA fragment for polyadenylation signal of the mutant gene. Then, the fragment was digested with Sac II and Sac I, which was inserted into pBluescript II KS vector. PsisCATβa vector

(Sasahara, M. et al. , Cell, 1991, 64 (1) :217-27) was digested with Xba I, linearized with Klenow enzyme, and digested with Hind III, resulting in human platelet- derived growth factor-beta (PDGF-beta) promoter fragment. The obtained PDGF-beta promoter fragment was inserted into pBluescript II KS vector which was digested with Sal I, linearized with Klenow enzyme and then digested again with Hind III. The pBluescript II KS vector bearing the above PDGF-beta promoter fragment was digested with Kpn I and Hind III, resulting in 1.5 kb sized PDGF-beta promoter fragment. And the fragment was inserted into Kpn I and Hind III recognition sites of pBlescript II KS vector containing SV40 pA region. The resulting vector, thus, has a structure that has PDGF-beta promoter and SV40-pA region respectively at each side of multicloning site of pBluescript II KS vector. In the vector, APP751 mutant gene containing Kozac sequence (GACC) in front of starting codon was inserted, resulting in an expression cassette for transformation. Particularly, the first half of APP751 cDNA prepared in the above <Example 1-1> was amplified by PCR using primer sets of app-Koz-f primer represented by SEQ. ID. No 15 (5¹- qctctaqaccatqctqcccqqtttqqcactqctc, the underlined part presented Xba I restriction enzyme recognition site) and app-2r primer represented by SEQ. ID. No 9. Then, the PCR product was digested with Xba I and Xho I. pBluescript II KS vector bearing the second half of mutant APP751 cDNA prepared in the above <Example l-2> was digested with Sac II and Xho I, which was fused to the vector pre-digested with Xba I and Sac II, resulting in a vector construct harboring Kozac sequence and the full length of mutant APP751 cDNA. The vector construct was digested with Hind III and Sac II to obtain DNA fragment bearing Kozac sequence and the full length of APP751 cDNA. The fragment was inserted into Hind III and Sac II restriction enzyme recognition sites of pBluescript II KS vector containing PDGF-beta promoter and SV40-pA sequence. Finally, the expression cassette was constructed to possess PDGF-beta promoter, Kozac sequence, APP751(Sw, V717F) , and SV40-pA in that order, and named "PDGF-APP(Sw, V717F)-pA" (Fig. 1) .

<Example 3> Construction of an expression cassette containing intron and APP751 mutant gene for transgenic animal

The intron elevates expression efficiency of a mutant gene and increases transcription stability. Thus, in order to introduce a mutant gene into an animal model effectively, the present inventors introduced the intron B gene (918 bp) (Choi et al. , Molecular and cellular biology, June 1991, p.3070-3074; Palmiter et al._r PNAS, 1991, 88:478-482) of human beta-globin gene into the expression cassette prepared in the above <Example 2>. Precisely, the intron B of human beta-globin gene was amplified by PCR using the primer sets of hglob-f primer represented by SEQ. ID. No 16 (5 ' -gatcctgagaacttcagg-3 ' ) and hglob-r primer represented by SEQ. ID. No 17 (5'- tctttgccaaagtgatgg-3 ' ) and genomic DNA, which was obtained from the human neuroblastoma cell line SH-SY5Y, as a template. The amplified intron B gene of human beta- globin gene was sub-cloned into pGEM-T Easy vector (Promega, Madison, WI, USA) , and the vector was digested with EcoR I to obtain DNA fragment. The fragment was linearized with Klenow enzyme, and then inserted into EcoR V restriction enzyme recognition site located between PDGF-beta promoter gene of PDGF-APP (Sw,V717F) -pA expression cassette constructed in the above <Example 2> and APP(Sw, V717F) mutant gene. The resulting expression vector for transformation was named "PDGF-intron- APP ( Sw, V717 F) -pA" ( Fig . 1 ) .

