KR101649214B1 - Generation of Drosophila DSCR1-GAL4, and DSCR1-GAL4, UAS-GFP double mutants preparation method of manufacturing the transgenic mutants - Google Patents

Abstract

The present invention relates to a mutant capable of expressing a fluorescent protein at a tissue-specific site of a Down Syndrome critical region 1 (DSCR1) gene of Drosophila and a preparation method thereof, and more particularly, A dual mutant of a DSCR1- expressing tissue-specific expression Drosophila mutant and a fluorescent protein overexpressing mutant prepared by isolating the promoter region of the DSCR1 gene from the GAL4 system and the Drosophila genome, which are expression systems capable of overexpressing in a specific tissue, .
The DSCR1-GAL4 and GFP overexpressing mutants of the present invention can be used as an animal model which is very useful in the screening of the DSCR1-related factors as well as in the study of the functions of the fruit fly genes and human degenerative brain disease genes.

Description

(DSCR1) -GAL4 Drosophila and DSCR1-GAL4, a UAS-Green fluorescent protein (GFP) double mutant Drosophila strain and a method for producing the Drosophila DSCR1-GAL4 and DSCR1-GAL4, mutants preparation method of manufacturing the transgenic mutants}

The invention Down syndrome risk region of the fluorescent protein in Drosophila 1 (Down syndrome critical region 1, DSCR1) genes can be expressed in the tissue to be expressed, by using this, Drosophila DSCR1 gene expression DSCR1-GAL4 to learn the position and Green fluorescent ( DSC1-GAL4) , which is an expression system capable of expressing a specific gene by isolating the promoter region of the DSCR1 gene, and a method for producing the DSCR1-GAL4 mutant. A dual mutant of a DSCR1-GAL4 mutation and a fluorescent protein (GFP) overexpressing mutation, and a method for producing the same.

Drosophila is the most studied insect in the field of molecular biology. It was not until the 1910s that the fruit fly was used as a material for genetic research by Dr. Morgan of the University of Columbia in the 1910s to be. Since then, mutation induction method, gene mapping method using outline chromosome, and utilization of balancer chromosome to hold single mutation have been enhanced as a biological experiment material.

The pPTGAL vector was studied and constructed by Daniel F. Eberl (Sharma Y., Genesis, pPTGAL, a Convenient Gal4 P-Element Vector for Testing Expression of Enhancer Fragments in Drosophila, 2002). This vector contains the GAL4 gene, which is expressed in a tissue-specific manner by a gene promoter.

Green fluorescent protein (GFP) has been reported by Shimomura O., Shimomura O., University of Boston, USA, Aqueous, It is a fluorescent protein that functions as a secondary fluorescent protein that receives energy from Aequorin and emits green fluorescence at 508 nm. Since the fluorescent protein has no toxicity and is easy to observe, it is used in various fields such as the presence or absence of gene expression, place and time measurement.

Down Syndrome is caused by the presence of three (all or a part) of human chromosome 21, but not two, of the normal, the most frequent genetic abnormality occurring in about one in 700 newborns.

As a study of the causative genes of Down syndrome, samples of patients with Down syndrome partially overlapping human chromosome 21 were analyzed to compare the presence of overlapping chromosomes and their symptoms. In the study, we found that a gene called Down Syndrome Critical Region1 (DSCR1) is likely to be involved in various symptoms including brain dysfunction. A study on the DSCR1 gene has been carried out.

Korean Patent Publication No. 10-2011-0117518 discloses a method for producing a Downs Syndrome model cell line overexpressing Down's syndrome Dangerous Region 1, comprising the steps of: preparing a cell line overexpressing the Down's syndrome Dangerous Area 1 (DSCR1) gene; There is one. However, since the gene itself is over-expressed, it is necessary to include a regulatory region such as a promoter region in order to know a specific mechanism of regulating the expression of DSCR1. In addition, there was a problem that the expression and regulation of DSCR1 in the tissues could not be visually confirmed.

