KR101447560B1 - method for screening agent using transgenic drosophila melanogaster expressing human DYRK1A gene - Google Patents

Abstract

The present invention relates to a method for screening a therapeutic agent for Down's syndrome associated with Down Syndrome using the transformed Drosophila melanogaster DYRK1A (DYRK1A) or DYRK1A, Specifically, the human Down's Syndrome gene (DYRK1A) or the minibrain transformed Drosophila of the present invention overexpresses hDYRK1A or minibrain specifically in the nervous system, resulting in a shortened life span and a decrease in exercise capacity, , Which is easy to manipulate, has excellent economic efficiency, and exhibits a phenotype that can easily observe Down syndrome symptoms, and thus can be effectively used for screening for a phenotype therapeutic agent related to Down syndrome.

Description

Methods for screening agents using transgenic drosophila melanogaster expressing human DYRK1A gene using human DYRK1A gene-

The present invention relates to a method for producing a transformed Drosophila melanogaster DYRK1A (DYRK1A) or a Drosophila homologue of DYRK1A, a method for producing the transformed Drosophila melanogaster, and a method for the treatment of Down's syndrome related phenotype using the transgenic Drosophila melanogaster And a method for screening a therapeutic agent.

Down Syndrome is a chromosomal disorder caused by a trisomy of human chromosome 21, which includes mental retardation, learning and memory loss, early onset of Alzheimer's disease, and craniofacial disorders. In particular, it is presumed that changes in the expression level of genes present in a specific region designated as 'Down syndrome critical region (DSCR)' of human chromosome 21 lead to the development of Down syndrome. Among these, DYRK1A (Dual-specific tyrosine (Y) Regulated Kinase 1A) plays an important role in the development and function of the neuronal central nervous system and is involved in the phosphorylation of various proteins. In particular, understanding the biochemical, functional, and molecular biologic effects of this protein as a gene involved in learning disorders and memory loss, altered flexibility of synapses, abnormal cell cycle and neuropathic symptoms similar to Alzheimer's dementia is associated with Down's syndrome It is essential to develop a therapeutic agent for neurodegenerative diseases related to Down syndrome and to identify the etiology of the related diseases.

In particular, in the case of Down syndrome, it is one of the most frequent chromosomal aberration syndromes in about 700 babies. Regardless of race, environment, and socioeconomic differences, the incidence occurs at a certain rate. When the mother is 35 years old or older, the incidence is higher. When the mother is 40 years old or older, birth rate is 1 per 100. There is no way to prevent this genetic abnormality in modern medicine. Only genetic testing can be done to confirm the fetal down syndrome before birth.

Disease prognosis in patients with Down syndrome has the following common physical features. Symptoms appear in all of the body, which causes a delay in behavioral development due to decreased brain function in Down syndrome children. 30 ~ 40% of patients are born with congenital heart disease such as heart valve abnormalities, and the incidence of pneumonia and leukemia, intestinal obstruction, enteritis due to immunity depression is high. With the development of medical technology, most symptoms can be treated or prevented, and the average lifespan of Down syndrome is increasing to 50 years old. However, there have been no developments in the treatment of cognitive decline and neurodegenerative symptoms until now, and the effect of Piracetam on improving cognitive ability has not been shown to be effective in Down syndrome children (Lobaugh, NJ et al. (2001) "Piracetam therapy does not enhance cognitive functioning in children with down syndrome." Arch Pediatr Adolesc Med 155: 442-448). Therefore, it is urgent to develop a therapeutic agent through the identification of underlying mechanism of the brain nervous system in Down syndrome patients.

To do this, we need molecular biologic pathologic studies on the major genes in chromosome 21, rather than whole human chromosome 21, which is thought to be the cause of Down syndrome. Genes located in the 4 to 5 Mb region called the Syndrome Critical Region (DSCR) are known to be involved in a variety of symptoms including brain dysfunction, and research on the genes located here is required. In a previous study on the brain dysfunction of Down's syndrome, we found that at least 225 genes were present in 2000 by decoding the base sequence of chromosome 21 (Hattori, M. et al. chromosome 21. "Nature 405: 311-319). In particular, a number of candidate genes, which are predicted to be a cause of brain dysfunction due to overexpression, have been discovered based on temporal and spatial expression patterns and roles in the nervous system (brain) (Benavides-Piccione, R. et al. Altafaj, X. et al. (2001). "Neurodevelopmental delay, motor abnormalities and cognitive deficits in transgenic mice overexpressing. &Quot; Prod Neurobiol 74: 111-126; Dyspnea, Dyskinesia, Dyskinesia, Dyskinesia, Dyskinesia, Dyskinesia, Dyskinesia, Dyskinesia, Dyskinesia, Nat Genet 5: 22-30 (1993), " Introduction and Expression of the 400 kilobase Amyloid Precursor Protein Gene in Transgenic Mice. "Nat Genet Acad Sci USA 84: 8044-8048; Chrest, R. et al. (2000). "Mice trisomic for a bacterial artificial chromosome with the single-minded 2 gene (Sim 2) show phenotypes similar to some of those present in the partial trisomy 16 mouse models of Down syndrome. "Hum Mol Genet 9: 1853-1864).

Among these genes, transgenic mice are known to be one of the major genes in DYRK1A that show decreased learning and memory capacity in Down syndrome (Smith, DJ et al. (1997). "Functional screening of 2 Mb of human chromosome 21q22. 2 in transgenic mice implicated in the developmental defects associated with Down syndrome. "Altafaj, X. et al. (2001)." Neurodevelopmental delay, motor abnormalities and cognitive deficits in transgenic mice overexpressing Dyrk1A ), a murine model of Down's syndrome. "Hum Mol Genet 10: 1915-1923).

Previously, DYRK1A transgenic mice were presented as two models. First, DYRK1A was constructed by introducing YAC (Yeast Artificial Chromosome) vector containing adjacent region (Smith, DJ et al. Nat Genet 16 (1): 28-36), and the second was a transgenic mouse with only DYRK1A cDNA introduced into it (1990). "Down's syndrome is a neurodevelopmental delay, motor abnormalities and cognitive deficits in transgenic mice overexpressing Dyrk1A (minibrain), a murine model of Down's syndrome. Hum MoI Genet 10: 1915-1923). However, in the transgenic mice into which the YAC vector is introduced, many genes other than DYRK1A are contained in the YAC vector, so that there is a problem that the gene-specific effect and disturbance of other genes can not be excluded in the screening of the therapeutic agent. The expression pattern of the DYRK1A cDNA was not identical to that of the original gene in the transgenic mice expressing the DYRK1A cDNA, and the disease-specific phenotype of the transgenic mice was weak, which is considered to be limited for the screening of the drug. Recently, transgenic mice using the YAC vector of hDYRK1A in humans have been developed and presented as a screening model for the development of Down syndrome (Ahn, KJ, et al. (2006).) DYRK1A BAC transgenic mice show altered synaptic plasticity with learning and memory defects. "Neurobiol Dis. 22 (3): 463-72). However, it appears that there are limitations in the cost and time of the mouse model used for drug screening.