<Example 4> Generation of transgenic animals

PDGF-APP(Sw, V717F)-pA expression cassette constructed in the above <Example 2> and PDGF-intron-

APP(Sw, V717F)-pA expression cassette constructed in the above <Example 3> were digested with a restriction enzyme

{BssH E-), resulting in 5.0 kb sized linearized DNA fragment. The product was microinjected into the pronuclei of fertilized eggs prepared from inbred C57BL/6 mice. After the microinjection, the fertilized eggs were transferred to the oviducts of pseudopregnant female (ICR) mice. The methods for transformation of animals used in the present invention including microinjection were in accordance with the conventional methods (Games efc al., Nature, 1995, 373:523-527; Hsiao et al., Science, 1996, 274:99-102) .

<Example 5> Confirmation of the insertion of a mutant gene into chromosomal DNA

Genomic DNA was extracted from the tails of Fl mice generated from the animal transformation procedure performed in the above <Example 4>, and PCR was performed to confirm whether or not a mutant gene was rightly inserted into nuclei of fertilized eggs. Particularly, PCR was performed with primer sets of APP(-intron) -f primer represented by SEQ. ID. No 18 (5¹- gcttgatatcgaattcctgcagc-3' ) and APP (-intron) -r primer represented by SEQ. ID. No 19 (5'-gaggaggaacagcctgcagag- 3') , in order to investigate the insertion of a mutant gene into the Fl mice generated by using PDGF-APP(Sw, V717F) -pA expression cassette excluding intron. In the meantime, another PCR was performed with primer sets of APP (+intron) -f primer represented by SEQ. ID. No 20 (5^!- aatgtatcatgcctctttgcacc-3' ) and APP (+intron) -r primer represented by SEQ. ID. No 21 (5'-gcacttgtcaggaacgagaaggg- 3"), in order to investigate the insertion of the mutant gene in the Fl mice generated by using an expression cassette including intron (PDGF-intron-APP(Sw, V717F)-pA) .

As a result, among the Fl mice generated by the introduction of an expression cassette excluding intron (PDGF-APP(Sw, V717F)-pA), 19 mice were confirmed to bear the expression cassette. In the case of Fl mice generated by the introduction of an expression cassette including intron PDGF-intron-APP (Sw, V717F)-pA), 8 mice were confirmed to bear the expression cassette.

Southern blot analysis was also performed to confirm the introduction of the expression cassette of the present invention. Particularly, genomic DNA was extracted from the tails of Fl mice generated from the animal transformation procedure taken in the above <Example 4>, and then 15 βg of the genomic DNA was digested with restriction enzyme Spe I. The resulting products were electrophorezed on agarose gel, and then transferred onto nitrocellulose membrane. Hybridization was performed using a ³²P-labeled probe prepared from the 320 bp BgI JI /Spe I fragment at the C-terminus of APP cDNA, and the results were developed on X-ray film.

The results resembled those of the above PCR with the genomic DNA, that is, among the Fl mice generated by the introduction of an expression cassette excluding intron (PDGF-APP(Sw, V717F)-pA), 19 mice were confirmed to bear the expression cassette. In the case of those Fl mice generated by the introduction of an expression cassette including intron (PDGF-intron-APP (Sw, V717F) -pA) , 8 mice were confirmed to bear the expression cassette (Fig. 2) . In the Figure, the number on each lane presents the ID number of each individual mouse.

<Example 6> Investigation of the transgene expression

In order to confirm whether or not APP mutant gene was successfully introduced and expressed in the transgenic mice of the present invention, total RNA was extracted from the brains of the transgenic mice, followed by Northern blotting. Northern blot analysis was performed according to the method of Lee, et al. (Lee et al._f J Neurosci, 2002, 15:7931-7940) . Particularly, total RNA was extracted from the brains of wild type and transgenic mice at 2 months, which were confirmed to have APP mutant gene transducted in the above <Example 4> and <Example 5>. Trizol^® reagent (Sigma, St. Louis, MO, USA) was used for the extraction of the total RNA.- A membrane blot carrying 30 βg of total RNA was prepared after separating on denaturing agarose gel (1% agarose, 6.2% formaldehyde in 1* MOPS) , and hybridized with a ³²P- labeled probe prepared from the BgI II /Spe I-digested fragment (320 bp) of APP cDNA, which was also used in the above <Example 5> for Southern blot analysis, and then the results were developed on X-ray film. The probe was able to recognize both internal APP transcript (~3.5 kb) and mutant APP transcript (-2.6 kb) .

