KR20120021316A - A novel dopaminergic neuronal dj-1 knockout cell line for diognostic markers - Google Patents

Abstract

PURPOSE: A DJ-1 knockout cell line which is able to detect pathogenetic marker of Parkinson's disease is provided to treat, prevent and relieve Parkinson's disease. CONSTITUTION: A method for preparing DJ-1 knockout dopaminergic neural cell line KCTC 11472BP comprises: a step of crossbreeding TH-TagA58 transgenic mouse and DJ-1 gene targeting mouse to prepare a dual gene targeting mouse; a step of collecting and establishing DJ-1 gene knockout cell line from the substantia nigra of the dual gene targeting mouse; and a step of culturing the cell line at pH7.0 and 33°C.

Description

A novel dopaminergic neuronal DJ-1 knockout cell line for diognostic markers for securing pathogenetic markers applicable to the diagnosis of Parkinson's disease.

The present invention is a novel DJ-1 (DJ-1) loss mutant that can detect a Parkinson's disease pathogene marker, and a therapeutic drug for the treatment, prevention and alleviation of the disease progression from Parkinson's disease from the pathogene marker detected using the same. The present invention relates to a diagnostic kit that can be used for screening and clinical diagnosis, and a method for screening Parkinson's disease inhibitors using Parkinson's pathogenetic markers.

Parkinson's disease (Parkinson's disease) is a disease first reported by James Parkinson in 1817, and loss of neurons and striatum of substantia nigra (SN), which secretes dopamine, a neurotransmitter that regulates muscles due to aging. It is a type of degenerative brain disease in which exercise capacity is lost due to dopamine deficiency of.

Parkinson's disease affects 1% of the population over 65 and 5% of the over 85 (Twelves et al. Mov Disord 18, 19-31). Characteristic clinical symptoms include resting tremor, bradykinesia, rigidit and postural instability, and selective loss of dopaminergic neuron in substantia nigra (SN). selective loss), and the presence of an intraeuronal proteinous inclusion known as the Lewy body is a representative pathological characteristic (Olanow et al., Annu Rev Neurosci 22,123-44).

The exact cause of Parkinson's disease is not known. In the case of the sporadic form, which is most Parkinson's disease, the cause is rarely known as idiopathic, but a complex interaction between environmental factors and genetic susceptibility, which has not yet been fully identified, is caused. It is presumed to be an important cause (Langston et al. Ann Neurol 44(3 Suppl 1): S45-52).

Parkinson's disease is pathologically characterized by a specific loss of dopaminergic neurons and neurite fibers, including melanin pigments present in the substantia nigra, and a behavioral abnormality due to a lack of dopamine in the striatum. Is displayed. In addition, in neurons of Parkinson's disease patients, the Lewy body, a protein aggregate, is observed as a marker of disease.

Representative pathogenesis mechanisms of Parkinson's disease include oxidative stress, mitochondrial dysfunction, ubiquitin-proteasome dysfunction, and accumulation of misfolded proteins.

Parkinson's disease is caused not only by the above environmental pathogenesis but also by genetic factors.Up to now, the genes related to Parkinson's disease are α-synuclein (Mutation in the alpha-synuclein gene identified in families with Parkinson's disease, Science) by Polymeropoulos. 276 (1997), p2045-2047), LRRK2 (Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease, Neuron 44 (2004), pp. 595-600) by C. Paisan-Ruiz et al., AR by Kitada et al. -JP was first reported by positional cloning (Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism, Nature, 392 (1998), pp.605-608) of a genetic defect in Japanese households. In addition, PINK1 (Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase, Nat. Genet. 38 (2006), pp. 1184-1191) by Valente et al. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism, Science 299 (2003), pp. 256-259), and ATP13A2 (Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type) by Ramirez et al. 5 P-type ATPase, Nat. Genet. 38 (2006), pp. 1184-1191) has been reported.

Currently, genetic mutations causing genetic Parkinson's disease, such as DJ-1, Parkin, and PINK1, have been discovered and studied. Although these genes are expressed in almost all cells, inherited Parkinson's disease ) It is known that only dopaminergic neurons in the patient's midbrain substantia nigra are specifically killed.

Recently, mutations in the genes that cause familial Parkinson's disease among Parkinson's disease have been discovered, and in particular, crypt dysfunction and oxidative stress caused by the loss of the recessive gene, DJ-1, are being studied. . In addition, studies are being conducted to discover therapeutic agents and biomarkers for neuroprotection through changes that appear when these genes lose their function.

Dopaminergic neurons that are currently commonly used to search for biomarkers and neuroprotective targets that change in these dopaminergic neurons include MES23.5 cells, MN9D cells, PC12 cells, SHSY5Y cells and CATH.a cells. have. However, these cell models cannot maintain the intracellular environment of dopaminergic neurons alone, and their origins are completely different, or they have the genetic characteristics of fused cells.

In addition, studies are being conducted through specific gene-targeted cell models. Most of these are cell models that cause loss of gene parts such as siRNA, rather than gene loss, and are insufficient in discovering targets for changes that may occur when complete gene function is lost.

In addition to cell models, there is a DJ-1 gene knockout animal model that has been produced and used in research as an animal model for Parkinson's disease. After the human DJ-1 mutation was discovered (Science, 299:256-259,2003), a DJ-1 gene transgenic mouse was constructed based on this (Journal of biology and chemistry, 180:21418-21426, 2005). ).

This animal model was a model in which the promoter portion of DJ-1 and the first exon portion were deleted. At the same time, another team of researchers created a mouse model that destroyed the second exon of DJ-1 (Neurons, 45:489-496), and both groups of models showed that the number of dopaminergic neurons in the melanoma was Showed no decrease. In addition, an animal model was created in which both the second and first exon of DJ-1 were deleted (Proceedings of the National Academy of Sciences, 102:5215-5220,2005).