Drosophila models are well preserved in more than 80% of human disease genes, and many previous studies have shown that Drosophila has the same symptoms as human diseases and can screen large quantities of new drugs at low cost, (Giacomotto J et al. (2010) Br J Pharmacol. 160 (2): 204-16).

In particular, the minibrain gene of Drosophila melanogaster DYRK1A is a Drosophila homologous gene of DYRK1A, which is first reported to be related to neurodevelopment. It is known to be expressed in the adult nervous system from the embryonic stage. Especially, Including the mushroom body in charge of the neurons. In addition, minibrain mutant Drosophila has been shown to have significantly reduced brain tissue size and behavioral defects compared to normal Drosophila, suggesting that Drosophila minibrain gene Drosophila DYRK1A possesses important functions in brain nervous system function (Tejedor, F., et al. (1995). "Minibrain: a new protein kinase family involved in postembryonic neurogenesis in Drosophila." Neuron 14: 287-301).

As a result, the inventors of the present invention have made efforts to develop a therapeutic agent for Down Syndrome symptomatically. As a result, the inventors of the present invention found that the DYRK1A (Dual-specific tyrosine (Y) Regulated Kinase 1A) or the minibrain gene After the transgenic Drosophila was produced, the transgenic Drosophila expresses hDYRK1A in a nervous system specific manner to shorten the lifespan and decrease the exercise capacity. Thus, the transformed Drosophila can be used for the treatment of the Down Syndrome related phenotype The present invention has been accomplished on the basis of these findings.

It is an object of the present invention to provide a method of producing a vector comprising: 1) preparing a vector comprising human DYRK1A (Dual-specific tyrosine (Y) Regulated Kinase 1A) or a minibrain gene; And

2) a step of transforming the vector of step 1) into Drosophila to produce a Down's syndrome gene-transgenic Drosophila.

Another object of the present invention is to provide Down Syndrome gene transgenic flies produced by the method of the present invention.

Another object of the present invention is to provide a method for screening a composition for treating Down's syndrome-related phenotype using the Down's syndrome gene transgenic flora of the present invention.

In order to achieve the above object,

1) preparing a vector comprising human DYRK1A (Dual-specific tyrosine (Y) Regulated Kinase 1A) or a minibrain gene; And

2) transforming the vector of step 1) into Drosophila to produce a Down's syndrome gene-transgenic Drosophila.

The present invention also provides Down Syndrome gene transgenic flies produced by the above method.

In addition,

1) treating the test substance with the Down Syndrome gene transgenic flies of the present invention as an experimental group;

2) measuring the hDYRK1A gene expression change in the transfected Drosophila treated with the test substance of step 1); And

3) screening a test substance that reduces hDYRK1A gene expression in step 2) as compared with the untreated control group.

In addition,

1) treating the test substance with the Down Syndrome gene transgenic flies of the present invention as an experimental group;

2) measuring the lifespan of the fruit fly in the transgenic fruit fly having been treated with the test substance of step 1); And

3) screening a test substance which prolongs the lifespan of the fruit fly measured in the step 2) compared to the non-treated control fruit fly, and screening the composition for prevention and improvement of life span.

In addition,

1) treating the test substance with the Down Syndrome gene transgenic flies of the present invention as an experimental group;

2) measuring the athletic performance in the transgenic flora treated with the test substance of step 1); And

3) screening a test substance which increases the exercise capacity of the fruit fly measured in the step 2) as compared with the non-treated control group.

The human Down's Syndrome gene (DYRK1A) or the minibrain gene-transformed Drosophila expresses hDYRK1A specifically over the nervous system, resulting in a shortened life span and reduced exercise capacity, and is easier to manipulate compared to the existing mouse model, And can exhibit a phenotype that can easily observe Down's syndrome symptoms. Therefore, it can be effectively used for screening a phenotype therapeutic agent related to Down's syndrome.

Brief Description of the Drawings Fig. 1 is a diagram showing a method for producing a DYRK1A (Dual-specific tyrosine (Y) Regulated Kinase 1A) expression vector.
Fig. 2 shows a method for producing a minibrain E expression vector.
Fig. 3 shows a method for producing a minibrain H expression vector.
FIG. 4 is a diagram showing gene over-expression effects of hDYRK1A and minibrain transformed Drosophila:
Elav - Gal4 : control Drosophila;
Elav > hDYRK1A : human down syndrome hDYRK1A transgenic flies;
Elav > minibrain : Drosophila minibrain Transgenic Drosophila; And
Elav > minibrain- H : Drosophila minibrain-H Transgenic Drosophila.
FIG. 5 shows the shortening effect of hDYRK1 and minibrain genes on fruit flies;
ElvaGS > hDYRK1A-RU: control Drosophila not inducing expression of hDYRK1A gene;
ElvaGS > hDYRK1A + RU: hDYRK1A gene overexpressing Drosophila;
ElvaGS > mnb-RU: control Drosophila without inducing minibrain gene expression; And
ElvaGS> mnb + RU: minibrain gene overexpressed Drosophila;
Figure 6 shows the change in the ability of Drosophila movement due to hDYRK1 and minibrain genes:
ElvaGS > hDYRK1A-RU: control Drosophila not inducing expression of hDYRK1A gene;
ElvaGS > hDYRK1A + RU: hDYRK1A gene overexpressing Drosophila;
ElvaGS > mnb-RU: control Drosophila without inducing minibrain gene expression; And
ElvaGS> mnb + RU: minibrain gene overexpressed Drosophila;
Figure 7 is an analysis of the developmental pattern of the Drosophila neuron by minibrain gene expression:
Elva > syt-GFP: Control Drosophila; and
Elva>syt-GFP; minibrain: minibrain gene transgenic flies.

Hereinafter, the present invention will be described in detail.

The present invention

1) preparing a vector comprising human DYRK1A (Dual-specific tyrosine (Y) Regulated Kinase 1A) or a minibrain gene; And

2) transforming the vector of step 1) into Drosophila to produce a Down's syndrome gene-transgenic Drosophila.

Also provided is a Down Syndrome gene transgenic flora produced by the method of the present invention.

In the above method, the DYRK1A gene of step 1) preferably has the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

The minibrain gene of step 1) preferably has the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3, but is not limited thereto.

The vector of step 1) preferably includes a human DYRK1A gene or a minibrain gene, but may also include a DYRK1B, DYRK2 or DYRK4 gene which is a subtype of the DYRK gene.

 Preferably, the vector of step 2) is a UAS vector, and the UAS vector comprising human DYRK1A or minibrain gene is introduced into Drosophila using P-element-mediated germ line transformation But is not limited thereto.

The fruit flies of step 2) above are yellow flies ( drosophila melanogaster ), but is not limited thereto.

In a specific embodiment of the present invention, the present inventors prepared an expression vector comprising DYRK1A and a minibrain gene to produce a transformed Drosophila containing the human DYRK1A gene, which is known as Down's syndrome gene, or the minibrain gene thereof, And then transformed with UAS-hDYRK1A and UAS-minibrain expression vectors by P-element-mediated germ line transformation (see FIGS. 1 to 3).