As a result, the expression of APP mutant transcript was much higher (2.6+0.1 fold) as a whole than that of the endogenous APP transcript in transgenic mice. Comparison of APP mutant gene expressions was made between offsprings generated by the insertion of mutant gene expression cassette including intron (PDGF-intron-APP (Sw, V717F) -pA) and those generated by the insertion of mutant gene expression cassette ■ excluding intron (PDGF-APP(Sw, V717F) - pA) . As a result, the expression of APP mutant gene was much increased along with the increase of stability of gene-drived transcript in offsprings generated by PDGF- intron-APP (Sw, V717F)-pA than that in others generated by the expression cassette not including intron (Fig. 3) . Thus, the transgenic mice (PDGF-intron-APP (Sw, V717F)-pA) showing elevated expression of APP mutant gene was named "Tg-APP/Bβ" and was ready for further experiments.

The fertilized egg of the transgenic mouse Tg-APP/B6 was deposited at Korean Collection for Type Cultures

(KCTC) of Korea Research Institute of Bioscience and Biotechnology (KRIBB) on March 10, 2004 (Accession No: KCTC 10608BP) .

<Example 7> Protein production by the transgene

It was confirmed in the above <Example 6> that Tg- APP/B6 transgenic mice of the present invention expressed APP mutant gene successfully. In order to investigate the possibility of protein production by the expressed gene, total protein was extracted from the brains of wild type controls and Tg-APP/B6 mice at 4 - 5 months, followed by Western blotting. The Western blot analysis was performed according to the method of Lee, et al. (Lee et al. , Brain

Res MoI Brain Res, 1999, 70:116-124) . Particularly, mouse brain tissue was homogenized in 4^°C lysis buffer (50 mM

Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) containing 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor (Complete™; Roche,

Mannheim, Germany) . Centrifugation was performed with the homogenized brain tissue samples at 13,000 rpm, for 20 minutes at 4^°C to obtain supernatant. The protein in the supernatant was guantified by BCA quantification kit

(Sigma, St. Louis, MO, USA) . Each lane was loaded with 30 βg of the protein, and acrylamide gel electrophoresis was carried out. The separated proteins were transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA) and the membranes were blocked with 5% non-fat dry milk, 2% BSA, 4% FBS, 4% horse serum, 4% goat serum in Tris-buffered saline and 0.1% Tween^® 20. Monoclonal or polyclonal anti- APP antibody (Zymed, San Francisco, CA, USA, polyclonal antibody: 51-2700, monoclonal antibody: 13-0200) was used for the Western blot analysis to confirm the production of the APP mutant protein. The monoclonal and the polyclonal antibodies are the antibodies each recognizing N-terminal and C-terminal of human APP. Immunoblots were detected using ECL detecting reagents (Santa Cruz, CA, USA) . As a result, approximately 120 kb sized APP751 protein was detected in the brains of Tg-APP/Bβ transgenic mice (Fig. 4) . For the comparison of mutant gene insertion between the mice in which the mutant gene of the present invention was inserted as a homozygote (referred as "homo" hereinafter) and the mice in which the mutant gene of the present invention was inserted as a heterozygote (referred as ^λλhetero" hereinafter) , Western blot analysis was performed by the same method as described previously using human APP Abeta specific monoclonal antibody (6ElO, Signet, Dedham, MA, USA) . As a result, the expression of APP was at least 2-fold increased in homo transgenic mice, compared to that of the hetero transgenic mice (Fig.5) . The production of C- terminal fragments (betaCTF and αCTF) digested with beta- secretase and α-secretase in the brain of Tg-APP/B6 transgenic mice of the present invention generated by the insertion of APP mutant gene was 2.55 +0.18 and 2.3+0.43 fold increased, respectively, compared to those of the wild type controls (Fig. 6) .