These animal models mentioned above are very effective animal models for prior experiments of gene therapy in Parkinson's disease, which occurs when the DJ-1 gene is deficient in humans, but these animal models are also sufficient to conduct molecular and biochemical studies on them. It is still difficult to obtain pure dopaminergic cells. These problems are also true for patients suffering from Parkinson's disease. In fact, it is very difficult to select patients due to functional abnormalities of DJ-1, and it is even more difficult to obtain experimental samples.

Korean Patent Application No. 2007-110102 discloses that it is partially involved in protein aggregate formation, and international patent PCT/EP2004/008609 discloses that it is involved in arthritis, but DJ-1 as an etiological gene for Parkinson's disease The detailed mechanism of this is not known at all, and it is still unclear that if the protein is not normally produced by a mutation in the DJ-1 gene, Parkinson's disease is caused.

The function of DJ-1 and particularly its role in the degeneration of dopaminergic neurons is not known for certain. DJ-1 was first reported as an oncogene in 1997 (Biochemical and Biophysical Research Communications, 231:509-513, 1997). DJ-1 is expressed in most parts of the brain and is located not only in the cytoplasm of nerve cells, but also in the inter-membrane space and matrix of the mitochondria (Human Molecular). Genetics, 14:1063-2073, 2005). It is known that DJ-1 moves to mitochondria by oxidative stress, and DJ-1 in this mitochondria plays a protective role in cells.

Parkinson's disease is caused by complex genetic abnormalities and environmental factors such as DJ-1 mentioned above, but the current treatment for Parkinson's disease is very limited, and detailed treatments according to the pathogenesis are not found. Until recently, the most effective treatment method known is a method of converting to insufficient dopamine by administering L-dopa, a dopamine precursor. However, this treatment has many side effects.

For this reason, the need to study more specifically the cause of Parkinson's pathogenesis and the mechanism of apoptosis has emerged, and the inventors of the present invention aimed to study the genetic and environmental complex mechanisms resulting from the loss of DJ-1, an autosomal recessive gene -1 By creating a new mutant cell line that has lost the gene, the pathogene markers detected using the same can be used for the treatment, prevention, and alleviation of the disease progression of Parkinson's disease, confirming that it can be used for the search and clinical diagnosis Completed the invention

An object of the present invention is to provide a more efficient model for screening for changes in another genetic expression pattern when the DJ-1 gene is lost for the treatment, prevention and diagnosis of Parkinson's disease.

In addition, an object of the present invention is to provide a method for screening Parkinson inhibitors using the above model.

In order to achieve the above object, the present invention provides a DJ-1 gene loss dopaminergic cell line KCTC 11472BP.

Hereinafter, the present invention will be described in detail.

The present invention provides a DJ-1 gene loss dopaminergic cell line KCTC 11472BP as a more efficient model for screening for a change in another genetic expression pattern when the DJ-1 gene is lost for the treatment, prevention and diagnosis of Parkinson's disease. .

The present invention analyzes other genetic mutations in Parkinson's disease caused by the loss of the DJ-1 gene through screening, compares it with this analysis, and observes it, so that it can be applied to clinical diagnosis, treatment, prevention, and prognosis evaluation of Parkinson's disease.

Currently, it is being analyzed through a number of experimental methods to find out the changes in the expression of a large amount of genes. A typical example is a DNA chip in which a large number of genes are hybridized. In order to analyze these genetic changes, most of them are currently done through RNA and proteins extracted from the actual brain tissues of Parkinson's disease animals, but these have very little specificity as dopaminergic neurons. Therefore, the present invention provides a rapid analysis of large amounts of molecular and biological mutations through the DJ-1 gene loss dopaminergic cell line KCTC 11472BP.

Specifically, the present invention relates to cytoskeleton related genes, energy metabolsim related genes, signal transduction related genes, stress/detoxification related genes, protein folding. It was observed that genes related to folding), genes related to protein degradation, and genes related to neurotransmitter metabolism were changed by the selective loss of DJ-1.

In particular, Pax 8 (Pax8, the nucleotide sequence is shown in SEQ ID NO: 1), glutaredoxin (Glrx, the nucleotide sequence is shown in SEQ ID NO: 2), which is important for tissue and organ formation in embryonic development, and mitochondrial fat Acyl-Coenzyme A dehydrogenase (acyl-Coenzyme A dehydrogenase: Acadsb, nucleotide sequence is shown in SEQ ID NO: 3), which catalyzes the beta-oxidation process, a gene related to the developmental sub-process (developmentally down-regulated gene 9: Nedd9, base) The sequence is shown in SEQ ID NO: 4). A pathogenetic biomarker that can be applied to the clinical diagnosis of Parkinson's disease can be applied to the clinical diagnosis of Parkinson's disease.

The sequence information of the Parkinson's disease marker gene identified in the present invention is shown in the following table.

[table]

The four Parkinson's disease marker genes of the present invention can be used alone or in combination in a Parkinson's disease diagnostic kit.

In addition, since the four types of Parkinson's disease marker genes are likely to be Parkinson's disease-related inheritance, small molecule compounds that bind to proteins encoded by these target genes can be candidates for compounds that inhibit or promote the target protein. It can be used as pharmaceuticals such as Parkinson's disease treatment.

In the present invention, a customized neuroprotective substance for degenerative brain diseases may be developed through a cell-based therapeutic method, and the developed therapeutic agent can be used independently or as a drug additive with a pharmaceutically acceptable nanocarrier or excipient according to the application. Or it can be used as a customized neuroprotective drug therapy that can be used in all other formulations and formulations suitable for administration to humans. Carriers that may be contained in the therapeutic agent for degenerative brain diseases of the present invention may include polyethylenic glycol, polyethylenic fibers, bulking agents, high fiber additives, chitosan weights, lipolipids, encapsulating agents and lipids, and examples of such carriers. Are commercially known.

