KR102012812B1 - Vector comprising a combined dual Parkinson gene and disease model using the same - Google Patents

Abstract

The present invention relates to a vector comprising a complex Parkinson gene and a Parkinson's disease model using the same. More specifically, a vector for producing Parkinson's disease model comprising a human-derived PINK1 (G309D) mutant gene and an alpha-synuclein (α-synuclein (A53T)) mutant gene, a transformed complex gene into which the vector is introduced. Mutant cell line (Accession Number: KCTC1330BP), the cell line relates to a Parkinson's disease model transformed using somatic cloning techniques.

Description

Vector comprising a combined dual Parkinson gene and disease model using the same}

The present invention relates to a vector comprising a complex Parkinson gene and a Parkinson's disease model using the same. More specifically, a vector for producing Parkinson's disease model comprising a human-derived PINK1 (G309D) mutant gene and an alpha-synuclein (α-synuclein (A53T)) mutant gene, a transformed complex gene into which the vector is introduced. Mutant cell line (Accession No .: KCTC1330BP), the cell line relates to a Parkinson's disease disease model transformed using somatic cloning techniques.

Parkinson's disease is a type of neurodegenerative disorder that causes tremors (tremors), muscle stiffness (rigidity), and motion slowdown (slowness). , bradykinesia) is a typical symptom. Most Parkinson's disease develops tremors from one arm and leg, then slows down and becomes stiff and gradually progresses to the opposite limbs.

Patients with Parkinson's disease show a decrease in mitochondrial respiratory complex I activity in substantia nigra in more than 85% of patients at postmortem necropsy. Parkenson's environmental causative factors, such as rotenone, paraquat, and MPTP, are known to target neurons, mitochondria, and induce neuronal death.

Mitochondria can be easily damaged by free radicals compared to other organelles because of their lack of intracellular reactive oxygen (ROS) -removing enzymes compared to the cytoplasm and lacking histone proteins unlike nuclear DNA. have. In addition, since more than 90% of the ROS generated in the cell is produced in the mitochondria, when the mitochondria are damaged, intracellular reactive oxygen species are further increased, leading to cell death.

Thus, there is a method of preparing a mouse model by directly injecting 6-OHDA (6-hydroxydopamine), which causes rotenone, parakuwat, MPTP, or reactive oxygen species, or systemically. However, this method causes toxicity to other sites or tissues other than dopaminergic neurons and has a problem in performing repetitive experiments due to poor reproducibility.

In order to study intractable diseases in humans, model animals with intractable diseases are needed, and experimental animals, such as model animals, are being used for mechanism identification, treatment development, new drug development, diagnostic technology development, and related medical device development.

As a model for mimicking human Parkinson's disease, 1) Parkinson's disease can be predicted according to the desired time and age, 2) Parkinson's disease caused by the complex action of various causes, and 3) Human Parkinson's disease Since the progression of the pathology and the pathological mechanisms coincide with each other, there is an urgent need for an animal model that can be applied to the treatment of Parkinson's disease and its application.

There are various experimental models for Parkinson's disease, ranging from fruit flies to mouse and rat models, but there are no animal models showing similar symptoms and pathology to human Parkinson's disease.

Because neurodegenerative diseases are caused by various causes, animal models using a single gene are known to be very different from human Parkinson's disease. It is best to produce animal models of primate Parkinson's disease that are similar to humans, but there are very limited problems in terms of price and facilities. Therefore, the production of disease animals using dogs among small and medium animals that can replace primates has become very important.

Background art of the present invention is Korean Patent Publication No. 10-2015-0145201 (2015.12.29.), The patent discloses a transgenic pig for Alzheimer's disease model and its use.

Under these technical backgrounds, the present inventors have made a diligent effort to develop a cell line incorporating the human-derived Parkinson mutant genes PINK1 and α-synuclein together.

Therefore, an object of the present invention is to provide a vector for producing Parkinson's disease model.

In addition, an object of the present invention is to provide a transformed complex gene mutant cell line by introducing a vector for producing the Parkinson's disease animal model.

In addition, an object of the present invention is to provide a transformed Parkinson's disease model using the complex gene cell line using somatic cloning techniques.