<Example 8> Immunohistochemical analysis of the transgenic mice

For immunohistochemical experiments, the mice were perfused with 0.9% saline through ascending aorta, and then perfused again with 4% paraformaldehyde in 0.1 M phosphate buffer (referred "PB" hereinafter, pH 7.4) . The brain was removed and fixed in the fixative at 4^°C. The fixed brain was coronally cut into 40 ^m-thick sections with a vibratome. The sections were reacted in 3% hydrogen peroxide solution dissolved in 0.1 M PB (pH 7.4) for 30 minutes and washed with PB. The sections were blocked by 5% normal goat serum, 2% BSA, 2% FBS and 0.1% triton X-100 for 2 hours at room temperature. The primary antibody was added to the blocking buffer, which was left at 4^°C for overnight for reaction. After washing with PB solution, the secondary antibody, which was biotinylated by being diluted 1:200 fold, was added. Then, 1:100 fold diluted avidin and biotinylated HRP complex (Vector Laboratories, Burlingame, CA) were also added for one more hour reaction. 0.05% 3, 3' -diaminobenzidine and 0.001% hydrogen peroxide in 0.1 M Tris (pH 7.4) were used for the color development. Cerebral cortex (referred "CX" hereinafter) , pyramidal cells of CA1-CA3 regions (referred "CA1"-"CA3" hereinafter), hippocampus (referred "HP" hereinafter) and dentate gyrus (referred "DG" hereinafter) were used for the analysis.

As a result, the expression of APP751 was observed in various brain regions including CX and HP of Tg-APP/B6 of the present invention (Fig. 7) . However, plaque-like Abeta-deposition was not clearly observed in the brains of the hetero transgenic mice at .12-18 months and at 14 months, either.

Tg257β transgenic mice expressing mutant APP695 that is able to express APP approximately 5.6 fold of the internal APP level could not survive over 150 days after the inbred with C57BL/6 (Carlson et al. , Hum. MoI. Genet., 1997, 6:1957-1959). In the meantime, hetero and homo Tg- APP/B6 transgenic mice of the present invention showed 2.6 and 5.2 fold higher expressions of APP, respectively, compared to the internal APP level. Hetero and homo Tg- APP/B6 transgenic mice of the present invention were inbred with C57BL/6 by the same manner as previously described. Even though chance of stillbirth was higher than that of controls, 83% of homo Tg-APP/B6 mice survived upto 420 days, indicating that the transgenic mouse of the present invention is more useful as an animal model.

<Example 9> Expressions of other proteins in the brains of transgenic mice

Expressions of other proteins that might be affected by APP mutant gene introduced into Tg-APP/B6 transgenic mice of the present invention were investigated. Immunohistochemical assay was performed in pyramidal cells of CAl - CA3, HP and DG regions in the analogy to the procedure as described in the above <Example 8>. The antibodies used for the analysis were anti-calbindin antibody (C9848; Sigma, St. Louis, MO, USA), anti- parvalbumin antibody (P3088; Sigma, St. Louis, MO, USA), anti-calretinin antibody (AB5054; Chemi-Con, Temecula, CA, USA) and anti-c-Fos antibody (sc-52; Santa Cruz Bio- Technology, Santa Cruz, CA, USA) .

As a result, the expression of calbindin, a calcium- binding protein, was reduced in CAl and DG regions of Tg- APP/B6 transgenic mice of the present invention at 13 - 15 months, compared to that of the age-matched controls. In addition, the expressions of parvalbumin and calretinin in HP region of Tg-APP/B6 transgenic mice were similar to those of age-matched controls (Fig. 8) . C-Fos is an initial gene used as a neuronal function marker, which is synthesized in neurons with the increase of levels of internal calcium, cyclic AMP or other secondary messengers. The expression of the c-Fos was also reduced in whole brain regions of Tg-APP/Bβ transgenic mice of the present invention, compared to that of^'the wild type controls (Fig.

9) • <Example 10> Cognitive function of the transgenic mice

Histopathological characteristics of AD brain are

(1) the deposition of extracellular senile plaques, (2) the formation of intracellular neurofibrillary tangle, (3) the degeneration of axons and synapses, and neuronal loss, and (4) malfunction of the brain by neuronal loss, which are all detectable by histological test. In particular, cognitive deficit is the most characteristic and important morphological and clinical symptom. Thus, it is important for an AD animal model to show not only histological characteristics including senile plaques deposition, but also, in fact more importantly, cognitive deficits. - The present inventors performed Morris water maze test, passive avoidance test, and open field test to judge the cognitive deficits in candidates for AD models.