In order to confirm whether hDYRK1A and minibrain transgenic flies can induce the expression of hDYRK1A and minibrain gene, respectively, we overexpressed DYRK1A and minibrain genes using Gal4 / UAS system, respectively. As a result, The minibrain gene-transformed Drosophila showed significantly increased expression of the hDYRK1A gene (see FIG. 4A) and the minibrain gene (see FIG. 4B) by about 4-5 times as compared to the control group (see FIG. 4).

In addition, to overestimate the effect of hDYRK1 and minibrain gene shortening on the lifespan of hDYRK1, the present inventors overexpressed DYRK1A and minibrain genes using Gal4 / UAS system, respectively. As a result, DYRK1A and minibrain gene expression- It was confirmed that the life span shortening effect was similar to that of the human Down Syndrome patients as compared with the control group (see FIG. 5).

In addition, in order to confirm the change of Drosophila movement ability by hDYRK1 and minibrain gene, we overexpressed DYRK1A and minibrain gene using Gal4 / UAS system, respectively. As a result, DYRK1A and minibrain gene expression-induced transgenic Drosophila And the decrease in the exercise capacity was significantly increased as compared with the control group (see FIG. 6).

In addition, the present inventors measured GFP fluorescence to measure the development of the Drosophila melanogaster by the minibrain gene expression. As a result, when the minibrain gene was overexpressed, various neurogenic abnormalities were confirmed, and the nervous system abnormality due to the minibrain gene overexpression was associated (see FIG. 7).

Therefore, the Drosophila melanogaster transformed with the Down Syndrome gene (DYRK1A or minibrain) according to the present invention overexpresses hDYRK1A or minibrain specifically in the nervous system, resulting in a shortened life span and a decrease in exercise capacity, and is easier to manipulate compared to the existing mouse model And is excellent in economic efficiency and exhibits a phenotype that can easily observe Down syndrome symptoms, so that it can be effectively used for screening a phenotype therapeutic agent related to Down syndrome.

Further, according to the present invention,

1) treating the test substance with the Down Syndrome gene transgenic flies of the present invention;

2) measuring the hDYRK1A or minibrain gene expression change in the transfected Drosophila treated with the test substance of step 1); And

3) screening a test substance that reduces hDYRK1A or minibrain gene expression in step 2) as compared to the untreated control group.

In the screening method, the hDYRK1A or minibrain gene expression change is measured by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), but is not limited thereto.

In a specific example of the present invention, the present inventors used the Gal4 / UAS system to determine whether the hDYRK1A transformed Drosophila and the minibrain transgenic Drosophila can induce the expression of hDYRK1A and minibrain genes, respectively, As a result, it was confirmed that the expression of hDYRK1A gene (see FIG. 4A) and the minibrain gene (see FIG. 4B) were significantly increased by about 4-5 times as compared with the control group in Drosophila transformed DYRK1A and minibrain genes 4).

Further, according to the present invention,

1) treating the test substance with the Down Syndrome gene transgenic flies of the present invention as an experimental group;

2) measuring the lifespan of the fruit fly in the transgenic fruit fly having been treated with the test substance of step 1); And

3) selecting a test substance that prolongs the lifespan of the fruit fly measured in step 2) as compared with the untreated control group.

In a specific example of the present invention, the present inventors overexpressed DYRK1A and the minibrain gene using Gal4 / UAS system to confirm the shortening effect of hDYRK1 and minibrain gene on the lifespan of Drosophila, respectively. As a result, expression of DYRK1A and minibrain gene Derived transgenic flies were found to have the same shortening of life span as those of the human Down syndrome patients (see FIG. 5).

Further, according to the present invention,

1) treating the test substance with the Down Syndrome gene transgenic flies of the present invention as an experimental group;

2) measuring the athletic performance in the transgenic flora treated with the test substance of step 1); And

3) screening a test substance which increases the exercise capacity of the fruit fly measured in the step 2) as compared with the non-treated control group.

In a specific example of the present invention, the present inventors overexpressed DYRK1A and the minibrain gene using Gal4 / UAS system to confirm the change in the ability of Drosophila movement by hDYRK1 and minibrain gene, respectively. As a result, expression of DYRK1A and minibrain gene Induced transgenic Drosophila showed a significant increase in athletic capacity reduction compared to the control group (see FIG. 6).

Therefore, the Drosophila melanogaster transformed with the Down Syndrome gene (DYRK1A or minibrain) according to the present invention overexpresses hDYRK1A or minibrain specifically in the nervous system, resulting in a shortened life span and a decrease in exercise capacity, and is easier to manipulate compared to the existing mouse model And is excellent in economic efficiency and exhibits a phenotype that can easily observe Down syndrome symptoms, so that it can be effectively used for screening a phenotype therapeutic agent related to Down syndrome.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

< Example  1> Drosophila preparation and breeding

Drosophila was maintained at 25 ° C, and Elav-Gal4 and Elav-GeneSwitch-Gal4 transformed Drosophila used as a control were described in Lee, Ks., Et al. (Lee, KS, et al., (2009), J. Biol. Chem 284 (43): 29454-61). In addition, UAS-hDYRK1A and UAS-minibrain transformed Drosophila were prepared and used according to the method described in <Example 2>.

< Example  2> UAS - hDYRK1A  And UAS - minibrain  Production of Transgenic Drosophila

<2-1> DYRK1A  Of the expression vector Cloning

To construct a transgenic Drosophila having the human DYRK1A gene (Dual-specific tyrosine (Y) Regulated Kinase 1A) gene, the human DYRK1A gene (Genebank number: BC156309; SEQ ID NO: 1 ) CDNA clone (Openbiosystem, USA) was purchased and cloning was carried out by the following method using a gateway system.

Specifically, 150 ng of the pENTR223.1-DYRK1A vector and 150 ng of the UAS vector were mixed and filled up to 8 μl with a TE buffer. Then, 2 μl of LR Clonase II enzyme (Invitrogen, USA) was added and reacted at 25 ° C. for 1 hour To construct a UAS-DYRK1A vector (Fig. 1).

<2-2> minibrain  E expression vector Cloning

Drosophila , the Drosophila homologue of the DYRK1A gene minibrain E, cloning was performed as follows based on the nucleotide sequence of mnb E (Genebank number: NM_167581.2; SEQ ID NO: 2).

