KR101431783B1 - Transgenic cloned caninds as models for alzheimer's disease and producing method thereof - Google Patents

Abstract

The present invention relates to a transgenic canine for Alzheimer's model that overexpresses a mutant human amyloid precursor protein (APP) and a method for producing the same. Transgenic canines for Alzheimer's model overexpressing the mutant human amyloid precursor gene according to the present invention are capable of production of live plants through crosses and are capable of subsequent delivery of the foreign gene and are capable of producing an expanded cerebral ventricle It has Alzheimer-like symptoms such as hippocampus, and the expression of APP, amyloid beta and tau is increased in the brain tissue. Thus, the immune response related to Alzheimer is induced, which can be used as a canine animal for the first Alzheimer model have.

Description

Transgenic canine models for Alzheimer's model and methods for producing the same

The present invention relates to a transgenic canine for Alzheimer's model that overexpresses a mutant human amyloid precursor protein (APP) and a method for producing the same.

Dementia is characterized by a general disorder of mental function such as learning and memory impairment and loss of judgment. It is a severe central nervous system disorder that causes human life to be devastated. It is mainly caused by old age, , Followed by stroke. Causes of dementia vary, but about 50% are classified as Alzheimer's disease, 20-30% as vascular dementia, 15-20% as Alzheimer's disease and vascular complex dementia, and other causes of dementia. The incidence of dementia in elderly people aged 65 or older is about 10%, and the incidence of dementia increases with age.

Dementia patients worldwide are rapidly increasing year by year, and it is expected that approximately 37 million patients will develop in 2025. Although the global dementia treatment market is estimated to reach 8 trillion won per year, there is no dementia treatment developed so far, and only dementia symptom relievers are sold worldwide.

Aricept (Pfizer), Exelon (Novartis) and Reminly (Janssen) are typical examples of estherase inhibitors that have been developed and marketed for the treatment of Alzheimer's disease. Acetylcholinesterase inhibitors do not cure the underlying cause of Alzheimer's disease, and some patients (about 20%) show a temporary relief of symptoms and their efficacy is not long-lasting, so they are defined as therapeutic agents in a strict sense Is difficult. Also, due to the nature of the disease, a long-term use of the drug is required. In the case of the above-mentioned medicines, it is revealed that accompanied with various side effects including liver toxicity. Therefore, it is urgent to develop a therapeutic agent that can prevent the progress of Alzheimer's disease.

The beta amyloid protein produced by APP, which is a precursor, aggregates and becomes toxic. The important enzyme involved in the production of this protein is beta or gamma secretase, and alpha secretase is known as an inhibitory enzyme. Therefore, the development of therapeutic agents targeting beta amyloid is aimed at the root cause of Alzheimer's disease, and has invested heavily in this field, including multinational pharmaceutical companies.

In order to identify the cause of Alzheimer's disease and to develop therapeutic substances, development of animal models is essential before clinical trials. Two types of demented mouse models such as 'Tg2575' and 'London type mouse' have been developed and used in the United States. However, experimental dogs and animals as a dementia model in which the human amyloid precursor gene is overexpressed cause Alzheimer It is not.

Accordingly, the present inventors have made efforts to develop transgenic canines as an animal model in which a human amyloid precursor gene is over-expressed to induce Alzheimer's disease, thereby completing the present invention.

Domestic Patent Application Number: 10-2004-0022562

The present invention provides nuclear transfer embryos necessary for producing transgenic canines for Alzheimer's model that overexpress the mutant human amyloid precursor protein (APP) of the present invention.

It is another object of the present invention to provide a method for producing transformed canine for Alzheimer's model using nuclear transfer embryos.

It is another object of the present invention to provide a transgenic canine for Alzheimer's model produced by the above method.

It is still another object of the present invention to provide a method for screening a therapeutic agent for Alzheimer's disease using the transgenic canine for Alzheimer's model.

In order to solve the above problem, the present invention provides a canine-derived somatic cell (accession number: KCTC 12242BP) transformed with a recombinant vector comprising a nucleic acid encoding a mutant human amyloid precursor protein (APP) of SEQ ID NO: 1, And the nucleus of the embryo is transferred to the enucleated oocyte to provide a canine embryo of the canine.

Also, the present invention provides a method for producing a transformed canine for an Alzheimer's model that overexpresses a mutant human amyloid precursor protein (APP) comprising a nuclear transfer embryo transferred to a tubulus of a surrogate mother to produce an embryo do.

The present invention also provides transgenic canines for Alzheimer's model that overexpress the mutant human amyloid precursor gene produced by the above method.