Mice were housed in cages in a temperature- and humidity-controlled environment with a 12 hour-light/dark cycle (light switched on at 7 a.m.). All animals were handled in accordance with the animal care guideline of Ewha Womans University School of Medicine. To track the animals' behavior, a computerized video-tracking system (SMART; Panlab S. I., Barcelona, Spain) was used.

Two-sample comparisons were carried out using the Student t-test, while multiple comparisons were made using one-way ANOVA followed by the Newman-Keuls multiple range test. All data were presented as the mean+S.E.M. The statistical differences were accepted at the 5% level unless otherwise indicated.

<10-l> Open field test

Locomotor activity was measured in the open field of a white Plexiglas chamber (45x45x45 cm) . Illumination in the chamber was adjusted to 70 lux. The mice were all placed in the same environment as that of the chamber 30 minutes prior to the test. Each mouse was placed individually in the middle of the open field and locomotion was recorded for 60 minutes. The horizontal locomotor activity was judged according to the distance the animal moved. The inner 30 percentage of the open filed was defined as the center of the chamber.

As a result, the locomotor activities or total traveled distances shown by homo (homozygous, APP-Homo) and hetero (heterozygous, APP) Tg-APP/B6 transgenic mice of the present invention at 13 months were similar to those of wild type controls. (Fig. 10A). The approaches to the center of the open field (a sign of anxiety) were also similar to those of control mice.

<10-2> Rota-rod test Rota-rod test was performed to evaluate motor coordination and motor learning. Rota-rod consists of a rotating cylinder (4.5 cm in diameter) with a speed controller attached. Mice were placed on the top of the cylinder where they have access to tight grip. Rota-rod was spinned at the speed of 5-20 rpm, and the speed was gradually increased. Cut-off time was set as 3 minutes and intertrial interval was 60 minutes. Hang-on time on rod was measured.

As a result, homo Tg-APP/Bβ transgenic mice at 11 months did not stay longer on the rod spinning at 15rpm (Fig. 10B) .

<10-3> Morris water maze test

Morris water maze test is a hippocampus-dependent analysis method that depends largely on the capability of an animal to learn and remember the relation between stimulus at a distance and hidden platform for escape (Morris et al., Behav. Neural. Biol., 1987 47 (3) : 333-345) . That is, in this test, forced swimming or the latency to find a hidden platform by taking advantage of spatial indices memorized during placing on the platform was observed. Based on this observation, cognitive function of a mouse was investigated and quantified by comparison of the distance and the time that the mouse swam. In order to investigate memory retention of a mouse, the locations of the entrance to the pool and the hidden platform were changed often, while spatial indices were still located same. Particularly, water maze consisted of a 90 cm-diameter cylinder pool filled with 22^°C opaque milky water. A 10 cm-diameter hidden platform was placed in a quadrant 1.5 cm below the surface of the opaque water. The pool was placed in a room with abundant environmental and artificial cues including a window, a chair and posters. In the course of daily testing, mice were admitted successively into each of the quadrants and allowed to swim for 90 seconds maximum. On locating the platform, the animals were permitted to remain on it for 30 seconds before the session was terminated. The latency to find the platform for each of two trails and the average of the two trails were recorded for each mouse.

As a result, wild type controls at 7 months could recognize the indices of the hidden platform, and this achievement improved trial after trail. The hetero Tg-

APP/B6 transgenic mice at 7 months showed similar ability to recognize the indices of the hidden platform. On the other hand, the hetero Tg-APP/B6 mice at 14 months showed longer latency to find the hidden platform, compared to that of the age-matched controls (Fig. 11A) .

In the meantime, swimming speeds of Tg-APP/B6 transgenic mice at 7 and 14 months were similar to those of age-matched controls (Fig. 11B). Those results indicate that Tg-APP/Bβ transgenic mice show elevated cognitive deficits age-dependently, compared to the wild type controls.