Specifically, total RNA was first extracted from Drosophila using an easy-BLUE RNA extraction kit (iNtRON Biotechnology, Korea), and then 3 μg of total RNA and 1 μl of Oligo-dT (Invitrogen, USA) were filled up to 13 μl of distilled water , SuperScript First-Strand Synthesis System (Invitrogen, USA). Then, cDNA was synthesized by reacting at 65 ° C for 5 minutes, 4 ° C for 2 minutes, 50 ° C for 60 minutes, and 70 ° C for 15 minutes. (1'-CCATGTATAGATTAGAGGATACGAATAG-3 ') and SEQ ID NO: 5 (5'-ACCGCCTGTTTCCACTCCGAGGATG-3') for the separation of mnb E (N-term) Primer pairs of SEQ ID NO: 6 (5'-AGTATCTGCTCGACCAGGCGCACAAG-3 ') and SEQ ID NO: 7 (5'-GTGGAGGCGTTGGTTGGCGGTGCAAT-3') were isolated using PCR mastermix (iNtRON Biotechnology, Korea) The mixture was denatured at 95 ° C for 5 minutes, rotated 30 times at 95 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 ° C for 1 minute and 30 seconds, and then elongated at 72 ° C for 5 minutes. PCR was carried out in the following order. PGEMEasy-mnb E (N-term) and pGEMEasy-mnb E (C-term) vectors were constructed using the pGEMEasy vector system (Promega, USA). HindIII restriction enzyme at 1281 bp was used for vector construction with full-length ORF of mnb E. pGEMEasy-mnb E (N-term) was cut out with HindIII restriction enzyme, and mNb E (C-term) was prepared by cutting pGEMEasy-mnb E (C-term) with HindIII and SpeI (Roche, USA). The prepared fragments were ligated using DNA ligase (Bioneer, Korea) to construct pGEMEasy-mnb E (full-length) vector.

In addition, UAS-mnb E (full-length) was prepared in the following manner to prepare a transformed Drosophila having mnb E (full-length).

Specifically, pGEMEasy-mnb E (full-length) vector and UAS vector were cut with restriction enzyme EcoRI (Roche, USA) and mnb E (full-length) fragment and linearized UAS vector were ligated using DNA ligase To construct a UAS-mnb E (full-length) vector (Fig. 2).

<2-3> minibrain  H expression vector Cloning

Drosophila , the Drosophila homologue of the DYRK1A gene In order to prepare a transformed Drosophila having minibrain H, cloning was carried out based on the nucleotide sequence of mnb H (Genebank number: NM_170659.2; SEQ ID NO: 3). (5'-GCGATTCCGATTGCCAGTGATATGT-3 ') and SEQ ID NO: 9 (5'-ACCGCCTGTTTCCACTCCGAGGATG-3') primer pairs were amplified by PCR mastermix (iNtRON Biotechnology, Korea) for 5 minutes at 95 ° C, and 30 revolutions were performed at 95 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 ° C for 1 minute and 30 seconds, followed by extension at 72 ° C for 5 minutes The PCR was carried out in the order of cooling at 4 캜. This PCR fragment was constructed with pGEMEasy-mnb H (N-term) vector using pGEMEasy vector system (Promega, USA). Since the C-term portion of mnb H consists of a base sequence such as mnb E (C-term), mnb E (C-term) was used instead of mnb H (C-term). mnb H (full-length) was prepared as follows using a HindIII restriction enzyme present in the 1717 bp portion of mnb H. After digesting pGEMEasy-mnb H (N-term) with HindIII restriction enzyme and preparing pMEMEasy-mnb E (C-term) with HindIII and SpeI (Roche, USA) Each fragment was constructed with pGEMEasy-mnb H (full-length) vector using DNA ligase (Bioneer, Korea).

In order to construct a transformed Drosophila having mnb H (full-length), a full-length pGEMEasy-mnb H vector and a UAS vector were cut with a restriction enzyme EcoRI (Roche, USA) And UAS-mnb H (full-length) vector were constructed by ligating the linearized UAS vector with DNA ligase (Bioneer, Korea) (FIG. 3).

<2-4> hDYRK1A  And minibrain  Drosophila Transformation of Expression Vectors

In order to transform the hDYRK1A and minibrain expression vectors prepared in Examples <2-2> and <2-3> into Drosophila, a known method, P-element-mediated germline transformation transformed with UAS-hDYRK1A and UAS-minibrain expression vectors [Rubin GM et al . (1982) Genetic transformation of Drosophila with transposable element vectors. 357 Science 218: 348-353].

<2-5> hDYRK1A  And minibrain  Overexpression of Transgenic Drosophila

To determine whether hDYRK1A and minibrain transgenic flies produced in Example <2-4> could induce the expression of hDYRK1A and minibrain genes, the Gal4 / UAS system of Drosophila was used to identify the nervous system-specific Elav- Elav> hDYRK1A and Elav> minibrain obtained after crossing UAS-hDYRK1A transformed Drosophila and UAS-minibrain transformed Drosophila into Gal4 were separated and their cDNAs were synthesized, and hDYRK1A primer and minibrain primer Were used for PCR.

Specifically, the total RNA was extracted using an easy-BLUE RNA extraction kit, and then 1 μg of total RNA and 1 μl of Oligo-dT (Invitrogen, USA) were filled up to 13 μl of distilled water. Synthesis System (Invitrogen, USA). CDNA was synthesized by reacting for 5 minutes at 65 ° C, 2 minutes at 4 ° C, 60 minutes at 50 ° C, 15 minutes at 70 ° C, and 5 minutes at 4 ° C. (5'-CGTGACTTCAATTGGATAAATAGCT-3 ') and the hDYRK1A primer pair of SEQ ID NO: 10 (5'-TGATTGCACCAACAGGTCCAGA-3') and SEQ ID NO: 11 (5'-TTCTCCCAATTCAACCGCTCA- ) And SEQ ID NO: 13 (5'-GCTGAAATTGACCCTCAGGTCC-3 ') minibrain primer pairs and PCR Premix (Bioneer, Korea). The PCR was denatured at 95 ° C for 5 minutes, and the resulting mixture was subjected to 30 rotation at 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds, followed by extension at 72 ° C for 5 minutes, .

As a result, as shown in FIG. 4, the hDYRK1A gene (FIG. 4A) and the minibrain gene (FIG. 4B) significantly increased expression about 4-5 times or more as compared with the control group (FIG.

< Experimental Example  1> hDYRK1  And minibrain  Identification of gene shortening of fruitfly life

In order to confirm the effect of hDYRK1 and minibrain gene shortening on the lifespan of Drosophila, we used the Gal4 / UAS system and the gene switch (GeneSwitch), an inducible gene expression system, to express the hDYRK1A and minibrain gene Drosophila life span was measured using 100 transgenic Drosophila mosquitoes within 2 days of breeding in the pupa after crossing with ElavGeneSwitch-Gal4 (ElavGS-Gal4) and UAS-hDYRK1A to induce expression. Life span was measured using a medium supplemented with RU486 (GeneSwitch) activating gene and a control medium not supplemented. Lifetime was measured by repairing carcass in the incubator every 3 days.

As a result, as shown in FIG. 5, overexpression of the hDYRK1A gene (ElavGS> hDYRK1A + RU, Maxiam lifespan = 45, Midian Lifespan = 22) significantly shortened lifespan (Fig. 5A). It was also confirmed that the lifespan was significantly shortened by minibrain overexpression (ElavGS> minibrain + RU, Maxiam lifespan = 36, Midian Lifespan = 19) (Fig. 5B) And it is confirmed that the life span of the patient is shortened.