By screening for such compounds, proteins encoded by the four Parkinson's disease marker genes are immobilized on an affinity column and contacted with a test sample to purify the protein [Pandya et al., Virus Res 87:135-13,2002], Method using the two-hybrid method [Fields, S and Song, O., Nature 340:245-246,1989], Western brotting method [“Molecular Cloning-A Laboratory Manual” Cold Spring Habor Laboratory, NY, Maniatis, T. at al. (1982) section 18.30-18.74], a high-throughput screening method [Aviezer et al., J Biomol Screen 6:171-7,2001], and other known methods can be used.

The present invention provides a DJ-1 gene loss dopaminergic cell line KCTC 11472BP for the search for pathogenetic biomarkers, and by providing gene markers in which gene expression is increased or decreased due to the mutation of the DJ-1 gene, which is the cause of Parkinson's disease, these The marker can be used for clinical diagnosis, which is used as a target gene used in the prevention, alleviation, and treatment of degenerative brain diseases, and a customized neuroprotective substance for degenerative brain diseases from the DJ-1 mutant-derived dopaminergic neuron cell line KCTC 11472BP cells It can be developed through a method of searching for -based treatments.

1 is a diagram schematically illustrating the manufacturing process of the DJ-1 gene loss dopaminergic cell line KCTC 11472BP for the search for pathogenetic biomarkers.
2 is a Western blot figure to confirm the DJ-1 loss of the DJ-1 gene loss mice and the DJ-1 loss dopaminergic cell line KCTC 11472BP at the protein level.
FIG. 3 is a diagram illustrating gene types according to biological classifications for confirming genetic changes due to loss of DJ-1 of the dopaminergic cell line KCTC 11472BP, the dopaminergic cell line KCTC 11472BP, of the mouse on the DNA chip.
Figure 4 shows the type of gene for each metabolic pathway for confirming the gene change due to the loss of DJ-1 of the dopaminergic cell line KCTC 11472BP, the dopaminergic cell line KCTC 11472BP, of the mouse on the DNA chip.

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

However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

< Example 1> Pathogenetics Biomarker For navigation DJ -1 gene loss Dopamine Sex cell line construction

1) DJ -1 gene loss dopaminergic cell line production

After crossing the TH-TagA58 transgenic mouse (The Journal of Neuroscience, January 1, 1999, 19 (1):10-20) and the DJ-1 gene hit mouse to create a double gene hit mouse, the tissue of the black matter of the mouse The pure DJ-1 gene loss neuronal cell line is established through the process of selecting the cells obtained from the cells with specific antibiotics. This cell line was selected for about 10 days or more in a medium (pH7.0) of the composition shown in Table 1 below, and then semi-permanently self-proliferating cell line, a novel DJ-1 hit dopaminergic neuron cell line KCTC 11472BP. Was produced. Fig. 1 shows the process of producing a novel DJ-1 gene-losing dopaminergic cell line for the detection of the pathogenetic biomarker. The present inventors deposited the DJ-1 gene-losing dopaminergic cell line with the Korea Research Institute of Bioscience and Biotechnology Gene Bank, which is an international depository, on March 6, 2009, and received the accession number KCTC 11472BP therefrom.

The DJ-1 gene loss dopaminergic cell line KCTC 11472BP secured as described above is a temperature sensitive cell line, has a culture temperature of 33° C., unlike the existing 37° C. culture cell lines, and must be cultured in a _{5% CO 2 incubator.} Cell lines cultured in a 100 mm culture dish of DMEM medium as shown in 1 were treated with Trypsin-EDTA to remove the cells, centrifuged for 3 minutes at 1000 rpm to discard the supernatant and discard the concentrated number of cells. To 2X10 ⁶ , add 0.1 ml of DMSO, 0.5 ml of DMEM medium containing 10% bovine serum, and 0.4 ml of FBS, and dispense into a vial for freezing storage, store at -20℃ for 3 hours for the first time, and then store at -80℃ overnight. After that, store it in a nitrogen tank containing liquid nitrogen at minus 280℃ for a long time.

2) Confirmation of DJ- 1 gene loss at the gene level and protein level

In this experiment, the novel DJ-1 gene loss dopaminergic cell line KCTC 11472BP produced in Example 1 was compared with wild cell lines, embryos, and DJ-1 mutants by performing protein separation by gene typing and Western blot. This is to check if there is a difference.

To confirm the quantitative increase or decrease of the DJ-1 gene and protein, the marker protein was isolated to determine the DJ-1 loss at the protein level in the embryonic and D-1 loss dopaminergic cell line KCTC 11472BP of the gene-losing mouse. Western blot was performed using an antibody (Chemicon, USA). Protein change was performed by Western blot by adding a protein lysate from the tissue at the time of embryonic 13.5 days and the tissue of the mice that lost DJ-1. Cool the tissue in lysis buffer (20 mM Hepes, pH 7.9, 150 mM (NaCl; sodium sodium), 1 mM MgCl ₂ , 5 mM EDTA, pH 8.0, 1% Nonidet P -40), 0.5% sodium deoxycholate, 0.1% SDS, 50 mM NaF, 5 mM sodium orthovanadate] for 20 minutes and recovering only the protein to measure the protein content It was quantified by measuring with a bio-red protein quantitative reagent kit (Bio-Rad Dc protein assay kit, Bio-Rad).

50 µg of the lysate was subjected to electrophoresis on SDS-polyacrylamide gel. After that, it was transferred to the PVDF membrane and reacted with the DJ-1 primary antibody, a specific antibody, and the primary β-actin antibody at 4°C overnight, and then the secondary antibody, horse radish peroxidase (HRP), was transferred. It is shown in FIG. 2 by analysis by the CEL chemiluminescence method (Amersham Corp.).