The present invention also provides a screening method for treating Alzheimer's disease using the transgenic canine for Alzheimer's model.

Transgenic canines for Alzheimer's model overexpressing the mutant human amyloid precursor gene according to the present invention are capable of production of live plants through crosses and are capable of transferring the external gene in the future, And the expression of APP, amyloid beta and tau is increased in the brain tissue. Thus, the immune response related to Alzheimer is induced, which can be used as a canine for the first Alzheimer model.

1 is a schematic diagram showing the structure of an open Thy-1 promoter.
Figure 2 shows the activity of a recombinant expression vector using various Thy-1 promoter portions.
Fig. 3 is a diagram showing a vector map of pGL3-Thy1-mhAPP-pCMV-EGFP / Neo recombinant expression vector.
FIG. 4 shows hAPP gene expression according to the type of vector in IMR-32 cells.
5 is a graph showing the expression of green fluorescence in transformed cells.
FIG. 6 is a diagram showing the transformed cells through PCR. FIG.
Fig. 7 is a diagram showing PCR-confirmed transgenic cloned dogs.
8 is a graph showing green fluorescence protein expression in transgenic cloned dogs. (A: appearance of transgenic cloned embryo, B: green fluorescence expression in claw, toe and white coat, C: pathological autopsy of cerebellum and brain stem, and D: green fluorescence expression in brain tissue)
9 is a diagram showing the expression pattern of mutant human APP gene and the APP gene in various tissues.
FIG. 10 is a diagram showing PCR results confirming the transfer of transgenic cloned dogs (male) and normal dogs (female), and the mutant human APP gene.
11 is an MRI diagram showing the brains of the control and transgenic cloned dogs.
12 is a view showing the brain phenotype of the control group and transgenic cloned dogs.
Fig. 13 shows the expression of Alzheimer-related molecular markers in the cerebrum and cerebellum of the control and transgenic cloned dogs.
Figure 14 shows mutant human APP expression in the cerebrum and cerebellum of the control and transgenic cloned dogs.
15 shows the expression of amyloid beta in the cerebrum and cerebellum of control and transgenic cloned dogs.
16 shows the expression of tau in the cerebrum and cerebellum of the control and transgenic cloned dogs.
FIG. 17 is a view showing immunohistochemical staining of the frontal region of the control and transgenic cloned dogs.
FIG. 18 is a view of immunohistochemical staining of hippocampal regions of the control and transgenic cloned dogs.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method for screening a nucleus of a canine-derived somatic cell (accession number: KCTC 12242BP) transformed with a recombinant vector comprising a nucleic acid encoding a mutant human amyloid precursor protein (APP) of SEQ ID NO: 1, Lt; RTI ID = 0.0 > of canines. ≪ / RTI >

The term " nuclear transfer " as used in the present invention refers to a genetic engineering technique in which nucleus-free cells are artificially bound to nuclear DNA of other cells to have the same traits.

As used herein, the term " nuclear transfer embryo " refers to an oocyte into which a donor nuclear cell has been introduced or fused.

The term " enucleated oocyte " as used herein means that the nucleus of the oocyte is removed.

The term "canine" in the present invention includes, but is not limited to, dogs, wolves, foxes, jackals, coyotes, snakeheads, raccoon dogs and the like.

Since the recombinant vector should be capable of overexpressing the APP gene in neural cells of canine animals, it is preferable that the recombinant vector is in the form of a recombinant expression vector. The recombinant expression vector may further comprise a promoter, secretory sequence, enhancer, upstream activation sequence, transcription termination sequence (for example, a promoter, ≪ / RTI > factor, etc.). The recombinant expression vector may include a selection marker, and the selection marker includes an antibiotic resistance gene such as a kanamycin resistance gene and a neomycin resistance gene, and a fluorescent protein such as a green fluorescent protein and a red fluorescent protein. Do not.

1, the nucleotide sequence of the mutant human APP gene of SEQ ID NO: 1, and the nucleotide sequence of SEQ ID NO: 2 (SEQ ID NO: 2) using commercially available pGL3-Basic (Promega Co., Madison, WI, EGFP and neomycin gene-introduced recombinant expression vector pGL3-pThy1-mhAPP-pCMV-EGFP / Neo, which contains Thy-1 promoter of EGFP and is regulated by the CMV promoter as a selection marker.