Another object of the present invention is to provide a method for producing a Parkinson's disease model using the cloned eggs.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, there is provided a vector for producing Parkinson's disease model comprising a PINK1 (G309D) mutant gene and an alpha-synuclein (α-synuclein (A53T)) mutant gene.

According to one embodiment of the present invention, the PINK1 (G309D) mutation is Gly substituted with Asp at position 309 of the PINK1 gene, and the alpha-synuclein (α-synuclein (A53T)) mutation is alpha-synuclein (α) -synuclein (A53T)) Ala is a mutation in which Ala is substituted with Thr at position 53 of the gene for Parkinson's disease model production vector is provided.

According to one embodiment of the invention, the vector is provided with a vector for Parkinson's disease model production, characterized in that the retroviral vector.

According to one embodiment of the present invention, Parkinson's disease animal model is provided with a vector for producing Parkinson's disease model, characterized in that the individual.

According to another aspect of the present invention, a complex gene mutant cell line (Accession Number: KCTC13310BP) transformed with the vector for producing the Parkinson's disease animal model is provided.

According to one embodiment of the present invention, the cell line (Accession No .: KCTC13310BP) is provided with a transformed complex gene mutant cell line (Accession No .: KCTC13310BP), which is derived from fibroblast cells of a mammal.

According to one embodiment of the invention, the mammal is provided with a transformed complex gene mutant cell line (Accession Number: KCTC13310BP), characterized in that the individual.

According to one embodiment of the present invention, the PINK1 (G309D) gene and alpha-synuclein (α-synuclein (A53T)) is a transformed complex gene mutant cell line (Accession Number: KCTC13310BP), characterized in that derived from human Is provided.

According to another aspect of the present invention, transformed Parkinson's disease model is provided using the complex gene cell line using somatic cloning techniques.

According to another aspect of the invention, the step of introducing a PINK1 (G309D) and α-synuclein (A53T) mutant gene into a vector; Transforming a mammalian cell using the vector; And a nuclear transfer step of introducing the transformed cells into denuclearized embryos.

According to an embodiment of the present invention, there is provided a method for producing a cloned egg, comprising the step of fusing the nucleus and the cytoplasm with a fusion voltage of 35 ~ 55V in a fusion method using an electrode.

According to one embodiment of the invention, there is provided a method for producing a cloned egg comprising a nuclear substitution step exhibiting a fusion rate of at least 55%.

According to another aspect of the invention, there is provided a method for producing a Parkinson's disease model comprising the step of transplanting the cloned eggs into surrogate mothers.

According to an embodiment of the present invention, there is provided a method of producing a Parkinson's disease model, characterized in that the vector is a retroviral vector.

According to an embodiment of the present invention, there is provided a method for producing a Parkinson's disease model, characterized in that the mammal is an individual.

According to another aspect of the present invention, there is provided a method for screening a Parkinson's disease preventive or therapeutic agent using the Parkinson's disease model produced according to the production method.

The present invention can provide an animal model using a complex gene capable of mutating two Parkinson-related human genes to show symptoms and pathological findings similar to human Parkinson's disease.

The present invention can provide the basic data for the expansion of new neurodegenerative technology and development of new neurodegenerative disease model by the successful development of human complex Parkinson gene loaded dog cell line technology.

Furthermore, the Parkinson's disease model of the present invention can be used in various ways, such as the identification of Parkinson's disease mechanism, the search for a therapeutic substance, the development of a diagnostic method, it will be very useful as a Parkinson's disease model.