3) Changes in gene expression patterns and identification of functional classification of marker genes

As a result of comparing the level of gene expression in the control group SN4741 cell line and the experimental group, DJ-1 loss dopaminergic neuronal cell line KCTC 11472BP, RNA obtained from these cell lines was hybridized with the Illumina bead array, and the level of expression was determined by Illumina bead. The gene expression signals on the array read by the array reader were compared and analyzed through the Benzamini-Hochberg FDR p-value, and classification was performed through the Support Vector Machine Program of Arravassist® (Stratagene). The classification of biological pathways, metabolism, and molecular function was made based on the Panther database (http://www.pantherdb.org ), and DJ-1 showed a significant change in the amount of expression, and about 151 times higher. It was reduced (Table 2). Through this, it can be confirmed that the DJ-1 gene in the DJ-1 loss cell line KCTC 11472BP according to the present invention was completely lost.

First, among the 24,000 genes present on the gene chip, about 9,500 genes showing changes in the DJ-1 loss dopaminergic neuron cell line KCTC 11472BP, an experimental group, were analyzed, and then the Panther database ( http://www.pantherdb.org ). It is shown in FIG. 3 by classifying it by molecular biological function based on the website database.

Table 3 shows the genes whose expression level increased by 20 times or more in the level of gene expression in the DJ-1 loss dopaminergic neuron cell line KCTC 11472BP, and 20 times the gene expression level in the DJ-1 loss dopaminergic neuron cell line KCTC 11472BP. The genes reduced below are shown in Table 4.

4) Comparative identification of pathogenetic biomarker genes

The gene expression levels in the experimental group DJ-1 hit dopaminergic neuron cell line KCTC 11472BP and the control group SN4741 cell line were compared in terms of pathogenetics.

As can be seen in FIG. 2, the experimental results showed that Pax8 (Pax8), glutaredoxin (Glrx), which are important for the formation of tissues and organs in embryonic development, and acri-coemzyme catalyze the fat beta-oxidation process of mitochondria. This is a pathogenetic biomarker that can be applied to clinical diagnosis of Parkinson's disease, consisting of a dehydrogenase (acyl-Coenzyme A dehydrogenase: Acadsb) and a gene related to the developmental sub-process (developmentally down-regulated gene 9: Nedd9). It was confirmed that the gene was lost or amplified at the expression level.

In addition, as can be seen in Table 2 above, the RNA of the DJ-1 hit dopaminergic cell line KCTC 11472BP according to the present invention and the previously established wild dopaminergic neuron SN4741 as a control were extracted respectively, and the results of the analysis after hybridization on the gene chip Compared to the control group (fold value; the level of expression in the dopaminergic cell line hit by DJ-1 (experimental group) / the level of expression in wild dopaminergic neuron SN4741 (control group)) it can be seen that the expression is markedly reduced.

Therefore, Pax8 (Pax8), glutaredoxin (Glrx), which are important for the formation of embryonic tissues and organs in the onset of Parkinson's disease due to malfunctions such as DJ-1, The catalytic acyl-Coenzyme A dehydrogenase (Acadsb) and genes involved in the developmental subprocess (developmentally down-regulated gene 9: Nedd9) are Parkinson's searched by DJ-1 gene loss dopaminergic cell line KCTC 11472BP. As a disease-specific expression gene, it can be seen that it is a pathogenetic biomarker that can be applied to clinical diagnosis of Parkinson's disease.

< Example 2> Parkinson's gene DJ -1 Screening experiment to observe changes in the expression of mouse genes due to gene loss

This experiment is a preceding experiment to investigate what kind of change occurs in the expression of the gene when DJ-1, one of the causative genes of Parkinson's disease, is knocked out.

In the experiment, the DJ-1 gene loss dopaminergic neuron cell line KCTC 11472BP and the control SN4741 cell line were previously cultured in a culture dish of 100 mm (diameter), washed twice with phosphate buffer, and Trizol (invitrogen, USA) 0.5 ml is added, the cells are removed using a scraper, extracted, and purified using RNeasy columns (Qaigen, USA), and then measured in the wavelength band (260/280 nm) with a UV spectrophtometer (Molecular designer) to lose the DJ-1 gene. This experiment was performed after confirming whether high-purity RNA was extracted from the sex neuron cell line KCTC 11472BP and the control cell line.

The DJ-1 loss dopaminergic neuron cell line KCTC 11472BP, the experimental group, and the mRNA of the SN4741 cell line as a control, were isolated with an RNA isolation kit (RNAeasy kit, QIAGEN, USA), and an RNA solution (10 to 100 μg of mRNA, 8 microliters, 50 picomoles). 4 µl of random primer, 2 µl of 40 unit RNAse inhibitor, and 34 µl of sterilized water were added to a 1.5ml Eppendorf tube and reacted in a thermostat for 10 minutes at 70℃, followed by 5 minutes. It was left on ice, and a mixture of 32 µl of RTase solution (4 units of Supercrete RTase, 16 µl of 5x RT buffer, 8 µl of DTT (dithiothreitol), 4 µl of 25mmol DNT) in advance. Then, react at 37°C for 1 hour, incubate in a 70°C thermostat to stop the reverse transcription reaction, and then add 320 µl of sterilized DiethylenePyrocarbonate (DEPC) and use it as mRNA, using a UV spectrophtometer (Molecular designer) to stop the reverse transcription reaction. /280 nm), cDNA was synthesized as a template with an absorbance of 10 to 100 μg at a concentration of 1.8 or higher.In particular, to increase the mRNA purity, amplification was performed using the Ambion Illumina RNA amplification kit (Ambio, USA) and then 550 ngRNA. Was reverse transcribed into cDNA using a T7 oligo(dT) primer, cDNA was synthesized, and the cDNA labeled with biotin NTP was purified and quantified.