< Experimental Example  2> hDYRK1  And minibrain  Identification of Drosophila exercise capacity change by gene

In order to confirm the change in the ability of hDYRK1 and the minibrain gene to express Drosophila, the expression of hDYRK1A and minibrain gene specifically in the adult nervous system using the Gal4 / UAS system and the gene switch (GeneSwitch), an inducible gene expression system (ElavGS-Gal4) and UAS-hDYRK1A were crossed with ElavGeneSwitch-Gal4 (ElavGS-Gal4) and the transformed Drosophila grown within 2 days after waking from the pupa was cultured in medium supplemented with RU486 After 30 days of incubation, the animals were placed in a 10-ml pipette with a 30-day-old ad libitum, and the distance of the flies was measured within a unit time (30 seconds) And locomotion activity changes due to overexpression of minibrain.

As a result, as shown in FIG. 6, it was confirmed that the decrease in exercise capacity due to aging was significantly increased according to hDYRK1A and minibrain overexpression (FIG. 6).

< Experimental Example  3> minibrain  Analysis of Drosophila neural development by gene expression

To analyze the development of the Drosophila neuron by minibrain gene expression, UAS-minibrain transformed Drosophila melanogaster was transfected using Elav-Gal4, UAS-GFP, which is a driver that expresses nerve system specific fluorescent protein using Drosophila Gal4 / UAS system The embryos obtained after crossing and Elav> GFP: minibrain and control Elav> GFP Drosophila were collected for 24 hours, and the embryos were defrosted and immobilized in fixative fluid to analyze the developmental pattern of GFP through GFP expression.

As a result, as shown in Fig. 7, the development of the central nervous system and the peripheral nervous system of the fruit fly (Elav> sytGFP) and the formation of the nervous network structure were well performed (Fig. 7A) In the case of overexpression, various neurogenic abnormalities such as developmental abnormalities of optic lobe and ventral nerve cord (Figs. 7B and 7C) and central-adrenal system neural network abnormalities (Fig. 7D) (Fig. 7). These results suggest that the phenotypes of neuronal outgrowth induced by minibrain overexpression are related to the phenotypic abnormalities of the nervous system.