<10-4> Elevated- plus maze test Increased anxiety is a ■problematic symptom of human

AD patients (Folstein and Bylsma, 1999, Alzheimer Disease

(Eds by Terry et al.,) 2nd. Lippincott Williams & Wilkins,

Philadelphia). Thus, the present inventors needed to investigate the possibility of increased anxiety by the introduction of APP mutant gene in Tg-APP/B6 transgenic mice. Elevated plus maze apparatus consisted of four arms

(30x7 cm) made of black Plexiglas, which were placed at right angles to each other and elevated 50 cm above the floor. Two of the arms had 20 cm high walls (enclosed arms) , while other two had no walls (open arms) . The illumination at the center was adjusted to 40 lux. For the test, the mouse was initially placed at the center of the platform and left to explore the arms for 5 minutes. The number of entries in the open and in the enclosed arms and the time spent in each arm was recorded. Entry into each arm was scored as an event if the animal placed all four paws into the corresponding arm.

As a result, the number of entries into open and enclosed arms for hetero Tg-APP/Bβ transgenic mice at 7- months was similar to that of age-matched controls (Fig. 12A and B) . However, the number of entries and the time spent in the open arm for the homo and hetero Tg-APP/B6 transgenic mice at 13 months was less than that of age- matched controls (Fig. 12C and D) . The results indicate that Tg-APP/Bβ transgenic mice of the present invention show increased anxiety. In particular, the above results encouraged researchers in using the transgenic mice of the invention as a better animal model for AD. That is, the transgenic mice of the present invention are very useful as an animal model for the study of anxiety, one of the most characteristic pathological symptoms of AD, and for the study of emotional disorder in general.

<10-5> Passive avoidance test

Mice prefer darkness to lightness. When mice are allowed to choose one of the two chambers, one is lighted and the other is dark chamber, they have no hesitation to go for the dark chamber. Mice are once placed in a lighted chamber and then allowed to move to a dark chamber but a strong electric shock is given then (that is, a training) . After the training, when mice are forced to select a chamber to enter, most of wild type mice try to stay in a lighted chamber without the electric shock, even though unwillingly. Passive avoidance test is designed based on the above idea, and so the test is to investigate learning and memory retention through spatial information such as a lighted and a dark chamber, and an electric shock. Particularly, the test apparatus of the invention consisted of a brightly lit and a dark compartment (15^χl5^χl5 cm each) , each equipped with a shock-grid floor, and a door between the two chambers. During the first day of testing, each mouse was placed in the lighted chamber and left to habituate to the apparatus for 5 minutes, while allowing it to explore the light and dark rooms. On the second day, the mice were placed in the lighted chamber. After 30 seconds, the middle door was opened and the latency for the mouse to enter the dark chamber was measured. When the mouse entered the dark room, the door was closed and two successive electric foot-shocks (100 V, 0.3 mA, 2 seconds) were delivered through the grid-floor. After training, mice were individually replaced in the lighted chamber and the latency to enter the dark chamber was measured. As a result, wild type control mice at 13 months hesitated to enter the dark chamber, whereas the age- matched hetero Tg-APP/B6 transgenic mice entered the dark chamber again without hesitation (Fig. 12E and F) . And, homo Tg-APP/Bβ transgenic mice of the present invention at 13 months also showed similar cognitive deficits to the above heterozygous transgenic mice.

<Example 11> Analysis of the anxiety causing mechanism <11-1> Microarray analysis

The transgenic mice of the present invention were confirmed to show anxiety in the above <Example 10>. Thus, in order to find out the anxiety inducing mechanism in Tg- APP/B6 mice of the present invention, microarray analysis was performed to observe the changes on genomic DNA of amyglada of the brains of the mice. The cDNA microarray chip (Digital Genomics, Seoul, Korea) bearing 14,000 mouse genes (identified genes: 9,052) was used for the analysis. Amyglada were removed from five mice of each experimental group (the transgenic mice) and control group (wild type mice) , and total RNA was extracted. 15 βg of the total RNA extracted from each mouse was used as a sample for microarray analysis. Aminoallyl-dUTP (Sigma, Mo, USA) was used to label the RNA samples indirectly by fluorescent dye. Cy3/Cy5 fluorescent dye (AmershamPharmacia, NJ, USA) labeling and hybridization were performed according to the manufacturer's instruction (Digital Genomics, Seoul,

Korea) . Probe labeled with Cy3 and Cy5 was used for the hybridization and signals were detected at 532 nm and 635 nm with ScanArray (ScanArray 3.0; Packard, USA) . The image data with signals were analyzed using GenePix Pro

(GenePix Pro 4.0; Axon Instrument, Inc., CA, USA) . The comparison of gene expressions between wild type control mice and the transgenic mice of the present invention was made by the method of Ha et al (Ha et al., Toxicol Lett.