The donor nucleic acid cells for producing somatic cell nuclei derived from canine animals include embryonic cells, fetus cells, juvenile cells, adult cells and the like derived from canine animals. More specifically, the cells of the present invention include cumulus cells, epithelial cells, fibroblasts, neurons, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, erythrocytes, macrophages, monocytes, muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, Cells, embryonic cells, embryonic cells, embryonic cells, and the like.

The somatic cells provided as the donating nuclear source cells can be obtained from a method for preparing a surgical specimen or a specimen for biopsy, and from the specimen, single cells can be obtained using known methods.

The transformation can be carried out according to a known method, for example, calcium phosphate transfection, electrophoresis, transduction, DEAE-dextran mediated transformation but are not limited to, transfection, microinjection, cationic lipid-transfection, ballistic introduction, and the like.

For the production of the enucleated oocyte, enucleation of the recipient oocyte may be carried out.

Methods for determining the ovulation-free period in canines for recovering the recipient oocytes include, for example, a method of determining when the keratinized epithelial cells are confirmed to be more than 70% by vaginal cell smear examination as the optimal period, A method of confirming the growth and development status of follicles in real time, and a method of determining the timely period by measuring plasma progesterone.

As the method for recovering the recipient oocyte, a surgical method including anesthetizing the subject animal and then lapping it can be used. More specifically, recovery of recipient oocytes in vivo can be accomplished by tubal resection, a method known in the art.

The nucleation process can be carried out using methods known in the art (U.S. Patent No. 4,994,384, U.S. Pat. No. 5,057,720, U.S. Pat. No. 5,945,577, European Patent Publication No. 0930009A1, Korean Patent No. 342437, Kanda Nagashima et al., Mol. Reprod. Dev. 48: 339-64, 1995, Willadsen, Nature, Prather et al., Biol. Reprod 41: 414-418, 1989, Prather et al., J. Exp. Zool. 255: 355 Saito et al., Assis Reprod Tech Andro, 259: 257-266, 1992; Terlouw et al., Theriogenology 37: 309, 1992). Preferably, a cumulus cell of a mature recipient oocyte is removed, and a portion of the zona pellucida of the recipient oocyte is cut using a micro needle to form a incision window, through which the nucleus and cytoplasm of the first polar body, , Removing the cumulus cells of the recipient oocyte, dyeing the oocyte, and removing the nucleus of the first polar body and the oocyte using a micropipette aspiration pipette. More preferably, The Enucleation (Squeezing) method can be used to form an incision in the oocyte.

The nuclear transfer can be performed by injecting donor nuclear cells between the cytoplasm and the zona pellucida of the enucleated oocyte using an implantable pipette. It is preferable that the enucleated oocytes in which the microinjection of the donor nucleic acid cells are completed are electrically fused with the donor nuclear cells using a cell manipulator. In electrical fusion, the current may be alternating current or direct current, and may be performed one to three times at a voltage of 1.5 to 4.0 kV / cm, for a time of 15 to 45 占 퐏.

The fused nuclear transfer embryo can be further subjected to an activation step.

Activation of fused nuclear transfer embryos implies a process of resuming the cell cycle that stops in the middle of the second meiosis in vitro maturation. For this purpose, high activity of MPF, CSF, etc., which stop the cell cycle, should be lowered, and low activity of APC which promotes metastasis from the middle stage of the second meiosis should be increased. In general, in order to enable the nuclear transfer embryos, and to increase the concentration of intracellular Ca ^{2 +} ions it should induce chromosomal condensation and embryonic development, the physical method To this end, chemical methods, or an electrical method is used. Physical methods include mechanical stimulation, heat, and direct current. By chemical means a substance, such as ethanol, inositol triphosphate, Ca ^{+ 2} or Sr ^{+ 2,} Saito collar Shin B, calcium child Ono fore, 6-dimethyl amino purine, cyclohexyl during mid, ball four 12-myristate 13-acetate . ≪ / RTI > As an electrical method, there is a method of performing 1-3 times for 30 to 60 으로 at a DC voltage of 1.5 to 2.5 kV / cm.

Also, the present invention provides a method for producing a transformed canine for an Alzheimer's model which overexpresses a mutant human amyloid precursor protein (APP) comprising the step of transplanting the nuclear transfer embryos into a tubal duct of a surrogate mother, to provide.

The present invention also provides transgenic canines for Alzheimer's model that overexpress the mutant human amyloid precursor gene produced by the above method.

Transgenic canines for Alzheimer's model overexpressing the mutant human amyloid precursor gene according to the present invention are capable of production of live plants through crosses and are capable of transferring the external gene in the future, And the expression of APP, amyloid beta and tau is increased in the brain tissue. Thus, the immune response related to Alzheimer is induced, which can be used as a canine for the first Alzheimer model.