<110> korea research institute of bioscience & biotechnology <120> method for screening agent using transgenic drosophila          melanogaster expressing human DYRK1A gene <130> 11p-10-73 <160> 13 <170> Kopatentin 1.71 <210> 1 <211> 2353 <212> DNA <213> Homo sapiens <400> 1 gtacaaaaaa gcagaagggc cgtcaaggcc caccatgcat acaggaggag agacttcagc 60 atgcaaacct tcatctgttc ggcttgcacc gtcattttca ttccatgctg ctggccttca 120 gatggctgga cagatgcccc attcacatca gtacagtgac cgtcgccagc caaacataag 180 tgaccaacag gtttctgcct tatcatattc tgaccagatt cagcaacctc taactaacca 240 ggtgatgcct gatattgtca tgttacagag gcggatgccc caaaccttcc gtgacccagc 300 aactgctccc ctgagaaaac tttctgttga cttgatcaaa acatacaagc atattaatga 360 ggtttactat gcaaaaaaga agcgaagaca ccaacagggc cagggagacg attctagtca 420 taagaaggaa cggaaggttt acaatgatgg ttatgatgat gataactatg attatattgt 480 aaaaaacgga gaaaagtgga tggatcgtta cgaaattgac tccttgatag gcaaaggttc 540 ctttggacagta gttgtaaagg catatgatcg tgtggagcaa gaatgggttg ccattaaaat 600 aataaagaac aagaaggctt ttctgaatca agcacagata gaagtgcgac ttcttgagct 660 catgaacaaa catgacactg aaatgaaata ctacatagtg catttgaaac gccactttat 720 gtttcgaaac catctctgtt tagtttttga aatgctgtcc tacaacctct atgacttgct 780 gagaaacacc aatttccgag gggtctcttt gaacctaaca cgaaagtttg cgcaacagat 840 gtgcactgca ctgcttttcc ttgcgactcc agaacttagt atcattcact gtgatctaaa 900 acctgaaaat atccttcttt gtaaccccaa acgcagtgca atcaagatag ttgactttgg 960 cagttcttgt cagttggggc agaggatata ccagtatatt cagagtcgct tttatcggtc 1020 tccagaggtg ctactgggaa tgccttatga ccttgccatt gatatgtggt ccctcgggtg 1080 tattttggtt gaaatgcaca ctggagaacc tctgttcagt ggtgccaatg aggtagatca 1140 gatgaataaa atagtggaag ttctgggtat tccacctgct catattcttg accaagcacc 1200 aaaagcaaga aagttctttg agaagttgcc agatggcact tggaacttaa agaagaccaa 1260 agatggaaaa cgggagtaca aaccaccagg aacccgtaaa cttcataaca ttcttggagt 1320 ggaaacagga ggacctggtg ggcgacgtgc tggggagtca ggtcatacgg tcgctgacta 1380 cttgaagttc aaagacctca ttttaaggat gcttgattat gaccccaaaa ctcgaattca 1440 accttattat gctctgcagc acagtttctt caagaaaaca gctgatgaag gtacaaatac 1500 aagtaatagt gtatctacaa gccccgccat ggagcagtct cagtcttcgg gcaccacctc 1560 cagtacatcg tcaagctcag gtggctcatc ggggacaagc aacagtggga gagcccggtc 1620 ggatccgacg caccagcatc ggcacagtgg tgggcacttc acagctgccg tgcaggccat 1680 ggactgcgag acacacagtc cccaggtgcg tcagcaattt cctgctcctc ttggttggtc 1740 aggcactgaa gctcctacac aggtcactgt tgaaactcat cctgttcaag aaacaacctt 1800 tcatgtagcc cctcaacaga atgcattgca tcatcaccat ggtaacagtt cccatcacca 1860 tcaccaccac caccaccatc accaccacca tggacaacaa gccttgggta accggaccag 1920 gccaagggtc tacaattctc caacgaatag ctcctctacc caagattcta tggaggttgg 1980 ccacagtcac cactccatga catccctgtc ttcctcaacg acttcttcct cgacatcttc 2040 ctcctctact ggtaaccaag gcaatcaggc ctaccagaat cgcccagtgg ctgctaatac 2100 cttggacttt ggacagaatg gagctatgga cgttaatttg accgtctact ccaatccccg 2160 ccaagagact ggcatagctg gacatccaac ataccaattt tctgctaata caggtcctgc 2220 acattacatg actgaaggac atctgacaat gaggcaaggg gctgatagag aagagtcccc 2280 catgacagga gtttgtgtgc aacagagtcc tgtagctagc tcgtcaggcc tcatgggccc 2340 agctttcttg tac 2353 <210> 2 <211> 5504 <212> DNA <213> Drosophila melanogaster <400> 2 atcatcgcga gtggctaacc cagctctgaa agggaagcaa gacacctaaa agcagcaaca 60 aaatgcgcag cgcggaaaca caggcggcgt gttaaataaa ataataatat atagtcggtg 120 cagctggccg tgtgtggccg ctggaaaaac gggaaaagcg tttacacagt gctttttatt 180 tagcattttc tccgtttcgc ccgtgtctgt gtatgcgtgt gttgcgtgcg tgagtgtgcg 240 tgtttttttt tcctcatttg gtgtgtgctt tttttttgta cattgcggag acgcgctctt 300 ttttgtggca gacgggcgga aaagatgaaa aaccgcagag agagcgtcga aaattgatgg 360 gaaaatgtat aaagtttgtt aattgatcgt tttcgaaatg aaagcataga aaaaggagca 420 gcaacggcgg cagcagcaac aacaacaaca acgagtaacg aaaaacggca aaattttttt 480 gtttgcctcg tatcgcattt tctcactctc tctctctccc gctgcgtgtg tgtgtgagtg 540 tgtgtgtgcg cgtcacatat cgacgtccct ctcttgccac cgacaacaac aacaaataca 600 aaacgaagga aggaaagaaa aaggggaata acgtgaaaca tgtttttctg tggcaacagc 660 aacaacataa gcggcagctg caactatcgg cagcaacaac aacagcagta tcataaaaca 720 tgtgtggaat aatcgcagcg aattgaacaa agcccaaagc ctggaaataa agtaaccaaa 780 aaagagaaac gtcgaaaatc aaatcaagca gcagcacaaa caacaacagc agcagtacag 840 ttttgtaaac aaagcgtggc gacaattttt taccattatc aattgcaaaa ggaaacattc 900 aaatttaaaa gtgcttttga aatgtttatg caaaatgttg agcatagcgg aagccaaaaa 960 ttaccataaa accaaaagta cattaacttg agaaaatcga tagattctag ctaacaaatc 1020 gataaagaga agacaaaaac aaaaaaacaa tccatacaac gcagaagaaa gacagatcgc 1080 aaaagttttt gctttcttct cagatttccc ccctgcgact gtgtgcgtgt gagttcgcat 1140 tcgcgtgtgt gtgtgtgtgt tttgttttca ttagagcaca cacacacgcc ccgttgtgtg 1200 ggagtgtgtg taattgcaat gtcgagcggt aaataaacgg aaagagcaat aacaataata 1260 acaacaagag cagcaggaga aacgtgtttt tgttttgttt gtttcataaa tgtttgacaa 1320 ttacactgta aaaaagaaag aaaagtaatt cgtgacaagt gcgcgtacaa acggtaaagc 1380 aaattcgaaa aaaagacttg aaacttgtgt tcataatttt aaacaaacaa caaaaagtat 1440 tcgacccatt taaaaatata tacgaaaaaa tatatattaa ccaataccac atacataaat 1500 ctatattaac tggtaatcac gaaaggaaat tcaaacaaaa acaagtaata aggcaacaac 1560 aacgacgcaa aggataacga aatgaatgca aaatacacaa aaaaaaatat ttaatttata 1620 aaaaaaaacc tacaaatcac aagcaaccaa aatccgacac aaaaacacaa tttcaaacta 1680 tccaaatcgc atgaacaaat aacgcaacat atatatattc atctatataa atatatataa 1740 gcaagagtat atatatgtca agtacatata gcgtaataca taggagccaa caaaccaagt 1800 aaagattcat taatcagcgc aaaaccaaca aacatcatcc ccatgtatag attagaggat 1860 acgaatagcg gcggcgttat ggataagaat aaacagaaac tgtccgctta tgggtccagc 1920 ggcggcagcg tcgacgcagc gcagggcagc ggaagcgggg gcggaagaca gcgccacgcc 1980 ccactctacg ggcgattcgt cgacgcagag gatctgcctg ccacgcaccg ggatgtcatg 2040 caccatcact ctagtccctc gtcttcgtcg gaggtgcgcg ccatgcaggc ccggattccc 2100 aaccactttc gggagcccgc ctcgggacct ttgcgcaagc tgagtgtcga tctgataaag 2160 acctacaagc acatcaacga ggtttactat gcgaaaaaga aacgtcgtgc ccagcagacg 2220 caaggagacg acgactcatc taacaaaaag gagcgaaaac tttataacga cggctacgac 2280 gacgataatc acgactatat aatcaagaat ggcgaaaagt ttttggatcg ctacgagatc 2340 gactctctga tcggcaaagg cagttttggc caggtggtga aggcttacga ccacgaggag 2400 cagtgccacg tggcgatcaa gataattaag aataagaaac cgttcctaaa ccaggcacag 2460 atcgaggtca agttgctcga gatgatgaac cgggcggatg ccgaaaacaa atactacatc 2520 gttaagctca agcggcactt catgtggcgc aaccacctgt gcctggtgtt cgagctgctc 2580 tcatacaacc tgtacgacct gctgcggaac accaacttcc ggggcgtttc gctgaacctc 2640 acgcgcaagt tcgcccagca gctgtgcacc gcactgctct tcctcagcac ccccgaactg 2700 aacataattc actgtgattt gaagcccgag aacatcctgc tctgcaaccc gaagcgctcg 2760 gcgatcaaga tcgttgactt tggcagctcc tgccagttgg gacagcggat ctaccactac 2820 atccagtcgc gcttctaccg ctcgccggag gtgctgcttg gcatccagta tgacctggcc 2880 atcgatatgt ggtcactggg ctgcatcttg gtcgagatgc acaccggcga gccgctcttc 2940 tccggctgca acgaggtgga ccagatgaac aagatcgtcg aggtgctggg catgccgccc 3000 aagtatctgc tcgaccaggc gcacaagacc cgcaagttct tcgacaagat cgtcgccgat 3060 ggctcctatg tgctgaagaa gaatcagaac ggcaggaagt acaagccgcc aggatcccgc 3120 aagcttcacg acatcctcgg agtggaaaca ggcggtccag ggggcaggcg tctcgatgag 3180 ccgggccact ccgtctcgga ctatctgaag ttcaaggatc tgatcctgcg catgctcgac 3240 tttgacccca agacacgggt cacgccctac tatgccctgc agcacaactt cttcaagcgc 3300 acggcagacg aggcaaccaa tacgagcggc gcgggcgcca cagcgaatgc aggagccggt 3360 ggctccggtt ccagcggagc aggaggctcc agcggcggag gcgtcggcgg tggcctcgga 3420 gcgagcaaca gcagtagcgg tgcggtctcc tcatccagtg cagcggctcc aacggcggcg 3480 acagcagcgg ccacggccgc aggatcctcc ggttcgggtt caagtgtcgg cgggggatcc 3540 tccgcagcgc agcaacagca ggcgatgccg ctgccattgc cgcttccctt gccgttgcca 3600 ccactcgcgg gtcctggcgg tgcgtcagat ggccaatgtc acggcctgct tatgcattcg 3660 gtggcagcca acgcgatgaa caacttctcc gctctgagcc tgcagtccaa cgcccatccg 3720 ccgccatcgc tggccaatag ccatcacagc accaacagcc tgggcagcct gaatcacatc 3780 agtcccggca gcaccggctg ccacaacaac aacagcaaca gcagcaacaa caacacacgc 3840 cacagtcgtc tgtacggcag caacatggtg aacatggtgg gccaccacaa cagcggcagc 3900 agcaacaacc acaacagcat cagctatccg cacgccatgg aatgtgatcc gccgcagatg 3960 ccgccaccgc cgccaaacgg acacggcagg atgcgggtgc cagcgataat gcaactgcaa 4020 ccgaacagct atgcgcccaa