On the other hand, the nucleotide sequence of the Drosophila DSCR1 gene (CG6072) was identified by Ejima A. (Ejima A., Expression level of sarah, a homolog of DSCR1, is critical for ovulation and female courtship behavior in Drosophila melanogaster, 2004). The inventors were able to prepare a DSCR1-GAL4 mutation by isolating up to 960 bp, including the first exon at which the DSCR1 gene begins to be transcribed, and inserting it into the pPTGAL vector.

Analysis of the nucleotide sequence of the Drosophila DSCR1 gene reveals that it encodes a protein very similar to that of the DSCR1 protein, which is expressed in human neurons and is an inhibitor of Ca ²⁺ -dependent dephosphorylase calcineruin (CN) have.

Currently, DSCR1 protein is known to be associated with degenerative brain diseases including Down syndrome. However, it is not known what function to perform through specific mechanism. Accordingly, the present inventors prepared a DSCR1-GAL4 mutant in order to investigate the specific function and mechanism of DSCR1 in order to identify the expression pattern of DSCR1 . In addition, a double mutation of a fluorescent protein overexpressing mutant UAS-GFP and DSCR1-GAL4 was produced, thereby completing the present invention. If we study the phenotype of this double mutant, we can reveal the role of DSCR1 in vivo and its significance.

It is an object of the present invention to contribute to the study of the function and mechanism of DSCR1 in degenerative brain diseases. However, since the antibody and RNA in situ hybridization system of Drosophila DSCR1 gene expression protein are not well-equipped, it is possible to provide a mutant capable of locating the DSCR1 expression position more simply, quickly and accurately, and thus the role and treatment of DSCR1 in degenerative brain diseases Lt; RTI ID = 0.0 > DSCR1-GAL4 < / RTI > mutants and methods for their production.

In order to achieve the above object, the present invention provides a DSCR1 gene-expressing tissue-specific fruit fly mutant comprising a tissue-specific expression mutation of Down's Syndrome Risk Zone 1 (DSCR1) gene.

The present invention also provides DSCR1 gene-expressing tissue-specific fluorescent protein-overexpressing Drosophila dual mutants that simultaneously contain Down's Syndrome Risk Zone 1 (DSCR1) gene tissue-specific expression mutations and fluorescent protein overexpression mutations .

In one embodiment of the present invention, the Down Syndrome Dangerous Region 1 (DSCR1) gene-specific expression mutation is detected by a Gal4-responsive pUAST expression vector (Brand AH. And Perrimon N., Development, Targeted gene expression as a means of altering cell fates and generating dominant phenotypes, 1993).

In one embodiment of the present invention, the fluorescent protein overexpressing mutation may be a gene overexpressed by the UAS-GAL4 system.

In one embodiment of the present invention, the fluorescent protein may be green fluorescent protein (GFP).

In one embodiment of the present invention, the Down Syndrome Dangerous Area 1 (DSCR1) gene-specific expression mutation is characterized in that a specific gene expression can be made tissue-specific by the UAS-GAL4 system .

In one embodiment of the present invention, the Down syndrome risk zone 1 (DSCR1) gene tissue specific (tissue-specific) expression mutations characteristic that can be specifically expressed in tissue DSCR1 gene is expressed by DSCR1 promoter Lt; / RTI >

In one embodiment of the present invention, the DSCR1 promoter region may be characterized in that up to 960 bp is isolated, including the first exon at which transcription of the Drosophila DSCR1 gene begins.

In one embodiment of the present invention, the DSCR1 gene-expressing tissue-specific fluorescent protein-overexpressing Drosophila double mutant specifically expresses a mutation in a fluorescent protein overexpression in a tissue expressing a DSCR1 gene-specific tissue-specific mutation .