1 is a diagram showing the results of RT-PCR with fibroblast specific markers in order to analyze the characteristics of individual cells.
Figure 2 shows the results of analyzing the similarity between the human PINK1 Parkinson gene and dog Parkinson gene species.
Figure 3 shows the results of analyzing the similarity between the human α-synuclein Parkinson gene and dog Parkinson gene species.
4 is a schematic diagram showing a retrovirus vector carrying a human complex Parkinson gene.
5 is a diagram showing the results of chromosome analysis (Karyotyping) analysis of human complex gene-loaded cells.
6 is a diagram showing the results of RT-PCR in order to analyze the expression of human complex Parkinson gene in the individual cells (cFF2, PS) loaded with the complex gene.
7 is a diagram showing the results of immunofluorescence staining in order to analyze the expression of human complex Parkinson's protein in individual cells (cFF2, PS) loaded with the complex gene.
8A and 8B are graphs showing the results of real-time PCR in order to analyze the number of Parkinson's complex genes inserted into chromosomes, respectively, and graphs of the number of Parkinson's complex genes copied.
9 is a view showing the results of the test whether the gene introduced Parkinson's disease model dog according to an embodiment of the present invention.
Figure 10 is a view showing the paternity discrimination of dogs Parkinson's disease model according to an embodiment of the present invention.
Figure 11 is a grown picture of dog Parkinson's disease model produced according to an embodiment of the present invention.

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. In addition, unless the context specifies otherwise, the singular forms "a", "an" and "the" also include their plural forms. In general, the nomenclature and related techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry, and hybridization are well known to those of ordinary skill in the art, and It is generally used in the technical field.

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, there is provided a vector for producing Parkinson's disease model comprising a PINK1 (G309D) mutant gene and an alpha-synuclein (α-synuclein (A53T)) mutant gene.

The PINK1 (PTEN-induced putative kinase 1) gene is a potential serine threonine kinase, and mutations are found in about 8-15% of Parkinson's patients. It has been reported that the PINK1 gene interacts with DJ-1 and is involved in mitochondrial dysfunction. Alpha-synuclein (α-synuclein (A53T)) is a protein that helps neurotransmission between brain cells is a major cause of degenerative brain disease Parkinson's disease.

In the present invention, among them, PINK1 and α-synuclein genes were examined for human and dog gene similarities. As a result, PINK1 showed high gene similarity of 86% (FIG. 1) and α-synuclein of 94% (FIG. 2). As such, the gene similarity was very high in the PINK1 and α-synuclein genes between humans and dogs, and the combination of the PINK1 and α-synuclein genes was adopted. Therefore, by using the Parkinson's disease dog according to the present invention, it is possible to provide a disease model that can replace primates, and to determine the induction of Parkinson's disease through behavior and to develop and use a more reliable Parkinson's disease animal model. .

According to one embodiment of the present invention, the PINK1 (G309D) mutation is Gly substituted with Asp at position 309 of the PINK1 gene, and the alpha-synuclein (α-synuclein (A53T)) mutation is alpha-synuclein (α) -synuclein (A53T)) Ala is a mutation in which Ala is substituted with Thr at position 53 of the gene for Parkinson's disease model production vector is provided.

According to one embodiment of the invention, the vector is provided with a vector for Parkinson's disease model production, characterized in that the retroviral vector.

According to an embodiment of the present invention, the Parkinson's disease animal model is provided with a vector for producing Parkinson's disease model, characterized in that the individual.

The term 'vector' or 'expression vector' of the present invention refers to a plasmid or viral vector known in the art to which a nucleic acid encoding a structural gene can be inserted and which can express the nucleic acid in a host cell. Or other mediator. Preferably it may be a viral vector.

The viral vectors include, but are not limited to, retroviral vectors, adenovirus vectors, herpes virus vectors, abipoxvirus vectors, lentiviral vectors, and the like. In particular, a method using a retrovirus is preferable. The retroviral vector is designed so that all of the viral genes have been removed or altered so that non-viral proteins are produced in cells infected by the viral vector. The main advantages of retroviral vectors for gene therapy are the delivery of large quantities of genes into cloned cells, the precise integration of genes transferred into cellular DNA, and no subsequent infection after gene transfection.

According to another aspect of the present invention, a complex gene mutant cell line (Accession Number: KCTC13310BP) transformed with the vector for producing the Parkinson's disease animal model is provided.

According to one embodiment of the present invention, the cell line (Accession No .: KCTC13310BP) is provided with a transformed complex gene mutant cell line (Accession No .: KCTC13310BP), which is derived from fibroblast cells of a mammal.