2003 146(1) :49-63, 2003) .

As a result, the expressions of TNF-α, T-LAK cell- originated protein kinase, hematopoietic cell specific Lyn substrate 1, staniocalcin, calmodulin 4, cytochrome c, type XVII procollagen alpha 1, prominin 1 and transthyretin were all increased at least 2-fold in amyglada of the transgenic mice of the present invention. On the contrary, the expressions of X- (inactive) -specific transcript, prostaglandin-endoperoxide synthase 1, crooked neck-like 1, dystrophin-associated glycoprotein alpha- sarcoglycan, chemokine (C-C motif) ligand 6, integrin alpha V, cullin 4B, klotho, cyclin F, amino acid N- acyltransferase and prostaglandin D2 synthase were reduced, compared to the wild type controls.

<ll-2> Investigation of a gene expression altered in the transgenic mice by RT-PCR RT-PCR was performed to re-confirm the result of microarray analysis carried out in the above <Example 11- 1>. Particularly, total RNA was extracted from amyglada of both Tg-APP/B6 at 13 months and age-matched wild type controls. RT-PCR was performed by the conventional method with primer sets that are able to amplify TNF-α, T-LAK cell-originated protein kinase, hematopoietic cell specific Lyn substrate 1, transthyretin, X- (inactive) - specific transcript, chemokine (C-C motif) ligand 6 and cullin 4B. Another RT-PCR was performed to quantify RNA by using primer sets that are able to amplify GAPDH.

As a result, the expressions of TNF-α, T-LAK cell- originated protein kinase, hematopoietic cell specific Lyn substrate 1 and transthyretin were increased in the transgenic mice of the present invention, compared to those in the wild type controls, while the expressions of X- (inactive) -specific transcript, chemokine (C-C motif) ligand 6 and cullin 4B were reduced, which were the same results as those produced by microarray analysis in the <Example 11-1> (Fig. 13) . [industrial Applicability]

As explained hereinbefore, the transgenic mouse generated in the present invention shows characteristic AD symptoms, such as cognitive and memory deficits, and increased anxiety, etc. Thus, the transgenic mouse can be used as an animal model for AD study. In particular, the transgenic .mouse of the present invention clearly shows increased anxiety, which is superior to any known conventional AD animal models, making it a promising model for the study of AD-related anxiety and also for the study of emotional disorder in general.

[Sequence List Text] The SEQ. ID. No 1 is a nucleotide sequence of a gene (hAPP751) coding amyloid beta precursor protein.

The SEQ. ID. No 2 is an amino acid sequence of amyloid beta precursor protein APP751 isoform that is encoded from the above nucleotide sequence represented by SEQ. ID. No 1.

The SEQ. ID. ^• No 3 is an amino acid sequence of amyloid beta precursor protein APP695 isoform.

The SEQ. ID. No 4 is an amino acid sequence of amyloid beta precursor protein APP770 isoform. The SEQ. ID. No 5 is an amino acid sequence of mutant amyloid beta precursor protein APP751 (Swe, VlIlF) of the present invention.

The SEQ. ID. No 6 is a nucleotide sequence of app-lf primer. The SEQ. ID. No 7 is a nucleotide sequence of app-lr primer.

The SEQ. ID. No 8 is a nucleotide sequence of app-2f primer.

The SEQ. ID. No 9 is a nucleotide sequence of app-2r primer.

The SEQ. ID. No 10 is a nucleotide sequence of^'app- swe primer.

The SEQ. ID. No 11 is a nucleotide sequence of app- 717-r primer. The SEQ. ID. No 12 is a nucleotide sequence of app- 717-f primer.

The SEQ. ID. No 13 is a nucleotide sequence of SV40pA-f primer.

The SEQ. ID. No 14 is a nucleotide sequence of SV40pA-r primer.

The SEQ. ID. No 15 is a nucleotide sequence of app- Koz-f primer.