(1) crossing a male Drosophila of the DSCR1-GAL4 mutant with an unmated female Drosophila of the UAS-GFP mutant; (2) selecting non-mature female Drosophila of the crossed gene having both the DSCR1-GAL4 mutant and the UAS-GFP mutant among the offspring of the step 1; (3) crossing a female Drosophila selected in step 2 above and a male Drosophila having a balancer CyO and having a phenotype of Curly (genotype Cy) and missing bristles (genotype noc); (4) a step of crossing a non-mating female Drosophila having the DSCR1-GAL4 mutant and the UAS-GFP mutant at the same time among the offspring of the above step 3, the male Drosophila having no noc and CyO having the balancer Cy and noc, ; And (5) selecting a double mutant that simultaneously exhibits a fluorescent protein overexpression and a DSCR1 gene-expressing tissue-specific expression mutation in the progeny of step 4 above, wherein the DSCR1 gene-expressing tissue-specific fluorescent protein-overexpressing Drosophila dual mutant ≪ / RTI >

In one embodiment of the present invention, the step (5) may include a selection by a phenotype.

In one embodiment of the present invention, the phenotype may be a phenotype of whether DSCR1 gene expression overexpresses a fluorescent protein in a tissue specific manner.

According to the invention as described above, the cloning and UAS-GAL4 and tissue-specific genes of the DSCR1 produced by a systemically expressing mutant DSCR1-GAL4, fluorescent protein overexpression mutants UAS-GFP in the double mutant UAS-GFP, DSCR1- By confirming the expression site and expression level of Drosophila DSCR1 using GAL4 , the function of DSCR1 can be deduced.

In addition, the fruit flies can be used for pre-clinical tests to test the performance and side effects of the brain disease drugs to be developed in the future, There is an effect that can be used as an animal model which is very useful not only in study of Drosophila gene function, but also in study of function by human mutation of gene, discovery of new functional gene and study of mental retardation disease.

Figure 1 is a drawing of the DSCR1-GAL4 vector, a DSCR1 tissue specific expression system.
Figure 2 is a promoter sequence up to 960 bp, including the first exon at which transcription of the DSCR1 gene begins.
FIG. 3 shows a genetic process for producing a fruit fly double mutant according to an embodiment of the present invention.
FIG. 4 is a photograph showing the position and expression pattern of DSCR1 gene using UAS-GFP, DSCR1-GAL4 double mutant.

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

In one aspect, the invention provides a DSCR1 gene-expressing tissue-specific Drosophila mutant comprising Down's Syndrome Risk Zone 1 (DSCR1) gene tissue-specific expression mutations.

In yet another aspect, the present invention provides a DSCR1 gene-expressing tissue-specific fluorescent protein-overexpressing Drosophila double mutant (Drosophila dual mutation) gene, which simultaneously contains a Down Syndrome Dangerous Area 1 (DSCR1) gene tissue-specific expression mutation and a fluorescent protein over- Provide sieve.

In one embodiment of the present invention, the Down Syndrome Dangerous Region 1 (DSCR1) gene-specific expression mutation is detected by a Gal4-responsive pUAST expression vector (Brand AH. And Perrimon N., Development, Targeted gene expression as a means of altering cell fates and generating dominant phenotypes, 1993).

In one embodiment of the present invention, the fluorescent protein overexpressing mutation may be a gene overexpressed by the UAS-GAL4 system.

In one embodiment of the present invention, the fluorescent protein may be green fluorescent protein (GFP).

In one embodiment of the present invention, the Down Syndrome Dangerous Area 1 (DSCR1) gene-specific expression mutation is characterized in that a specific gene expression can be made tissue-specific by the UAS-GAL4 system .

In one embodiment of the present invention, the Down syndrome risk zone 1 (DSCR1) gene tissue specific (tissue-specific) expression mutations characteristic that can be specifically expressed in tissue DSCR1 gene is expressed by DSCR1 promoter Lt; / RTI >

In one embodiment of the present invention, the DSCR1 promoter region may be characterized in that up to 960 bp is isolated, including the first exon at which transcription of the Drosophila DSCR1 gene begins.