(A) treating the transgenic canine with a test substance;

(b) analyzing brain tissue of the transgenic animal of step (a); And

(c) analyzing brain tissue in step (b), wherein the expansion of the ventricle, atrophy of hippocampus, expression of amyloid beta protein, or expression of tau protein is compared with normal canine animal and analyzed; .

The test substance of step (a) may be an antisense nucleic acid, an siRNA, a miRNA, an aptamer, a polypeptide, a protein, a compound, or the like which is presumed to have the possibility of treating Alzheimer's disease or randomly selected according to a conventional selection method Extracts or natural products.

Hereinafter, the present invention will be described in detail with reference to Examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example  1. Mutant human APP  Preparation of Recombinant Expression Vector Containing Gene

For selective expression in the neural tissue of the mutant human APP (mhAPP) gene, four Thy-1 promoters were used. The structure of the Thy-1 promoter with two tissue-specific expression sites is shown in FIG. Using genomic DNA isolated from beagle fibroblasts, various parts of the Thy-1 promoter (-2.0 to +2.3 (k nts), +1 = transcription start site) were subjected to long-range PCR (LA Taq; TaKaRaBio., Inc ., Shiga, Japan). This was inserted into a promoter-free pGL3-Basic vector (Promega Co., Madison, WI, USA) using the MluI restriction enzyme site.

In addition, cassettes expressing human APP genes, including two distinct mutations, Swedish (K670N and M671L) and London FAD (V717F), which are known to induce AD-like pathological symptoms in mouse models . This was inserted into the recombinant pGL3-Basic vector containing the Thy-1 promoter using NcoI and XbaI restriction enzyme sites.

For the selection of specific sites of the Thy-1 promoter, luciferase activity assays were performed in human neuroblastoma, IMR-32 cells. The results are shown in Fig.

As shown in FIG. 2, it was confirmed that the recombinant vector containing the Thy-1 promoter (SEQ ID NO: 2) of -1.5 kb to +2.3 kb showed the highest promoter activity.

Therefore, in the pGL3-pThy-1 (-1.5 kb to +2.3 kb) vector, EGFP (Enhanced green fluorescent protein gene) and neomycin gene (EGFP) linked with IRES (internal ribosome entry site) And these genes were designed to be regulated by the CMV promoter.

 The vector map of the final product pGL3-pThy1-mhAPP-pCMV-EGFP / Neo vector is shown in FIG. 3, and the activity in IMR-32 cells is shown in FIG.

As shown in Fig. 4, it was confirmed that the recombinant expression vector of the present invention showed mhAPP expression ratio twice as high as that of the control vector pGL3-Basic vector.

Example  2. dog Donating nuclear resource  Establish fibroblasts

Establishment of donor cells was established as reported by Hossein et al. (Animal reproduction science, 2009, 114 (4), 404-414). More specifically, fetal fibroblast was recovered from a fetus separated from cesarean section at 30 days of pregnancy through artificial insemination and replication, and the adult cells were biopsied in the abdominal skin tissue of the adult dog. The establishment of the repopulated fetal fibroblast was confirmed by pregnancy through the transplantation of the cloned column and ultrasound of the fetus at 30 days of age. When pregnancy was confirmed, fibroblasts were established from fetuses separated from cesarean sections in the dogs. The pGL3-pThy1-mhAPP-pCMV-EGFP / Neo vector prepared in Example 1 was cut into NotI and introduced into the fibroblast. Subsequently, the cells were cultured in a medium containing G-418 (350 ug / ml) to grow only the cells having the introduced gene. Cells resistant to G-418 were subcultured for 4 weeks and then the expression of EGFP was confirmed by fluorescence microscopy in order to confirm whether cell selection was normal. The results are shown in Fig.

As shown in FIG. 5, it was confirmed that fluorescence expression was observed in the recombinant fibroblasts into which pGL3-pThy1-mhAPP-pCMV-EGFP / Neo vector introduced in Example 1 was introduced.

PCR was also performed to confirm the insertion of intracellular pGL3-pThy1-mhAPP-pCMV-EGFP / Neo vector. Primer (A, B) for identifying the screening cassette and primer (C, D) sequence for identifying the expression cassette are shown in Table 1 below, and PCR results using the primer (C, D) sequence are shown in FIG.

Primer A Forward CCT TGT GCT GTC TCC CCC TC Primer B Reverse TCA CAA AGT GGG GAT GGG TC Primer C Forward CAT GAA GCA GCA CGA CTT CT Primer D Reverse CCT AGG AAT GCT CGT CAA GA

As shown in Fig. 6, it was confirmed that the screening cassette and the expression cassette were simultaneously inserted into the recombinant fibroblast.