Use the obtained cDNA as a template by a PerkinElmer PCR instrument (PerkinElmer Life Sciences, USA) at 95°C for 5 minutes, denaturation step 95°C, 30 seconds, annealing step 55°C, 1.0 minute, extension step 68°C , 2 minutes, and finally 68 ° C., synthesized at 2 minutes, 10 micrograms of each genomic DNA of the experimental group and the control group were respectively labeled with fluorescent dyes Cy3 (test group sample) and Cy5 (sample group sample) by a random priming method. . After hybridization of 750 ng of the labeled cRNA with the expression bead array at 58°C for 16 to 18 hours, the expression signal was detected using amersham fluorolink streptavidin-Cy3. The labeled DNA was hybridized in a humid chamber at 37° C. for 24 hours on Illumina Expression Chip/MouseRef-8, 24K (Illumina, USA) (Marcrogen, KOREA).

The array slides were washed with washing buffer and dried completely, and then the array slides were scanned with an Illumina bead array reader and analyzed using Illumina beadStudio v3.1.3. The fluorescence images obtained above were analyzed using MAC ViewerTM software (Macrogen Inc.) optimized for the array analysis. The fluorescence spot location was determined using the automatic grating function and adjusted manually. It was considered abnormal that the ratio of fluorescence intensity of all spots had a statistically significant difference from the average ratio of “0”.