Somatic cells that can be used in the present invention include, but are not limited to, cumulus cells, epithelial cells, fibroblasts, nerve cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, macrophages, monocytes, muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, fetal-derived cells, fetal cells and embryonic cells. More preferably, they may be fetal-derived cells, adult fibroblasts, and oocytes. Most preferably, fibroblasts isolated from the fetus and adult of the dog are used. The characteristics of these cells have the advantage that a large number of cells can be obtained at initial separation, the cell culture is relatively easy, and the culture and manipulation are easy in vitro.

According to one embodiment of the invention, the mammal is provided with a transformed complex gene mutant cell line (Accession Number: KCTC13310BP), characterized in that the individual.

According to one embodiment of the present invention, the PINK1 (G309D) gene and alpha-synuclein (α-synuclein (A53T)) is a transformed complex gene mutant cell line (Accession Number: KCTC13310BP), characterized in that derived from human Is provided.

According to another aspect of the present invention, transformed Parkinson's disease model is provided using the complex gene cell line using somatic cloning techniques.

According to another aspect of the invention, the step of introducing a PINK1 (G309D) and α-synuclein (A53T) mutant gene into a vector; Transforming a mammalian cell using the vector; And a nuclear transfer step of introducing the transformed cells into denuclearized embryos.

Vectors according to the invention can be introduced into cells by methods known in the art.

For example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran DEAEDextran-mediated transfection, polybrene-mediated transfection, electroporation, gene guns and other known for introducing nucleic acids into cells The method can be introduced into cells for the production of transgenic animals.

On the other hand, nuclear donor cells, for producing Parkinson's disease model according to the present invention can be proliferated and cultured according to methods known in the art.

Suitable media are any use that can be developed in the laboratory for the cultivation of animal cells and especially mammalian cells, or can be prepared in the laboratory with the appropriate components necessary for animal cell growth, such as anabolic carbon, nitrogen and / or micronutrients. Possible media can be used.

The medium may be any basic medium suitable for animal cell growth, and as a non-limiting example, a basic medium generally used for culture includes Minimal Essential Medium (MEM), Dulbecco modified Eagle Medium (DMEM), and Roswell Park Memorial Institute. Medium) and K-SFM (Keratinocyte Serum Free Medium), and any medium used in the art can be used without limitation. Preferably, α-MEM medium (GIBCO), K-SFM medium, DMEM medium (Welgene), MCDB 131 medium (Welgene), IMEM medium (GIBCO), DMEM / F12 medium, PCM medium, M199 / F12 (mixture) (GIBCO), and MSC expansion medium (Chemicon).

To this basal medium, anabolic sources of carbon, nitrogen, and micronutrients can be added, including but not limited to serum sources, growth factors, amino acids, antibiotics, vitamins, reducing agents, and / or sugar sources.

It will be apparent that one of ordinary skill in the art can appropriately culture by a known method by selecting or combining a medium most suitable for stem cells derived from various tissue origins.

In addition, it is apparent that the culture can be carried out while adjusting conditions such as a suitable culture environment, time, temperature, and the like based on common knowledge in this field.

According to an embodiment of the present invention, there is provided a method for producing a cloned egg, comprising the step of fusing the nucleus and the cytoplasm with a fusion voltage of 35 ~ 55V in a fusion method using an electrode. Although not limited thereto, the fusion rate (successful egg / fused egg) may be low when the fusion voltage is less than 35V, and when the fusion voltage is higher than 55V, the fusion rate (successful egg / fusion-fed egg) may be low. Rather it can be lowered.

Although not limited thereto, according to one embodiment of the present invention, there is provided a method for producing a cloned egg comprising a nuclear substitution step representing a fusion rate of 55% or more, preferably 60% or more, more preferably 65% or more. Is provided.

According to another aspect of the invention, there is provided a method for producing a Parkinson's disease model comprising the step of transplanting the cloned eggs into surrogate mothers.

According to an embodiment of the present invention, there is provided a method of producing a Parkinson's disease model, characterized in that the vector is a retroviral vector.

According to an embodiment of the present invention, there is provided a method for producing a Parkinson's disease model, characterized in that the mammal is an individual.