The SEQ. ID. No 16 is a nucleotide sequence of hblog-f primer. The SEQ. ID. No 17 is a nucleotide sequence of hblog-r primer.

The SEQ. ID. No 18 is a nucleotide sequence of APP (- intron) -f primer.

The SEQ. ID. No 19 is a nucleotide sequence of APP (- intron) -r primer.

The SEQ. ID. No 20 is a nucleotide sequence of APP (+intron) -f primer.

The SEQ. ID. No 21 is a nucleotide sequence of APP (+intron) -r primer.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

[CLAIMS]

[Claim l]

A vector, for transformation of animals to induce Alzheimer's disease, that contains a gene coding mutant human amyloid beta precursor protein (APP) bearing Swedish mutation and Indiana mutation simultaneously.

[Claim 2]

The vector for transformation of animals to induce Alzheimer's disease pathology as set forth in claim 1, wherein the mutant human amyloid beta precursor protein

(APP) is selected from a group consisting of APP751(Sw,

V717F) in which lysine (K) , the 651^st amino acid of APP751 represented by SEQ. ID. No 2, is replaced with asparagine (N) , methionine (M) , the 652^nd amino acid, is replaced with leucine (L) , and valine (V) , the 698^th amino acid, is replaced with phenylalanine (F), APP695(Sw, V717F) in which lysine (K) , the 595^th amino acid of APP 695 represented by SEQ. ID. No 3, is replaced with asparagines (N) , methionine (M) , the 596^th amino acid, is replaced with leucine (L) and valine (V) , the 642^nd amino acid, is replaced with phenylalanine (F) and APP770(Sw, V717F) in which lysine (K), the 670^th amino acid of APP770 represented by SEQ. ID. No 4, is replaced with asparagines (N) , methionine (M) , the 671^st amino acid, is replaced

48 with leucine (L) and valine (V) , the 717^th amino acid, is replaced with phenylalanine (F).

[Claim 3] The vector for transformation of animals to induce Alzheimer's disease pathology as set forth in claim 1, wherein the mutant human amyloid beta precursor protein (APP) is represented by SEQ. ID. No 5.

[Claim 4]

The vector for transformation of animals to induce Alzheimer's disease pathology as set forth in claim 1, wherein the vector additionally includes a promoter and polyadenylation region.

[Claim 5]

The vector design for transformation of animals to induce Alzheimer's disease pathology as set forth in claim 4, wherein the promoter is human PDGF-beta promoter.

[Claim 6]

The vector for transformation of animals to induce Alzheimer's disease pathology as set forth in claim 4, wherein the polyadenylation region is SV40 pA.

49 [Claim 7]

The vector for transformation of animals to induce Alzheimer's disease pathology as set forth in claim 4, wherein the vector is designed to include human PDGF-beta promoter gene, a gene coding mutant human amyloid beta precursor protein represented by SEQ. ID. No 5 and SV40 pA in that order, and represented by the cleavage map PDGF- APP(Sw, V717F)-pA.

[Claim 8]

The vector for transformation of animals to induce Alzheimer's disease pathology as set forth in claim 4, wherein the vector additionally includes intron between a promoter gene and a gene coding mutant human amyloid beta precursor protein.

[Claim 9]

The vector for transformation of animals to induce Alzheimer's disease pathology as set forth in claim 8, wherein the intron is intron B that is derived from human beta-globin gene.

[Claim IO]

The vector for transformation of animals to induce Alzheimer's disease pathology as set forth in claim 9,

50 wherein the vector is designed to include human PDGF-beta promoter gene, intron B gene of human beta-globin, a gene coding mutant human amyloid beta precursor protein represented by SEQ. ID. No 5 and SV40 pA in that order, and represented by the cleavage map PDGF-intron-APP (Sw, V717F) -pA.

[Claim ll]

A transgenic mouse with induced Alzheimer's disease pathology generated by introducing the vector for transformation of animals of claim 1.

[Claim 12]

A transgenic mouse with induced Alzheimer's disease pathology generated by introducing the vector for transformation of animals of claim 10, in which the mouse is Tg-APP/Bβ showing clinical symptoms of Alzheimer' s disease such as motor coordination deficit, impaired memory retention, cognitive deficits and increased anxiety (Accession No: KCTC 10608BP) .

51