Korea Research Institute of Bioscience and Biotechnology KCTC11472BP 20090306

<110> Ewha University-Industry Collaboration Foundation <120> A novel dopaminergic neuronal DJ-1 knockout cell line for diognostic markers <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 2527 <212> DNA <213> Mus musculus <400> 1 acttcagaag gaagagacgc ctgggccttg ggcaccctca ggggcagacc caggcagaaa 60 gggcctgagg ccagccggcc agggtagctg cgtggcagcc agagctgcca ggacctgcgt 120 aggaaagctg cgagtgtccc tcagtctgtg agcgactccc cggcgatgcc tcacaactcg 180 atcagatccg gccatggagg gctgaatcaa ctaggagggg cctttgtgaa tggcaggcct 240 ctgccagaag ttgtacgtca acgcattgtg gacttggccc accagggcgt gaggccctgt 300 gatatttctc gccagctccg tgtcagccat ggctgtgtaa gcaagatcct tggcaggtac 360 tacgagactg gcagcatccg gcctggagtg atagggggct ccaagcccaa ggtggccacc 420 cccaaggtgg tggagaagat aggagactac aagcggcaga accctaccat gtttgcttgg 480 gagatccggg accggctcct ggcagaaggc gtttgtgaca atgacactgt ccccagtgtc 540 agctccatca acagaatcat ccggaccaaa gtgcagcagc cattcaacct ccccatggat 600 agctgtgtgg ccaccaagtc tctgagccca ggacacacac tgatccccag ctcagctgta 660 acacccccag agtcacccca gtcggattcc ttgggctcta cctactctat caacgggctc 720 ctaggaattg ctcagcctgg caatgacaac aagagaaaga tggatgatag tgaccaggac 780 agctgtcggc taagcatcga ctcacagagc agcagcagtg gtcctcgaaa gcaccttcgt 840 acggacacct tcagccagca ccatctcgag gcccttgagt gcccgtttga gcggcagcat 900 tacccggagg cctatgcctc ccccagccac accaaagggg agcaggggct gtacccactg 960 cccttactca acagtgccct ggatgatggg aaggccaccc tgacatcttc caatacacct 1020 ctgggacgca acctctcgac tcaccagacc taccctgtgg tggcagatcc tcattcaccc 1080 ttcgccataa agcaggaaac cccagagctc tccagttcta gctccacccc ttcctcttta 1140 tctagctccg cctttttgga tctgcagcaa gtcggctctg ggggcccagc aggtgcctcg 1200 gtcccaccct tcaatgcctt tccccatgct gcctccgtgt atgggcagtt cacgggccag 1260 gccctcctct cagggcgaga gatggtgggg cccacgctgc ctggataccc accccacatc 1320 cccaccagtg gacagggcag ctatgcctct tctgctatcg caggcatggt ggcaggaagt 1380 gaatattctg gcaatgccta cagccacacc ccgtattcct cctacagtga ggcctggcgc 1440 ttccccaact ccagcctgct gagttctcca tattattaca gctctacatc aaggccaagc 1500 gcaccaccca cctctgccac agcctttgac catctgtagt tgtcatgggg acagtgggaa 1560 gaaccaggca agaatcagga ggacttggcc tgagacaggc cccagagagt cacacaaagg 1620 aatctttatt tattacatga aaaataacca caattccagc attgccgctc actccctgtg 1680 tggtcaatta aatgaaccat gaaagtcagg atgaccttgg agaaggccaa actgtcctcc 1740 aggattcatt ttgtgagtga caggagttcc aggaaacaag aaccacccat ttcaaaagag 1800 acacagagga gaaatgtacc gaagctggca cccaccaaag gagagaaaag gggttcacga 1860 agaacttaga gggagatgag tctgagggtg tacagggcca cctgtttggt cttcatctag 1920 acaactccag caattatgaa cagtatgcaa gtccttctgg cctacaatca cagcctaggg 1980 ccaaggctct acgtgtcagt catcctagca gttaccacct tgagcctcta ctacctgcat 2040 ctttctgggg attcagctga ccttctattt ccagttgctg tgcatttaca agcacccctt 2100 agcctctcaa ccagtggcta cccctttggc agcagcgaac ctgtcccctg gaaagttcag 2160 tgaggctcta cgtagaacat aggaagacac aacagatggg accagccatg tcctctcctc 2220 aacagccttg ccaattcaag tgtgctttct gcagttccag cccgcgcagc cactgaagag 2280 acccccaagg ccagcaacaa gattccctct ctggctcctc agctcctcc ccacatacaa 2340 cctcactgta aataccgtaa atgaaactct gtttcggtca agctttcctc tttccatccc 2400 ccagtctttg gtctctgaaa tatctcccta ctatgttggg gttttctcct tcatactttt 2460 tttttttaaa aaaaaaaaaa aagacaaccc accattacca catgactcaa taaaccatta 2520 ctcttgg 2527 <210> 2 <211> 1335 <212> DNA <213> Mus musculus <400> 2 agctgctggc taagcgccgc tgcattaccg gaccattact ttcaactgca cgttcctccc 60 tggagaagct gcagcctgtc agcatggctc aggagtttgt gaactgcaag atccagtctg 120 ggaaggtggt cgtgttcatc aagcccacct gcccctactg cagaaagacc caagaaatcc 180 tcagtcaact gcctttcaaa caaggtcttc tggagtttgt ggacatcaca gccactaaca 240 acaccagtgc gattcaagat tatttacaac agctcaccgg agcgagaaca gttcctcggg 300 tcttcatagg taaagactgc ataggcggat gcagtgatct aatctccatg caacagactg 360 gggagctgat gactcggctg aagcagattg gagctctgca gttataaaag gggtggcagg 420 cagagtccat gctgacacag ctgtctaacc atgctgatgg ccagtgcccc tgagagttga 480 tgtgcatcgc agaggatgtc agtatttcct ggtgactggg atttttcaac aaggcggcct 540 ttattcttct tttcctcagt gctaaaaact gttgcaattt gcccctaacc atggggccga 600 gaagcttaac agaccacact ggtttgatta tccattcttc atgtgccaac atgtctctac 660 ctctaagccc aggttttcca aatccagttg ctctaaatct ccagtggatc tgttgctggt 720 tttctgctac tgttcgtcag ctgaagtcat tttgcagaag tccactttct aaagaattat 780 tgaatcaatg gatatcgaaa atttgtttcc tgagtcatgc atcggctcct ctctccctcg 840 tgcacgcacc cttcccactc ctgcattcac tgcccttact tagccagtgt tctcagcctc 900 aacctcctac aacccgcaga cgtccacact ggtgtgagga tgctgtttga aaaatcagat 960 gaactttagc atagttggtc ctcacggagg ccacgttaac ttaggcggca gagcagatgg 1020 tgcatgcagc tccctctgta aaggtgatta attgtccaga aaatcccaag cagctgtgtg 1080 ttgatccgag ttagagggcc agaaaaatca aatgtgaaat acaaaattgc aaaattctcc 1140 ttccaagaat ttttgtgaaa gacgttgttt ctgaaacatt gtcctaaaca gtttcttcca 1200 tccaaacttt gacattttgc tttgatgtct tgctatgctg tttaattctc atggatctgt 