The transfected cells obtained from the above process were deposited with KCTC 12242 BP on July 17, 2012, under the provisions of the Budapest Treaty on the International Recognition of Microorganism Deposit, Korea Research Institute of Bioscience.

Example  3. mhAPP  Over-expression transformation Of a cloned dog  production

Donor nuts that will provide mature oocytes The surrogate mothers to be transplanted are 1-6 years old and 20-25kg in weight. The used dogs are fed free of feed for general breeding The breeding was managed. The management of all experimental dogs was carried out in accordance with the experimental animal ethics regulations of the Suan Institute of Bioscience and Biotechnology. The production process of mhAPP-overexpressed transgenic cloned dogs was carried out according to the procedure disclosed in Korean Patent Application No. 10-2009-0038315 filed by the applicant of the present invention.

More specifically, after checking the estrous cycle of the bitches every week, when the ovulation time approaches, 2 ml of blood is extracted daily to separate the serum. Serum progesterone concentrations were determined using ECLIA from Cobas E411 (Roche Diagnostics, USA). When serum progesterone concentration reached 4.5 ng / ml, mature oocytes were recovered by TCM 199 medium through a surgical procedure. As described by Hossein et al. (Animal reproduction science, 2009, 114 (4), 404-414), cumulus cells of recovered in vitro matured oocytes were isolated for somatic cell nuclear transfer. Using the BTX Electro-Cell Manipulator 2001 (BTX Inc., San Diego, Calif., USA), the donor nuclei prepared in Example 2 were injected into the worm cells of mature oocytes in which the cumulus cells were removed through micro- kV / cm for 15 μsec.

The nuclear transfer embryos were loaded on a Tom Cat catheter (Sherwood Medial, St. Louis, Mo., USA) and then transplanted into the surrogate embryo of the surrogate mother. A total of 17 dogs were transplanted. Ultrasonographic findings were confirmed at 4 and 30 days, and finally 6 cloned dogs (AM141 ~ 146) were obtained.

Example  4. mhAPP  Over-expression transformation Of a cloned dog  Confirm

PCR was performed using the primers A to D used in Example 2 to identify transgenic cloned dogs overexpressing mhAPP in the cloned dogs produced in Example 3. The results are shown in Fig.

As shown in Fig. 7, out of the six cloned dogs, except for AM145, five transgenic cloned dogs overexpressing mhAPP were identified.

Further, in order to re-verify the above results, it was confirmed whether EGFP was expressed in individuals identified as transgenic cloned dogs overexpressing mhAPP. The results are shown in Fig.

As shown in FIG. 8, it was confirmed that EGFP was normally expressed in the claws of the individual (AM144) identified as transgenic cloned dogs overexpressing mhAPP, white hair of hairy hairs, and brain.

In addition, RNA samples were extracted from various organs of transgenic cloned dogs (AM144) overexpressing mhAPP and wild-type cloned dogs (AM145), a littermate thereof, and RT-PCR was performed. The results are shown in Fig.

As shown in FIG. 9, the APP mRNA was detected in both organs of two individuals, but the mhAPP gene expression was observed only in the transgenic cloned dog (AM144) overexpressing mhAPP.

From the above results, it was confirmed that the transgenic cloned dog overexpressing mhAPP was normally produced.

Example  5. mhAPP  Over-expression transformation From cloned dogs  Verification of Alzheimer's disease

5-1. Lion  Production and Genetic Validation

AM166 and normal female dog (AF165), which were confirmed as transgenic cloned dogs overexpressing mhAPP in Example 4, were crossed to produce live weeds, and the mhAPP gene was stably transferred to the host A through D were used for PCR. Its pedigree and PCR results are shown in Fig.

As shown in FIG. 10, four mothers and three females were produced through crosses (SB 16 to 22), and all of them except SB21 were found to have mhAPP gene.

5-2. Brain tissue observation

In order to confirm whether the produced mhAPP overexpressed transgenic clone could be used as an animal model of Alzheimer's disease, the brain of a transgenic clone (SB17) overexpressing mhAPP and a general cloned dog (SB21) imaging, 7.0T). The results are shown in Fig.

As shown in FIG. 11, mhAPP transgenic cloned dogs showed enlarged cerebral ventricles and hippocampus, which are similar to Alzheimer's disease, compared with control mice.