As used herein, the term “disease model animal” refers to an animal having a disease that is very similar to a human disease. The significance of disease model animals in human disease research is due to physiological or genetic similarities between humans and animals. In disease research, biomedical disease model animals provide research materials for various causes, pathogenesis, and diagnosis of disease, and research on disease model animals to identify genes related to disease and to understand the interactions between genes. In addition, basic data for judging the possibility of practical use can be obtained by examining the actual efficacy and toxicity of the new drug candidate.

In the present invention, "animal" or "experimental animal" means any mammalian animal other than human. The animals include animals of all ages including embryos, fetuses, newborns and adults. Animals for use in the present invention can be used, for example, from commercial sources. Such animals may be laboratory animals or other animals, rabbits, rodents (e.g. mice, mice, hamsters, gerbils and guinea pigs), cattle, sheep, pigs, goats, horses, dogs, cats, birds (e.g. Chickens, turkeys, ducks, geese), primates (eg, chimpanzees, monkeys, rhesus monkeys).

According to another aspect of the present invention, there is provided a method for screening a Parkinson's disease preventive or therapeutic agent using the Parkinson's disease model produced according to the production method.

As described above, the present invention will be very useful for the development and practical use of Parkinson's disease treatment technology by providing Parkinson's disease animal model using dogs and various uses thereof in Parkinson's disease, which is considered as intractable disease.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the present invention is not limited by the following examples.

Example 1: Beagle cFF2 Cell Isolation

Beagle dog cancer, male 35-day-old fetus is isolated, the fetus's head, limbs, intestines and blood are removed, the remainder is collected and washed with PBS, and then placed in 20ml 0.1% trypsin / 0.04% EDTA solution for 10 minutes reaction and grinding. Cells were separated by washing three times in culture. The isolated beagle fibroblasts were cultured in a culture medium in which 10% fetal bovine serum was added to DMEM.

Example 2: Canine Somatic Cell Characterization

RT-PCR was performed with fibroblast-specific markers to analyze somatic cell characteristics used for somatic cloned dogs. cFF2 cells expressed all fibroblast markers except Mmp13 and Sox5 genes (FIG. 1).

Example 3: Confirmation of similarity between human and dog growth induction gene and complex Parkinson gene

Most of the genes are not very different between species, but if the gene difference is severe, it is difficult to apply human genes to dogs. PINK1 showed high gene similarity of 86% (FIG. 2) and α-Synuclein of 94% (FIG. 3).

 Among the human Parkinson genes, PINK1 used a gene whose 309th amino acid was mutated from glycine to aspartate, and α-Synuclein used a 53rd amino acid mutated from alanine to threonine.

Example 4: Construction of Human Complex Parkinson Gene Loading Vector

Retro retroviruses with the PINK1 (G309D) and α-synuclein (A53T) genes were constructed to produce transgenic dogs. The pQC lentiviral vector was used, which is controlled by the CMV promoter and contains long terminal repeat (LTR), virus packaging signal (ψ +) and puromycin resistance genes. Using the vector and PCR, the human PINK1 (G309D) and α-synuclein (A53T) genes were amplified by PCR and cloned into restriction enzymes KpnI and MluI, respectively (see FIG. 4). Retrovirus vector was cloned into 293T retrovirus vector cloned with PINK1 (G309D) and α-synuclein (A53T) genes for the production of transgenic dogs.

Example 5: Development of human complex Parkinson's mutant transgenic cell line

The produced human complex Parkinson gene present retrovirus was introduced into 293T cells to produce a complex Parkinson gene-loaded virus, and the virus was produced using a retrovirus enrichment kit.

cFF2 cells and complex Parkinson virus were infected by incubating for 48 hours.

After removal of the residual virus and the addition of puromycin for two weeks of selective culture, single cells were transferred to 96 well culture plates and cultured to select monoclonal complex Parkinson gene loaded dog cell lines.

RT-PCR was performed using RNA isolated from each cultured cell line, and the expression of the introduced human complex Parkinson gene was confirmed.

Cells for use in nuclear replacement were stored in liquid nitrogen in 1 vial of 1x10 ⁶ cells.