1260 agatcacttc tctggtctcc agtgaggagg attcattact attaaagatg tatctataga 1320 taactaaaaa aaaaa 1335 <210> 3 <211> 3243 <212> DNA <213> Mus musculus <400> 3 ggaaagcctt gcgagcttaa cagggccacc ctgcccgaag cgaggatggc ggtctctgcg 60 ctccagctgt ggcgtatggg cgggctgctg agaagacgct tcccaacctg cttgtctcct 120 tggaagattc ctcctcgtgt cctcaaatcc tcacaaccgg aagctctagt cagtctgaca 180 aacaatgcag tagcctttgc acctctgcag acacttactg atgaggaaat tatgatgaag 240 cagacagtca aaaaatttgc acaggagcac gttgctcctc tggtttcctc tatggatgag 300 aactcaaaaa tggagaaatc ggtgatccag ggattgttcc agcaagggct gatgggcatt 360 gaagttgaag cacaatatgg agggacagaa gcttcctttt tctgctctgt cctagtgata 420 gaggaactag ctaaggtgga tgcttcggtg gctctcctgt gtgacatcca gaacacaata 480 attaacaacc tgtttagaaa acacgcttca gaagaacaga aggccaccta tttgccaaag 540 ctggttacag aaaaattagg gagcttttgc ctctctgaag ctggagccgg tagcgactct 600 ttcgctatga aaacaagagc tgataaaagt ggaaattact acgtcctcaa tgggtcgaag 660 atgtggatca gccatgccga gcatgcagag ctcttcctgg tcttcgccaa tgtggacccc 720 agctctggct acagaggcat cacctgcttc ttagtagacc gagatacaga aggtttccag 780 atagggaaac gagaaaataa aatgggcatc agagcttcat ccacctgtca gttaacattt 840 gaaaatgtta aggttccaga gactaatatt ttggggaaaa ttgggcatgg ttataagtat 900 gccataggaa gtcttaatga aggtagaatc ggaattgctg cacagatgct aggactggcc 960 caaggatgtt ttgactacac tattccatac attaaagaaa ggatgcagtt tggcaaacga 1020 atatttgatt ttcaggggct ccaacaccaa gtggctcagg tggccaccca gctggaagcc 1080 acacggttgc taacatacaa cgctgctagg ctcgtagaag ccggaaggcc atttataaaa 1140 gaagcatcta tggccaaata ttatgcatct gaggtcgctg ggctaacaac aagcaagtgc 1200 atcgagtgga tgggaggggt cggctacacc aaagattacc ctgtggagaa attcttccga 1260 gatgccaaga tcggtacaat atatgaagga gcttccaaca tccagctgaa caccatcgcc 1320 aagcacatcg atgcagagta ctgatgactg tgggatgggc cctctgcgtc actgacaaac 1380 catttccagc tgctgtgcct tattgagcag ggtctagagc agtgcagggc ttccttggac 1440 ttcccttgtc ctggtctcag gcctggattt tgttcctgtc tctttccaat ctactctaag 1500 tctatttcta agcttctgaa cgcatatttc tcatcctagt tgcagagtat acaaaagttt 1560 cactctagca gcatttggga agaagaaacc atttgaggta ttggtattgc tgacagaaat 1620 agtcacttta tattcctact aaatctttgt actgtggtat gagccagagg agcgtttgtt 1680 acacttggca atttttattc aatactttat agattcagtg gtaagtggct aaacaaggta 1740 gaagctgata aaatttattt ggaaaaatct agaatcttag ttctaaatat caaaaatagt 1800 agaaaataaa aagtgtagct tgtaggccat ataattgaca ataacaaaaa gtcccaaatg 1860 ttattttaac caacaaaagg agtatgtttg gctgctataa ttgaactata tgaaagatgt 1920 attatgtggt ggcttttagc agcccattta aaaaacatgt ccctgtatta gttttcaact 1980 ataaagttta agtaatttgg cctaatcatc tgaaaataat ttattagatc atgcactatt 2040 ttttggtagt atgatttatt tctgtatttt aattgagtat tttccctcaa acttggttat 2100 tggactttat cctaaatagt aattaattga gaaattcaat tttggttttc aaaattgatt 2160 cttaagagaa atataccccc ataagaaaat aatatcacaa tctcataagg atagggaatg 2220 gagacttggt agtctgaaaa catacattca gtataaatat atgtagtcat ttatatagta 2280 tattagataa ttttatattt gtgaagacaa agatctatgt tttacaatgt aaatgaaaaa 2340 caggcaaagc ctaatcagat atccagctgg tgaagccatt gatcagtgtt agggatttac 2400 agtcgggaga agacgctcta gttgcagacc ctgagtcccc tggagagaga agtagacagt 2460 agacaaacga gtcgcacatt tccagaggaa acatcacaag ttagatgtga aaaatgcccg 2520 aggctcacgc agttgctgga aataatcgtt acttctagat agaaagtatt ttggtgcttt 2580 tgcaaaagga taatgtttat taagaaactt gacattttct aggtaatttt gctttgcaca 2640 gttaatgttt attgagctaa attaatttcc acaatgcaaa tcatagttaa atatgcaagg 2700 ttgtataaat acagttgaaa taggaattac attaaaacag taggaagaaa taaaacaaat 2760 ttagaccttg aatccaaaga gataaggtct acttgacttt caaatggggg aaatgatgaa 2820 aggcctcacc cagtctcaga acagacagat gagtgtgata agaaaggaag gggtggatgc 2880 agaccctgac agggcagaca ccttccactc ctgtaataat gggaaagagc agggcttaga 2940 gatgatgcag ccgcgggagt caggatgagt aaacagcctg ccctcctttc ctagactcat 3000 ggcaatcctc ctgcttcatg tgtaacctgg gctggccgca gattcatggc aatcctgctt 3060 tcaggcttcc agtgctgaga ttttatgtct gcaccccagc tcccatttct gacttgttga 3120 ttttgaagtc atgcccacga ttacagcttt gtcaattatt ctcatttatt tctatttgtt 3180 ttgctctatg tagttcccag ttatgttttt aggggtaata aagttcatgg ctgttggatc 3240 atc 3243 <210> 4 <211> 4384 <212> DNA <213> Mus musculus <400> 4 tcagagactc cgcagtgcgc gggaggcgaa ggcttctcat ttccatccag cccagcgctc 60 aatggagggg cggactctgc agtgcctcac tcactgtgtg attcgatagg accagggctc 120 atcagatcac cgccgagatg aagtacaaga atcttatggc aagggccttg tatgacaacg 180 tccctgagtg tgctgaggag ctggccttcc gcaagggaga catcttaact gtcatagagc 240 agaacacagg agggcttgag ggatggtggc tgtgttccct ccacggtcgc caaggcattg 300 tcccagggaa ccgggtgaag cttctgattg gtccagtgca agagaccccc ggtcatgagc 360 agcctactcc tggacctatg catcagacct ttggccaaca gaaactctat caagtgccaa 420 attcccaggc agcatctcgg gataccatct accaagtgcc accctcctac cagaatcagg 480 gaatttacca agtacccact ggccatggca ctccagaaca agatgtatat caagtaccac 540 catcagttca gaggaacatt ggcggcacta atggacccct tctaagcaaa aaggtgatca 600 ccccagtgag gacgggccat ggctatgtgt acgagtaccc atccagatac caaaaggatg 660 tctacgatgt