In one embodiment of the present invention, the DSCR1 gene-expressing tissue-specific fluorescent protein-overexpressing Drosophila double mutant specifically expresses a mutation in a fluorescent protein overexpression in a tissue expressing a DSCR1 gene-specific tissue-specific mutation .

In yet another aspect, the present invention provides a method for producing a UAS-GFP mutant strain, comprising: (1) crossing a male Drosophila of the DSCR1-GAL4 mutant with an unmated female Drosophila of the UAS-GFP mutant; (2) selecting non-mature female Drosophila of the crossed gene having both the DSCR1-GAL4 mutant and the UAS-GFP mutant among the offspring of the step 1; (3) crossing a female Drosophila selected in step 2 above and a male Drosophila having a balancer CyO and having a phenotype of Curly (genotype Cy) and missing bristles (genotype noc); (4) a step of crossing a non-mating female Drosophila having the DSCR1-GAL4 mutant and the UAS-GFP mutant at the same time among the offspring of the above step 3, the male Drosophila having no noc and CyO having the balancer Cy and noc, ; And (5) selecting a double mutant that simultaneously exhibits a fluorescent protein overexpression and a DSCR1 gene-expressing tissue-specific expression mutation in the progeny of step 4 above, wherein the DSCR1 gene-expressing tissue-specific fluorescent protein-overexpressing Drosophila dual mutant ≪ / RTI >

In one embodiment of the present invention, the step (5) may include a selection by a phenotype.

In one embodiment of the present invention, the phenotype may be a phenotype of whether DSCR1 gene expression overexpresses a fluorescent protein in a tissue specific manner.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1: DSCR1-GAL4  Production of mutants

In the present invention, a DSCR1-GAL4 mutation was constructed by using the pPTGAL vector DSCR1 promoter (FIG. 1) in order to examine the expression pattern of the DSCR1 gene by a tissue biomolecular design method.

First, the inventors of the present invention used a pPTGAL vector (pPTGAL) which was studied and manufactured by Daniel F. Eberl (Sharma Y., Genesis, pPTGAL, a Convenient Gal4 P-Element Vector for Testing Expression of Enhancer Fragments in Drosophila, From Daniel F. Eberl. This vector contains the GAL4 gene, which is expressed in a tissue-specific manner by a gene promoter.

GFP is a fluorescent protein found by Shimomura O. (Shimomura O., Boston Scientific University, USA), a bioluminescent protein from the Luminous Hydromedusan, Aequorea, 1962 It acts as a secondary fluorescent protein that receives energy from Aequorin and emits green fluorescence at 508 nm. Since the fluorescent protein has no toxicity and is easy to observe, it is used in various fields such as the presence or absence of gene expression, place and time measurement. GFP- overexpressing mutant UAS-GFP was obtained from the Bloomington Drosophila Stock Center (http://flystocks.bio.indiana.edu/), which mutants were overexpressed through a gene overexpression system called UAS-GAL4 system received.

In addition, Ejima A. The DSCR1 be revealed by sequencing (Ejima A., Expression level of sarah , a homolog of DSCR1, is critical for ovulation and female courtship behavior in Drosophila melanogaster, 2004) of genes in our team DSCR1 (PCR) and inserted into the pPTGAL vector up to 960 bp (see FIG. 2 and SEQ ID NO: 1) including the first exon from which the transcription starts, and inserted into the Drosophila to transform the Drosophila DSCR1- GAL4 system mutations were generated.
[SEQ ID NO: 1]

Example 2: Fabrication of Drosophila double mutants

In the present invention, a double mutation of a fluorescent protein overexpressing mutant UAS-GFP and DSCR1-GAL4 was constructed in order to more simply and effectively confirm the expression pattern of the DSCR1 gene.

First, by crossing a male Drosophila of the DSCR1-GAL4 mutant with an unmated female Drosophila of the UAS-GFP mutant according to the genetic design method of FIG. 3, the DSCR1-GAL4 mutant and the UAS-GFP The mutant female drosophila having crossed genes at the same time was screened, and the selected female Drosophila and male chromosome chromosomes (Cyo) and missing bristles (genotype noc) in the phenotype Curly (genotype Cy) Drosophila.