ttcggtgccc tattacggca acatgtcgtc ctcgtcggtg 4080 gctgcggcgg cggcagcggc agcagctgct gcctcgcacc taatgatgac cgattcgagt 4140 gtgattagcg cctcggcggc gggcggtggc cagggcggcg gcaatcccgg ccagaatccg 4200 gttacgccgt cagctgccgc ctttctcttc ccatctcagc ccgccggaac gctctatggc 4260 acggcgctcg gttcgctgag cgacctgcca ctgccgatgc cactgcccat gtccgtgccc 4320 ctgcaactgc cgccctcgtc gtcgtcgtcc gtctcctcgg gatcagcatc ggttggatcc 4380 ggaggagtgg gtgtcggcgt ggttggccaa aggcggcaca tcaccggacc cgccgcgcag 4440 gtgggcatct ctcaaagtgt cggcagtggc agcagcggct cggccaccgg agccagcagc 4500 agcgatgcct cctcgtcgtc acccatggtg ggcgtttgtg ttcaacagaa tcctgtagtt 4560 atacattagc ataaactgga agcaagcaaa gaacgttgga tgctcgctta aaacactact 4620 tttcacgccc tttgccgctc tctgctgcta tttattgcac cgccaaccaa cgcctccacc 4680 cgcccacaaa ccaaggaacc acaaattcac caacgcagca gcaccatagt tggtcggtaa 4740 tcgggggtct tgtccacacg tttttccaat tttacatacc atgtacgatg tacttcgatg 4800 tacatatact actaattttg acgaataatt gttaatattt tcatctgcgc ttttgtaaga 4860 tcttaagtga agaaaatttc tgttctatgt agacaatttt aaatgaaaat tcttttaatt 4920 agcgattgtg acaacgtgac ttcaattgga taaatagcga agatcatttt gttttgtttg 4980 agtggtaata gtcacacttt cagcttttcc tgaaaagaag ctgagattct tggttcattg 5040 taagacaaga cgcccgatga agatcactgc aacacagaca catgtccaaa ataaaaatac 5100 attaacatta actagctgaa attgaccctc aggtcccacg aaacacacga aaactcactc 5160 acctgcggtt cttactgatt agtcacttcg agctctcacc gccgcgccca cagcaccggc 5220 cacgccccca gtcgcccggg atcggggagg ggatgccgtg tgggcgcctt gatggattcg 5280 gcagagttta tttttttaca cgctagaacc tatttttttt tttggtctaa ttataatgtt 5340 taactttatg cagtaaccgt aacgacatac ttttacttta tataaacgaa tcaaaattaa 5400 actttattta attgttaatt taaattatgt aattttattc gacgagaaat cgctagtaat 5460 atagaaagca aacaaatatg cttaatttaa gcaaaactca aaaa 5504 <210> 3 <211> 4228 <212> DNA <213> Drosophila melanogaster <400> 3 aacagaccgc gcttcataca gccagatcca aatggatctt aaattactta aataaactat 60 ataaaaataa acgaaataag catatcattt tgattgaaaa aaggaagcgg ttagtgcaaa 120 gtgcgttcgc gattccgatt gcgattccga ttgccagtga tatgtcgacg gccctggttt 180 attggcagtc aatgaatggc ggcgatccgg ttgacaaggc caccgcggat tgggcgccat 240 tcgtggttgg tgcagcagct ggtggcagct tcagtagcag catcatcacc agcagcagca 300 gcagcttgaa ccaccatccg cacaacggcc accacaatca ccatcatccg cctcattaca 360 tcaattattt ggcgcaaatt ggctgccagc atcgtgtcag aaattggcca acaaatggca 420 atcaggatca ggcggatgac gacgacgctg gccatcagat catctctaaa ttcctgccca 480 aaagtgcctg ggacacggtg ctgttggatg tgaagcggga tgtagatgaa gatggagata 540 gagatagaga tcgggtcgat ggcaatgtgc tgatggttca gggatcgtca tcatcatcat 600 cgagaatgaa ggcggaacgg gcactctatt atactatgtc cgcttatggg tccagcggcg 660 gcagcgtcga cgcagcgcag ggcagcggaa gcgggggcgg aagacagcgc cacgccccac 720 tctacgggcg attcgtcgac gcagaggatc tgcctgccac gcaccgggat gtcatgcacc 780 atcactctag tccctcgtct tcgtcggagg tgcgcgccat gcaggcccgg attcccaacc 840 actttcggga gcccgcctcg ggacctttgc gcaagctgag tgtcgatctg ataaagacct 900 acaagcacat caacgaggtt tactatgcga aaaagaaacg tcgtgcccag cagacgcaag 960 gagacgacga ctcatctaac aaaaaggagc gaaaacttta taacgacggc tacgacgacg 1020 ataatcacga ctatataatc aagaatggcg aaaagttttt ggatcgctac gagatcgact 1080 ctctgatcgg caaaggcagt tttggccagg tggtgaaggc ttacgaccac gaggagcagt 1140 gccacgtggc gatcaagata attaagaata agaaaccgtt cctaaaccag gcacagatcg 1200 aggtcaagtt gctcgagatg atgaaccggg cggatgccga aaacaaatac tacatcgtta 1260 agctcaagcg gcacttcatg tggcgcaacc acctgtgcct ggtgttcgag ctgctctcat 1320 acaacctgta cgacctgctg cggaacacca acttccgggg cgtttcgctg aacctcacgc 1380 gcaagttcgc ccagcagctg tgcaccgcac tgctcttcct cagcaccccc gaactgaaca 1440 taattcactg tgatttgaag cccgagaaca tcctgctctg caacccgaag cgctcggcga 1500 tcaagatcgt tgactttggc agctcctgcc agttgggaca gcggatctac cactacatcc 1560 agtcgcgctt ctaccgctcg ccggaggtgc tgcttggcat ccagtatgac ctggccatcg 1620 atatgtggtc actgggctgc atcttggtcg agatgcacac cggcgagccg ctcttctccg 1680 gctgcaacga ggtggaccag atgaacaaga tcgtcgaggt gctgggcatg ccgcccaagt 1740 atctgctcga ccaggcgcac aagacccgca agttcttcga caagatcgtc gccgatggct 1800 cctatgtgct gaagaagaat cagaacggca ggaagtacaa gccgccagga tcccgcaagc 1860 ttcacgacat cctcggagtg gaaacaggcg gtccaggggg caggcgtctc gatgagccgg 1920 gccactccgt ctcggactat ctgaagttca aggatctgat cctgcgcatg ctcgactttg 1980 accccaagac acgggtcacg ccctactatg ccctgcagca caacttcttc aagcgcacgg 2040 cagacgaggc aaccaatacg agcggcgcgg gcgccacagc gaatgcagga gccggtggct 2100 ccggttccag cggagcagga ggctccagcg gcggaggcgt cggcggtggc ctcggagcga 2160 gcaacagcag tagcggtgcg gtctcctcat ccagtgcagc ggctccaacg gcggcgacag 2220 cagcggccac ggccgcagga tcctccggtt cgggttcaag tgtcggcggg ggatcctccg 2280 cagcgcagca acagcaggcg atgccgctgc cattgccgct tcccttgccg ttgccaccac 2340 tcgcgggtcc tggcggtgcg tcagatggcc aatgtcacgg cctgcttatg cattcggtgg 2400 cagccaacgc gatgaacaac ttctccgctc tgagcctgca gtccaacgcc catccgccgc 2460 catcgctggc caatagccat cacagcacca acagcctggg cagcctgaat cacatcagtc 2520 ccggcagcac cggctgccac aacaacaaca gcaacagcag caacaacaac acacgccaca 2580 gtcgtctgta cggcagcaac atggtgaaca tggtgggcca ccacaacagc ggcagcagca 2640 acaaccacaa cagcatcagc tatccgcacg ccatggaatg tgatccgccg cagatgccgc 2700 caccgccgcc aaacggacac ggcaggatgc gggtgccagc gataatgcaa ctgcaaccga 2760 acagctatgc gcccaattcg gtgccctatt acggcaacat gtcgtcctcg tcggtggctg 2820 cggcggcggc agcggcagca gctgctgcct cgcacctaat gatgaccgat tcgagtgtga 2880 ttagcgcctc ggcggcgggc ggtggccagg gcggcggcaa tcccggccag aatccggtta 2940 cgccgtcagc tgccgccttt ctcttcccat ctcagcccgc cggaacgctc tatggcacgg 3000 cgctcggttc gctgagcgac ctgccactgc cgatgccact gcccatgtcc gtgcccctgc 3060 aactgccgcc ctcgtcgtcg tcgtccgtct cctcgggatc agcatcggtt ggatccggag 3120 ggtgggtgt cggcgtggtt ggccaaaggc ggcacatcac cggacccgcc gcgcaggtgg 3180 gcatctctca aagtgtcggc agtggcagca gcggctcggc caccggagcc agcagcagcg 3240 atgcctcctc gtcgtcaccc atggtgggcg tttgtgttca acagaatcct gtagttatac 3300 attagcataa actggaagca agcaaagaac gttggatgct cgcttaaaac actacttttc 3360 acgccctttg ccgctctctg ctgctattta ttgcaccgcc aaccaacgcc tccacccgcc 3420 cacaaaccaa ggaaccacaa attcaccaac gcagcagcac catagttggt cggtaatcgg 3480 gggtcttgtc cacacgtttt tccaatttta cataccatgt acgatgtact tcgatgtaca 3540 tatactacta attttgacga ataattgtta atattttcat ctgcgctttt gtaagatctt 3600 aagtgaagaa aatttctgtt ctatgtagac aattttaaat gaaaattctt ttaattagcg 3660 attgtgacaa cgtgacttca attggataaa tagcgaagat cattttgttt tgtttgagtg 3720 gtaatagtca cactttcagc ttttcctgaa aagaagctga gattcttggt tcattgtaag 3780 acaagacgcc cgatgaagat cactgcaaca cagacacatg tccaaaataa aaatacatta 3840 acattaacta gctgaaattg accctcaggt cccacgaaac acacgaaaac tcactcacct 3900 gcggttctta ctgattagtc acttcgagct ctcaccgccg cgcccacagc accggccacg 3960 cccccagtcg cccgggatcg gggaggggat gccgtgtggg cgccttgatg gattcggcag 4020 agtttatttt tttacacgct agaacctatt tttttttttg gtctaattat aatgtttaac 4080 tttatgcagt aaccgtaacg acatactttt actttatata aacgaatcaa aattaaactt 4140 tatttaattg ttaatttaaa ttatgtaatt ttattcgacg agaaatcgct agtaatatag 4200 aaagcaaaca aatatgctta atttaagc 4228 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forword primer for mNb E <400> 4 ccatgtatag attagaggat acgaatag 28 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mNb E <400> 5 accgcctgtt tccactccga ggatg 25 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forword primer for mNb E <400> 6 agtatctgct cgaccaggcg cacaag 26 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mNb E <400> 7 gtggaggcgt tggttggcgg tgcaat 26 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forword primer for mnb H <400> 8 gcgattccga ttgccagtga tatgt 25 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mNb H <400> 9 accgcctgtt tccactccga ggatg 25 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forword primer for hDYRK1A <400> 10 tgattgcacc aacaggtcca ga 22 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for hDYRK1A <400> 11 ttctcccaat tcaaccgctc a 21 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forword primer for minibrain <400> 12 cgtgacttca attggataaa tagct 25 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for minibrain <400> 13 gctgaaattg accctcaggt cc 22