In addition, the brain of the individual was extracted to analyze whether it represents a pathological Alzheimer's phenotype. The results are shown in Fig.

As shown in Fig. 12, similar to the results of the MRI, mhAPP transgenic cloned dogs showed enlarged ventricles and atrophied hippocampus in comparison with the control group.

Through the above results, it was confirmed that the mhAPP transgenic cloned dog had Alzheimer-like symptoms.

5-3. Alzheimer-related molecules Marker  analysis

Immunoblotting was performed from cerebrum (cerebrum) and cerebellum (cerebellum) to analyze the characteristics of mhAPP transgenic cloned dogs and to analyze the molecular mechanisms associated with Alzheimer-like symptoms. The results are shown in FIG. 13 to FIG.

As shown in Figs. 13 to 16, mhAPP transgenic cloned dogs showed increased expression of amyloid beta (A [beta]) and tau protein, which are typical characteristics of APP and Alzheimer's in the cerebrum and cerebellum, Respectively.

From the above results, it was confirmed that Alzheimer-like symptoms such as enlarged ventricle, atrophied hippocampus, and abnormal behavior are caused by accumulation of Aβ and tau in the brain of transgenic cloned dogs overexpressing mhAPP.

5-4. Histological characterization

Frontal cortex and hippocampal region were examined by immunohistochemical analysis to analyze the histological characteristics of mhAPP transgenic cloned dogs. The results are shown in Fig. 17 and Fig.

As shown in Fig. 17, Aβ-plaque-like structure was observed in the frontal lobe portion of the mhAPP-overt transgenic cloned dog compared to the control group. As shown in FIG. 18, Aβ-specific staining of the hippocampal region of the mhAPP-overexpressed transgenic cloned dog revealed that a large amount of Aβ accumulation was observed in the frontal lobe of mhAPP-overexpressed transgenic cloned dogs compared to the control group, (microglial marker) Iba1 staining revealed that microglial cells were also observed.

From the above results, it was confirmed that an Alzheimer-related immune response was induced by accumulation of A [beta] in transgenic cloned dogs overexpressing mhAPP.

Thus, it was confirmed that the mhAPP overexpressed transgenic clone of the present invention can be used as the first Alzheimer canine animal model.