Example 6: Characterization of Parkinson's Complex Gene Loaded Canine Somatic Cell Line

 As a result of confirming the presence of chromosomal aberration in the cell when the gene was introduced by virus introduction, cFF2.PINK1 / α-synuclein (cFF2.PS) cells were analyzed as XX on 76 autosomal bodies and confirmed to have a normal karyotype (FIG. 5).

After incorporating the Parkinson complex gene into cFF2, it was verified by the RT-PCR technique that the strong expression of PINK1 and α-synuclein genes in the complex gene loaded individual cells (cFF2, PS) (Fig. 6).

In addition, it was verified that the expression of PINK1 and α-synuclein proteins were expressed in all cFF2.PS cells by immunofluorescence staining, indicating that the complex Parkinson mutant gene was introduced (FIG. 7).

Real time PCR was performed using gDNA to analyze the number of genes inserted into the chromosome. Comparing the PINK1 + α-synuclein gene expression Ct value and GAPDH and albumin gene Ct value, the relative gene copy was calculated and found to be inserted in one place in the genome (FIG. 8).

Example 7: Production and Assay of Dog Parkinson's Disease Model Using Somatic Cloning Technique

Parkinson's disease gene-loaded somatic cell utilization Nuclear substituted cloned egg production and transplantation Parkinson's disease model dog was produced by the following method.

Fertilized or naturally mated beagle dogs are recovered by caesarean section at 28 days of gestation. The recovered fetuses were transported to the laboratory to remove heads and limbs, and the body was washed three times with Dulbecco's phosphate buffered saline (DPBS).

After washing, the remaining tissue was chopped and incubated in a 100 mm petri dish in DMEM with 15% (v / v) FBS (invitrogen), 1% penicillin-streptomycin at 39 ° C, 5% CO ₂ and 95% air humidification conditions. It was. Tissues that did not adhere to the petri dishes were removed and attached cells were incubated to confluency. Trypsinized for 1 min using 0.25% trypsin-EDTA and aliquoted at 1 × 10 ⁵ cells / vial concentration and frozen and stored in liquid nitrogen at −196 ° C. using FBS with 10% DMSO.

Nucleus ovules were obtained using hybrid bitch 1-5 years old. Blood samples were collected daily from estrous dogs and serum progesterone and estrogen were measured using a Roche cobas e 411 immunofluorescence assay and an estrogen and progesterone assay kit (Roche Diagnostics, Mannheim, Germany). 72 hours after the estrogen peak in the oocyte donor dog was estimated as the ovulation day, and the blood progesterone concentration (6-15 ng / ml) was estimated as the ovulation day in the individuals whose estrogen peak measurement was impossible. Three days after the estimated ovulation date, the oocytes were recovered from the body by surgical operation. After removal of the oocytes using 0.1% hyalulonidase, only the oocytes were selected through stereomicroscope and used for the production of cloned eggs. Mature eggs were stained with 5 ug / ml Hoechst 33342, and then aspirated to remove the first polar and mesochromosomes with a micropipette, and thawed somatic cells containing the Parkinson's disease gene, which was cryopreserved in liquid nitrogen, and washed three times. After stabilizing by incubation at 37 ° C. for a minute, the denuclearized eggs were injected.

In order to fuse the eggs and somatic cells, the cells were fused under the conditions of DC49V, 15μsec pulse width, 2pulse and 0.1sec interval, and then the cloned eggs were activated for 4 minutes in 10μM Ca-ionophore. Then, the culture was performed for 4 hours in 6-DMAP (dimethylaminopurine).

For transplantation into surrogate mothers, surrogate mothers were prepared two to three days after the ovulation date estimated by the above-mentioned ovulation estimation method, and cloned fertilized eggs were implanted into the fallopian tube swell of the surrogate mother through surgical operation.

At 30 days after transplantation, gestation was measured, and 24 hours after day 57 (day 62) when blood progesterone was measured and blood progesterone levels were rapidly decreased, Parkinson's disease model dogs were produced by caesarean section.

During cesarean section, genomic DNA was extracted from the separated umbilical cord and the presence of mutant human PINK1 and α-synuclein was confirmed by PCR, confirming that two foreign genes were inserted together in the gDNA of the disease model. (FIG. 10).