Then, a male Drosophila having the phenotype DS of CyCR1-GAL4 mutant and UAS-GFP mutant was selected from the above-mentioned crosses and the phenotype was Cy, and the non-noc male Drosophila was selected, and the selected male Drosophila and the balancer, CyO, Cy was crossed with non-mating female Drosophila, noc.

Then, a double mutant that expresses overexpression of a fluorescent protein and a DSCR1 gene-expressing tissue in a double mutant obtained through the above-mentioned crossing step was screened and constructed to complete the invention. The step of screening for the mutant mutant is a step of expressing a fluorescent protein To compare the presence or absence of.

Example 3: Mutations in double mutants DSCR1  Gene expression analysis

The present inventors compared these DSCR1 gene-expressing tissue-specific site and expression pattern with GFP expression confirmation. The UAS-GAL4 system was constructed to overexpress the fluorescent protein in the DSCR1- expressing tissue-specific region. As a result of observing double mutation at larval stage and adult stage, they are expressed respectively in eye disc, fat body, ring gland and brain of adult larvae, , Lipid body, brain, and the like (see Fig. 4).

These results suggest that the DSCR1 gene is mainly expressed in the tissues of the oocyte , lipid, and intestine. It is strongly expressed in the brain in both the larval stage and the adult stage. Especially in the adult brain, (Fig. 4).

Therefore, the present invention contributes to further elucidation of the DSCR1 function, which is a lipid protein of the glia, by confirming the location and pattern of the expression of the DSCR1 gene, and by confirming the expression of DSCR1 in the brain, the association of DSCR1 with degenerative brain disease is enhanced . In addition, the double mutation can be used to examine the interaction with other factors, and thus can be used for screening DSCR1-related factors.

Suggests that it is possible to construct a model animal useful for studying gene function using mutants of Drosophila and related human gene function studies and screening related factors.

Having described specific portions of the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Konkuk University-Industry Cooperation Foundation <120> Generation of Drosophila DSCR1-GAL4, and DSCR1-GAL4, UAS-GFP          double mutants preparation method of manufacturing the transgenic          mutants <130> P14-E556 <140> 10-2014-0124740 <141> 2014-09-19 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 960 <212> DNA <213> Drosophila melanogaster <400> 1 cagctcgtag ttcgtcttac ttgtattcca cttataatta tgttcatact ccattgagtg 60 tcaagtgaaa gattaaaatg attagaatag atgaaagtga agaatcagta cggacgctgg 120 cgcctggcgg ttggcggcag tcggttgggc ggagtaatcg agatgtccag aaagtctcca 180 atgctgaatt tggcctgagc aagtgtctta ttatcatcga ttcctttttg acccgtgcag 240 gtcactccga tttcgcgcat ctggaagtga ttactccgga agttggggta cacgacagca 300 aagtcaaagt aggtgccctt cttccgggtg tccggattga cgtctcgcac cagagaggtc 360 agttcgtgca gggtggcgtc ttgccaggtg taaatctgaa gctcgttggt gggcacgttg 420 ccgaacatat actccgacac ggagtggtgt cgtcccgtag agcagaagac acggagcagc 480 ataggacagg tctggaaaat acataaactt attaacacat ctagagcaaa cgcgaagtaa 540 acatgaccgg ggtcacaaac aaaccttctc gcggtcaatc tgcttgacct gcgtcttttc 600 ctccacaatc atagattcca cgttggccat gtctccctat tctgcggtat atcgttacta 660 attttcttaa ttttaataca aaatgcactt ttaatgccgt atattccggc ttctctttgc 720 tcttttcgat aactatataa tcgaatgcag caatcctatc gcacatcgcc gagtgtgacc 780 gcttgtcgca tacttatcgg gttttagttt aatcgatgtt tccagaagca ccaaaattcg 840 ccgtttctca gctctagtcc gaaaacaaaa ttcgctgtga gcggttgtgt cgtcgaacgt 900 gt;                                                                          960