Claims (10)

1) a human DYRK1A (Dual Regulated Kinase 1A) having the nucleotide sequence shown in SEQ ID NO: 1 or a UAS-DYRK1A vector containing the minibrain gene having the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 Or a UAS-minibrain vector; And
2) transforming the vector of step 1) into Drosophila, wherein the vector of step 1) is selected from the group consisting of Down's Syndrome gene A method for producing a transformed Drosophila.
delete delete delete 2. The method of claim 1, wherein the vector of step 2) is introduced using a P-element-mediated germ line transformation.
delete 1) a human DYRK1A (Dual Regulated Kinase 1A) having the nucleotide sequence shown in SEQ ID NO: 1 or a UAS-DYRK1A vector containing the minibrain gene having the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 Or a UAS-minibrain vector;
2) transforming the vector of step 1) into Drosophila to produce Drosophila gene transgenic Drosophila;
3) treating the test substance with the transformed Drosophila in step 2) as an experimental group;
4) measuring the hDYRK1A or minibrain gene expression change in the transformed Drosophila of step 2) in which the test substance of step 3) has been treated; And
5) screening a test substance that reduces hDYRK1A or minibrain gene expression in step 4) as compared to the untreated control group.
The composition according to claim 7, wherein the change in hDYRK1A or minibrain gene expression in step 4) is measured by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Screening method.
1) a human DYRK1A (Dual Regulated Kinase 1A) having the nucleotide sequence shown in SEQ ID NO: 1 or a UAS-DYRK1A vector containing the minibrain gene having the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 Or a UAS-minibrain vector;
2) transforming the vector of step 1) into Drosophila to produce Drosophila gene transgenic Drosophila;
3) treating the test substance with the transformed Drosophila in step 2) as an experimental group;
4) measuring the lifespan of the fruit fly in the transgenic fruit fly of step 2) in which the test substance of step 3) has been treated; And
5) selecting a test substance that prolongs the lifespan of the fruit fly compared to the untreated control group as measured in step 4).
1) a human DYRK1A (Dual Regulated Kinase 1A) having the nucleotide sequence shown in SEQ ID NO: 1 or a UAS-DYRK1A vector containing the minibrain gene having the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 Or a UAS-minibrain vector;
2) transforming the vector of step 1) into Drosophila to produce Drosophila gene transgenic Drosophila;
3) treating the test substance with the transformed Drosophila in step 2) as an experimental group;
4) measuring the athletic ability in the transgenic flora of step 2) in which the test substance of step 3) has been treated; And
5) selecting a test substance which increases the exercise capacity of the fruit fly measured in the step 4) as compared with the non-treated control group.

Images