When confirmed by the PCR technique, the types of primers used are shown in Table 1 below.

In addition, genomic DNA is isolated from the donor cell, the umbilical cord tissue of the cloned dog, and the blood of the surrogate mother for paternity discrimination. To determine whether the produced transgenic model dogs are genetically matched with the donor donor, genomic DNA was extracted from the umbilical cord of the transgenic model dog, the blood of the donor dog, the blood of the surrogate mother, and the somatic cells of the donor donor. Sequencing was performed. First, microsatellite analysis used 18 canine-specific markers (FIG. 11). As shown in FIG. 11, the microsatellite patterns of cFF2 Parkinson's original cells and cFF2 Parkinson's disease model cloned dog 1 were found to be consistent in all locus.

The fusion rate according to the fusion voltage during nuclear replacement using the transgenic somatic cells was confirmed. When the fusion voltage was changed to 34 V, 49 V, and 59 V, Table 2 shows the percentages of fusion successful eggs / fused eggs as the fusion rate.

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13310BP

Deposit Date: 2017818

<110> RURAL DEVELOPMENT ADMINISTRATION <120> Vector comprising a combined dual Parkinson gene and disease          model using the same <130> NPF-31300 <160> 12 <170> PatentIn version 3.2 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 1 ccttctacgg ctagggcaag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 2 ttaagcgtac gaggcctacc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 3 caaggccccc aaagtagtga 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 4 ctctcttacc cgtcattggc t 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 5 agtagcccag aagacagtgg a 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 6 acgcgtacgc gtttaggctt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 7 ccaagctagg atggaggtgc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 8 atgaatggcc tgggtggttt 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 9 ttgtggctgc tgctgagaaa a 21 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 10 cagaattcct tcctgtgggg ct 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 11 gtcatccctg agctgaacgg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 12 tccgatgcct gcttcactac 20

Claims (16)

Parkinson's disease model comprising a PINK1 mutant gene with Gly substituted Asp at position 309 of the PINK1 gene and an alpha-synuclein mutant gene substituted with Ala Thr at position 53 of the alpha-synuclein gene Complex gene mutant cell line, transformed with a dragon vector and deposited with accession number KCTC13310BP. delete The method of claim 1,
Said vector is a retroviral vector, complex gene mutant cell line. The method of claim 1,
The Parkinson's disease animal model is characterized in that the individual, complex gene mutant cell line. delete The method of claim 1,
The cell line is characterized in that derived from fibroblasts of mammals, complex gene mutant cell line. The method of claim 6,
The mammalian gene complex cell line, characterized in that the individual. The method of claim 1,
The PINK1 mutant gene and the alpha-synuclein mutant gene are characterized in that the human, complex gene mutant cell line. A transformed Parkinson's disease model animal transduced by introducing the complex gene mutant cell line of any one of claims 1, 3, 4, and 6 to 8 into a human-excluded fertilized egg, which is denuclearized. Production replica. A transformed Parkinson's disease model dog produced by developing the cloned egg according to claim 9. Introducing the PINK1 mutant gene and alpha-synuclein mutant gene of claim 1 into a vector;
Transforming a mammalian cell using the vector; And
A nuclear transfer step of introducing the transformed cells into a human-excluded animal fertilized egg that has been denuclearized,
The method of manufacturing a cloned egg comprising the step of fusing the nucleus and cytoplasm with a fusion voltage of 35 ~ 55V in the fusion method using an electrode in the nuclear substitution step. The method of claim 11,
The method of manufacturing a cloned egg comprising a nuclear substitution step exhibiting a fusion rate of 55% or more in the nuclear substitution step. A method for producing a Parkinson's disease model animal comprising transplanting a cloned egg produced by the method according to claim 11 to a surrogate mother. The method of claim 11,
The vector is a production method of Parkinson's disease model animal, characterized in that the retroviral vector. The method of claim 11,
The mammal is characterized in that the individual, Parkinson's disease model animal production method. A method for screening a Parkinson's disease preventive or therapeutic agent using a Parkinson's disease model animal produced according to the production method of claim 13.

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