Claims (11)

Wherein the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of GAL4 gene are joined,
Down syndrome risk zone 1 (DSCR1) tissue-specific genes (tissue-specific) that contains the mutant expression, tissue-specific gene expression DSCR1 Drosophila mutants.
Characterized in that the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of GAL4 gene are joined
Down Syndrome Dangerous Area 1 (DSCR1) Drosophila double mutant overexpressing a DSCR1 gene-expressing tissue-specific fluorescent protein simultaneously containing a tissue-specific expression mutation and a fluorescent protein overexpressing mutation.
3. The method of claim 2,
The Down syndrome risk zone 1 (DSCR1) gene tissue specific (tissue-specific) expression of mutants, specifically DSCR1 gene expression tissue, characterized in that is made ever using the GAL4 responsive pUAST expression vector (GAL4-responsive pUAST expression vector) Fluorescent protein overexpressing Drosophila dual mutants.
3. The method of claim 2,
Wherein the fluorescent protein overexpressing mutation is overexpressed by the UAS-GAL4 system, wherein the DSCR1 gene-expressing tissue-specific fluorescent protein-overexpressing Drosophila double mutant.
3. The method of claim 2,
Wherein the fluorescent protein is a green fluorescent protein (GFP), and the DSCR1 gene-expressing tissue-specific fluorescent protein-overexpressing Drosophila double mutant.
3. The method of claim 2,
The DSCR1 gene-specific expression mutation in the Down's Syndrome Disease 1 (DSCR1) is expressed in a DSCR1 gene-expressing tissue-specific fluorescent light , which is capable of making the expression of a specific gene tissue-specific by the UAS-GAL4 system Protein Overexpressing Drosophila Dual Mutants.
3. The method of claim 2,
The Down syndrome risk zone 1 (DSCR1) gene tissue specific (tissue-specific) expression of mutants, specifically DSCR1 gene expression tissue wherein that can specifically expressed in tissue DSCR1 gene is expressed by DSCR1 promoter Fluorescent protein overexpressing Drosophila dual mutants.
3. The method of claim 2,
The DSCR1 gene expression tissue-specific fluorescent protein over-expression in Drosophila double mutants, DSCR1 gene tissue specific (tissue-specific) expression of mutations specific to the fluorescent protein over-expression mutations DSCR1 gene expression, characterized in that which is expressed in the tissue to be expressed Tissue - specific fluorescent protein overexpressing Drosophila dual mutants.
A method for producing a DSCR1 gene-expressing tissue-specific fluorescent protein overexpressing Drosophila double mutant comprising the steps of:
(1) crossing a male Drosophila of the DSCR1-GAL4 mutant with an unmutated female Drosophila of the UAS-GFP mutant;
(2) selecting non-mature female Drosophila of the crossed gene having both the DSCR1-GAL4 mutant and the UAS-GFP mutant among the offspring of the step (1);
(3) crossing a female Drosophila selected in step (2) and male Drosophila having a balancer CyO and having a phenotype of Curly (genotype Cy) and missing bristles (genotype noc);
(4) a non-mating female having a DSCR1-GAL4 mutant and a UAS-GFP mutant at the same time as the step (3), Cy having a male drosophila and a balancer Cyo having no phenotype, Cy and noc, Crossing the flies; And
(5) selecting a double mutant that simultaneously exhibits fluorescent protein overexpression and DSCR1 gene-expressing tissue-specific expression mutation in the progeny of step (4).
10. The method of claim 9,
Wherein the step (5) comprises screening by a phenotype. 2. The method of claim 1, wherein the step (5) comprises screening by a phenotype.
11. The method of claim 10,
Wherein said phenotype is a phenotype for whether DSCR1 gene expression overexpresses a fluorescent protein in a tissue specific manner. 2. A method for producing a double mutant of Drosophila melanogaster expressing a DSCR1 gene-expressing fluorescent protein.

Images