ccctccttcc cacagcactc aaggggtata tgacatccct ccttcctcag 720 taaaaggccc tgtgttttca gttccagtgg gagagataaa acctcaaggg gtatatgaca 780 ttccccccac ccaaggggtc tatgccattc caccatcggc ttgccgagat gaggcagggc 840 tcagggaaaa ggaatatgat ttccctcctc caatgaagca agatggaaaa ccagacacca 900 gacctgaggg ggtttatgac atccctccaa ccagcaccaa gacagcaggc aaggaccttc 960 acatcaaatt cccctgtgat gctccaggag gtgtcgaacc aatggcacga agacaccaga 1020 gcttttccct gcaccatgca ccctctcagc tgggacagtc tggggacact cagagtgatg 1080 cctatgatgt cccccgggga gttcagtttc tggaggtacc aacagaaacc agtgaaaagg 1140 caaatccgga ggaaagagac ggtgtctacg atgtccctct gcacaaccca gcagatgcca 1200 aaggctctcg ggacgtggta gatgggatca acagactgtc tttctccagc actggcagta 1260 ccaggagtaa catgtccacc tcttccacct cctcaaagga gtcttcactg tcagcctccc 1320 cgtctcaaga caaaaggctc cgactggacc cagacacagc catagagaag ctctatcggc 1380 tccagcagac cctggagatg ggtgtgtgca gcctcatgtc actggtcacc acagactgga 1440 ggtgctacgg atacatggaa aggcacatca atgagatccg caccgcggtg gacaaagtag 1500 agctgttctt acgagaatac ctccattttg ccaagggagc tttagccaat gcctcctgcc 1560 tcccagaact ggtcctccac aacaaaatga agcgggaact ccaaagagta gaagattccc 1620 accagattct aagccaaacc agccatgact tgaatgaatg cagctggtcc ctgaatattt 1680 tagctatcaa taagccccaa aataagtgtg atgacctaga ccggtttgtg atggtcgcca 1740 agacagtgcc agacgacgcc aaacaactga ccaccaccat cagcacctac gcggagaccc 1800 tctttagagc agatcctgcc aattcccatc tgaagaatgg gcccaacagc atcatgaact 1860 caagcgagta cacacatccg ggctcccaga tgcagccact gcatcctggt gactacaaag 1920 cccaggtcca cagtaagccg ttgcctccta gtctaagcaa ggaccagcca ccagactgcg 1980 gtagcagtga cggttctgag cggagttgga tggatgatta tgattatgtt cacctacagg 2040 gcaaggagga gtttgagcga cagcagaagg agctcttgga aaaggagaac atcatgaagc 2100 agagtaaggc gcagctggag catcaccagc tgagtcagtt ccagctgttg gaacaagaga 2160 tcaccaagcc tgtggagaat gacatctcta aatggaagcc ctctcagagc ctcccaacca 2220 ccaacaacag tgtgggtgct caggataggc agttgctttg cttctactat gaccagtgcg 2280 agacccattt catttcccta ctcaacgcca tcgacgccct cttcagctgc gtcagctcag 2340 cccaaccccc acggatcttt gtggcgcaca gcaagtttgt cattcttagt gcgcacaaac 2400 tggtgttcat tggagacact ctgacaaggc aggtggctgc ccaggacatt cgcaacaaag 2460 tcaggaactc cagcaaccag ctctgcgaac agctcaagac gatagtgatg gcgaccaaaa 2520 tggccgccct ccactacccc agtaccaccg ccttgcagga aatggtgcac caggtgacag 2580 acctgtccag aaatgctcag ctgtttaagc gttccttgct ggagatggcc accttttgag 2640 aagacaaaga agtggaagga actgggtgaa taattactaa ggaaaactgg aaatactatc 2700 tagtttttgt aaatgctatc tatttttgta gatattttat atgaaattga aatatttcga 2760 tgtttttgtg agttagtcga ttttcatcaa ttcagggagc tgaagcttgg atttattttg 2820 tttcccctgt gtggttctga tataaacata taagtatcta agacataagt tgtacagaac 2880 tgtgtccacg tttgtgtatg cctacatatc catatttgtt tatctgtgtg tctgatacag 2940 cccattaaaa acatgaattg agaagcacct tagtgagcac cttctaatgc tgcattgttt 3000 gggttttgta gaaaattata ccacttagtt gtaatattgc tcttcatgta gtagtagtcg 3060 tctgagccca gcacacccaa ctttcttgtc tgaaacctct ttcaaacttg acttgtcttt 3120 aacacggtgg taaatctgac caacttctgg ttgaaggggg gcagggaaag aaagaaagat 3180 tcaaaaaata tattaaccta aggtttcagg tgctagagag aggcaaaaca tgtgtgctgt 3240 actaagaggc catgtctaca gtagttattg tgttatttca attctgaaag gaactacaat 3300 aaagagagaa cacttgtttc cctggggcta catttgtgag tgattcagtc atggttttct 3360 gaaggatgtt tttagagttg aatttttttt taaaaaaaat tgcaacagga attcatgaag 3420 atacatcaga actgtgattg tgggcaaaaa gaagggctgt ttccaacagg cagtagttaa 3480 atcaagacct ctctaccctc attctctaat ggaaatggcc agttccttag tcactgaact 3540 gtggaaccac cgggccctgt cctgctctcg cattgcttga tggatcctac accagtctaa 3600 aaacttacat aagcagacca acactcaaaa cagctgccaa gagctgacct tcacgtccca 3660 ctgtggctat ggaccttgtg gatgtcagct agtcacagcc atgatcgtgt ggtctgtccc 3720 ttcacaccgg ctgtgtggat acagcccagg caccagattc atcgtgggca gataaactac 3780 ttccctcttc ctttatgacc aactcaagaa atatagtagt ctctgatcta tttcattcca 3840 gcctacttgg aaatgtgttt ttatttgtta tggatgtctt gactgagtta atgttatttg 3900 ttttaaacaa ccaaattaca aaggcaagga ggggcttaag aaggacatgt gatctcaatg 3960 tgattttttt ttaataaatg gaagatatca aaggaaaggt gcttttcaaa acaaactata 4020 attgtaattc tcaaagttct acatcgccag aagatgaaca gctgagctat tggagagcaa 4080 ttcactgtgt ctggcgtgtg gaagaagagg gtcctgaagc atcagtacca ttgcaggaag 4140 tcaggaaggg tatgcttgta gatcttacta cacaatgtgt atatgttatg tgatggctgc 4200 cttgtcctaa cctgattgat gacattccac attgtaaaaa ccaaactatt caatttctac 4260 tgtttattgc catcttgtat atttaagcca ttaaatgttt ggatatttct ctttatagcc 4320 actgggtttc ttttccttgt gtctctctta tcacagaatt aaatattgac ccatctgagt 4380 ataa 4384

Claims (1)

After cross-linking TH-TagA58 transgenic mice and DJ-1 gene targeting mice to produce double gene targeting mice, DJ-1 gene loss neuronal cell lines were obtained and established from cells of the mouse stromal tissue, pH7.0 and 33 Method for producing DJ-1 gene loss hit dopaminergic neuronal cell line KCTC 11472BP characterized by culturing at ℃.

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