Korea Biotechnology Research Institute KCTC12242BP 20120717

<110> HBION CO., LTD. <120> Transgenic cloned caninds as models for alzheimer's disease and          producing method thereof <130> suam <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 2256 <212> DNA <213> human Amyloid Precursor protein <400> 1 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt tcgagaggtg tgctctgaac aagccgagac ggggccgtgc 900 cgagcaatga tctcccgctg gtactttgat gtgactgaag ggaagtgtgc cccattcttt 960 tacggcggat gtggcggcaa ccggaacaac tttgacacag aagagtactg catggccgtg 1020 tgtggcagcg ccattcctac aacagcagcc agtacccctg atgccgttga caagtatctc 1080 gagacacctg gggatgagaa tgaacatgcc catttccaga aagccaaaga gaggcttgag 1140 gccaagcacc gagagagaat gtcccaggtc atgagagaat gggaagaggc agaacgtcaa 1200 gcaaagaact tgcctaaagc tgataagaag gcagttatcc agcatttcca ggagaaagtg 1260 gaatctttgg aacaggaagc agccaacgag agacagcagc tggtggagac acacatggcc 1320 agagtggaag ccatgctcaa tgaccgccgc cgcctggccc tggagaacta catcaccgct 1380 ctgcaggctg ttcctcctcg gcctcgtcac gtgttcaata tgctaaagaa gtatgtccgc 1440 gcagaacaga aggacagaca gcacacccta aagcatttcg agcatgtgcg catggtggat 1500 cccaagaaag ccgctcagat ccggtcccag gttatgacac acctccgtgt gatttatgag 1560 cgcatgaatc agtctctctc cctgctctac aacgtgcctg cagtggccga ggagattcag 1620 gatgaagttg atgagctgct tcagaaagag caaaactatt cagatgacgt cttggccaac 1680 atgattagtg aaccaaggat cagttacgga aacgatgctc tcatgccatc tttgaccgaa 1740 acgaaaacca ccgtggagct ccttcccgtg aatggagagt tcagcctgga cgatctccag 1800 ccgtggcatt cttttggggc tgactctgtg ccagccaaca cagaaaacga agttgagcct 1860 gttgatgccc gccctgctgc cgaccgagga ctgaccactc gaccaggttc tgggttgaca 1920 aatatcaaga cggaggagat ctctgaagtg aacctggatg cagaattccg acatgactca 1980 ggatatgaag ttcatcatca aaaattggtg ttctttgcag aagatgtggg ttcaaacaaa 2040 ggtgcaatca ttggactcat ggtgggcggt tttgtcatag cgacagtgat cgtcatcacc 2100 ttggtgatgc tgaagaagaa acagtacaca tccattcatc atggtgtggt ggaggttgac 2160 gccgctgtca ccccagagga gcgccacctg tccaagatgc agcagaacgg ctacgaaaat 2220 ccaacctaca agttctttga gcagatgcag aactag 2256 <210> 2 <211> 3780 <212> DNA <213> Thy-1 Promoter <400> 2 gcccctagac tggaccatcc ttgcagctca ttccagttag aaaggtccat cactctgtgt 60 tctgggaggt cacctaacct cccagggagg gagagggaag tgaatctccc acttgctggc 120 ccagggatga cttccaacag tagcactgcg gtaatggaaa ctgcagttaa ggtgccagga 180 ctgacttctg tgaagaaata ggagaatggg catcaggcgg cagagaggca gggctagccc 240 acaggatgcc ctggccagaa ttcaccaaga ggccatgggg atgcaaatca aacaaataaa 300 ccttcaaaaa acaatccact tcacctgtct gtgggagaga caagtcattg gcactggcca 360 tgaaaaaata atataaaata aaagatccaa gggcttatgg tgacttcaaa ttgatagggc 420 gagggcgggt gccccatggg atttaaggct ggtttcctct attccccttt cccacagttc 480 cacagcggcc cctcccacct ggctgggata tagcccatag agtggctcag gactgaggac 540 atggggttcc acagcccaga ccaatcggcc aggtctgcca cttactagcc atgaaacagg 600 atggggagtg gaatgggatg gacccagcat attggtgtgg ggtcaacaca gccatggatg 660 taatatgccc agcacagtgc ttggtggaaa acagtacccc tcttgtctga agaccaaaca 720 agcagacact ccccaggaac gaggagtagg aaagggagtg tcaggaggag aatatgagaa 780 gaaccttcta gctgtctgct ccaggagggg ccagcccagg aatgagggag actgagtgcc 840 ccagagcaaa agcccctgag gggtggggcg tcctgggacg gatgggggag tggagcaggg 900 ctcaggacca ggaaggacag gatgcagggc ctgccttgct tctgaagggc tgctccaatg 960 tggaaaaaca ccccaccatc ttcctttggg gaaagcctgg aatattccaa ctccaaaact 1020 tctcactgag gctgcaggga ggtgggctcc cgcagaaaag gaaaggagga ggcgtgagga 1080 ggcgtgggca cgggccaaag gggcgggttg ctggcctgca acccccactc cttataaccc 1140 cctcggtttt cccacaggct tctgaagcct aggtcagcag aagggattaa agccttaaaa 1200 ggggagcaat cgtcttgggg tttctgggag gcaactagcg ttcccggcac tgccccaccc 1260 tgatcctcgg ggccctgaac cctcccatcc cggcgcggcc tgccagcccg agggtgaggt 1320 cccccttctg cggcagccgg cggctcccgc atccccaccg ccgcctcccc ctcgctgggc 1380 caggctgcct cgccccccct cggcctctga ttggccgcgt cccgggccct ccccgctcct 1440 ccactcccac ccccggtgaa aactgcgggc gcgggcaggg agctgcaacc ggaggcggcg 1500 ggcgcggcgg ggaggctgcg gcggcggagg acgcgagccc aggtgcgagg ggagccgggc 1560 gggacccggg gcgccggaga ggccccagcc ctcccgaccc aggcttcggg gagcaggttc 1620 ggggcgcccc ggggaggagc cccgcggtgc tgggggaggg gtctgcctcc caggcagcgg 1680 gccgggccgg ccgcaccccg ccctgcaccc cgccctgcgc gcaaggcgcc ccccggcttt 1740 cacccgcccg agggccgccg cctgggccgg ggcgcagaag gggaaccggc acgaagggcg 1800 ccgagggcgc ctggcatccc gcgctccacg caggcgtccc caaacccagc tcgcgggggc 1860 ggggggcggg ggggcggtga aactgcggga ggccgcggat gagggagctg gggggctctc 1920 tgccccggac cccgcggggc gggctgggtg cggggcgtca aaggacaggg aaaccgcagt 1980 gccgcgggcg gggactggaa ggcgggcgga cgggggaggg ggggccggga aaggctgagg 2040 ggggaagggg gacctcggtt gggagaggag acgggtggaa gacggaagcg gaatgagggt 2100 cgggctagtg gaagggggca gggagcgcag aacttctccg ggttctgggc tggggacacc 2160 tggcagagga aatgcaaacc tctgtgcctc cgccgcagct ctccagccat cccttggctc 2220 tccatccctc tccagcccca gccctatttc tttccggttc tggcagaggc gccccctttt 2280 ccctcctaga tctcaagctc acacccctcc tccataactc gtggtctgca aaccaaagcc 2340 agatgccaac tccccgttcc ccgacagcac ccccacggcc cttttggtta tatttccgag 2400 agtgtacaga ctgcagcccg gggactgtgg gaaccccggc atcctcccca cccccacccc 2460 ccatggcctt ggatgcgcca gggtgcacct aggcatgggc atcagggcag ggcccctcct 2520 gtgtggcctc tccctggcca gcaccagggc agacgggcca ggctccctgc tctgtgtcac 2580 caggatgctg gtccagacta ggcttcgaaa aaaagggaga gcacagagct taggcaggga 2640 gtctattgaa gccggactgc aacactgcag gcccaggagg tctgcccaga gagctggaca 2700 tttggaaacg tctcggttcc tatgcaatca gagaacatat gcaatcagag agcactgtca 2760 caggccctta gctgtgtgcc aagtacaggc ccatctggaa gcaccttagc cacacagccc 2820 tttctagagg agggaaatta acatttgtgg ccccgctacg gagcagagca gtcatagtgc 2880 gacgtgatgt cagttttctt aatccccatc acaacaaccc tgtggccatc taaagctcat 2940 tcattcattc acacactcac tatcttttga gcatctatta tacaccagga actgtttggg 3000 cattggggat tttcttctga acaagaaaca gaaaagtccc caaatgggga gttcttgtgt 3060 aatgggtaga ggtttaagtg ttgcaagata aaaagagctc tggagattgc ctgcacaaca 3120 atgcgagtgc acttaacacc actgaattgt acactttaaa atgattaaga taggggcgcc 3180 tgggtggcac agttggttaa gcaaccgact cttggtttca gttcaggttg tgatctcagg 3240 gtcctgagat caagcaccga ggcacatcac gccaggcagg gggcagggaa tctccagatt 3300 ctctccccct ccttctcttc tccccctccc tccctccctc ctcccctctg ctccccaccc 3360 ccttgtgctg tctccccctc tctctctcta aaataaataa acaagtcttt ttaaaaaatg 3420 attaagatag taaattttgt gttagctctg ctttgcctca attaggaaat aggatgtctc 3480 tgccttcctg aagggaaaag gaaatggtcc aagtcactac tccaagcttt ccacactgac 3540 acacaccatc atagggtgtg ttctccctcg ccttcctttc ttccaggggg aaggcaccaa 3600 gtggcagtgt cacattcact cataagtggg aaggggggga accctaaggc catgcatgcc 3660 tgccatggtc atcatggcat gtgtcccaga ggcccccaac cctagtagta agtgtggaca 3720 cacctgccct atctcttggc tgattcccaa cctgtcccac ccccagatca aggactgagc 3780                                                                         3780

Claims (7)

The nuclei of somatic embryos (Accession No. KCTC 12242BP) transformed with a recombinant vector containing a nucleic acid encoding the mutant human amyloid precursor protein (APP) of SEQ ID NO: 1 were inserted into the enucleated oocyte, , Nuclear transfer eggs. 2. The nucleus-transferred embryo of claim 1, wherein the recombinant vector comprises the Thy-1 promoter of SEQ ID NO: 2. The nucleus-transferred oocyte as claimed in claim 1, wherein the recombinant vector is pGL3-pThy1-mhAPP-pCMV-EGFP / Neo vector represented by the following vector map.

delete A transgenic animal model for overexpressing a mutant human amyloid precursor protein (APP), comprising the step of transplanting a nuclear transfer embryo according to any one of claims 1 to 3 into a tubal duct of a surrogate mother, A method for producing transgenic dogs. Transgenic lines for Alzheimer's model over-expressing mutant human amyloid precursor genes produced by the method of claim 5. (a) treating the transgenic can of claim 6 with a test substance;
(b) analyzing the transformed brain tissue of step (a); And
(c) The brain tissue analysis in step (b) is a step of comparing cerebral ventricle enlargement, hippocampus atrophy, amyloid beta protein expression or tau protein expression with normal dogs A method for screening a therapeutic agent for Alzheimer's disease.

Images