JPWO2008102903A1 - RNA splicing detection system - Google Patents

Abstract

The present invention relates to a DNA fragment or an expression comprising the same, which can reveal how an exon of a gene defining a protein subject to alternative splicing is selectively spliced in a cell. It is an object to provide a DNA construct such as a vector. The inventors of the present invention insert an intervening fragment containing a cloning site into which an exon of a gene defining a certain protein to be subjected to alternative splicing can be inserted so as to cleave the reporter protein cDNA. Clarified that the above problems can be solved.

Description

  An object of the present invention is to provide a new system for detecting RNA splicing (alternative splicing). More specifically, the present invention aims to provide a new system for quantitatively detecting alternative splicing in situ.

In mammals, alternative splicing is thought to play a major role in protein diversity, and in humans at least 74% of all genes have been reported to undergo alternative splicing ( Non-patent document 1). Among the diseases known as genetic diseases, there are known those caused by alternative splicing abnormalities and those caused by mutations that cause abnormal alternative splicing.
Under such circumstances, it is desired to elucidate the details of the alternative splicing mechanism in vivo, but so far, the elucidation of the control mechanism of alternative splicing has not progressed sufficiently.
For the purpose of clarifying the alternative splicing control mechanism in vivo, a method for producing a transgenic animal using C. elegans to visualize cells and subsequent alternative splicing using a fluorescent protein as a reporter has been developed. (Non-Patent Document 2). However, this method can only identify the splicing phenomenon of a specific gene disclosed in the literature, and can also be used for analyzing the control mechanism of alternative splicing for other genes. As an analysis tool, there was a drawback that it could not be used.
Johnson J. M.M. , Et al. , Science. 2003 302: 2141-2144 Kuroyanagi H. et al. , Et al. Nat Methods. 2006 3: 909-915

  The present invention relates to a DNA fragment or an expression comprising the same, which can reveal how an exon of a gene defining a protein subject to alternative splicing is selectively spliced in a cell. It is an object to provide a DNA construct such as a vector.

The inventors of the present invention have clarified that the above problem can be solved by inserting an intervening fragment to be subjected to alternative splicing so as to cleave the reporter protein cDNA. This intervening fragment contains a cloning site into which an exon of a gene defining a protein can be inserted.
In one specific aspect of the present invention, the inventors of the present invention provide a first fragment and a second fragment of the coding region of DNA encoding a reporter protein; the first fragment and the second fragment An intervening fragment located between, having a splicing motif and comprising a cloning site; an exogenous alternative splicing cassette inserted into said cloning site; is provided.
In this aspect, the present invention can also provide a kit for detecting alternative splicing of a desired exogenous exon comprising the DNA fragment described above; and any eukaryotic expression vector. .
In another aspect of the invention, the inventors of the present invention provide a first fragment and a second fragment of a coding region of DNA encoding a reporter protein; between the first fragment and the second fragment. Cloning of a vector for protein expression comprising a DNA fragment comprising a located intervening fragment having a splicing motif and comprising a cloning site; an exogenous alternative splicing cassette inserted in said cloning site; Provided is a eukaryotic expression vector produced by insertion into a site.
In this aspect, the present invention can also provide a kit for detecting alternative splicing of a desired exogen exon comprising the above-described protein expression vector.
In yet another aspect of the invention, the inventors of the present invention provide a first fragment and a second fragment of the coding region of DNA encoding a reporter protein; between the first fragment and the second fragment. An intervening fragment having a splicing motif and comprising a cloning site; an exogenous alternative splicing cassette inserted into said cloning site; A eukaryotic cell expression vector produced by insertion into a cloning site is transfected into a eukaryotic cell; the transfected eukaryotic cell is cultured; a DNA fragment inserted into the expression vector in the cell And then select Monitoring in situ the functional reporter protein produced by direct ligation of the first and second fragments of the coding region of the DNA encoding the reporter protein as a result of alternative splicing; A method for in situ monitoring of alternative splicing in eukaryotic cells is provided.

  Whether or not alternative splicing occurs in the target exon within the cell by transfecting a eukaryotic cell using the DNA fragment having the above-described sequence structure inserted into an expression vector and expressing the DNA fragment Can be monitored in real time in situ. This monitoring system can be applied to any organism by changing the vector, and can be used for both in vivo experiments and in vitro experiments. In addition, since reporter gene expression can be detected using any of the conventional detection methods, it is possible to monitor in situ whether alternative splicing occurs without preparing a new device. be able to.

FIG. 1 is a diagram showing a splicing mechanism using a reporter gene as an example. FIG. 2 is a diagram showing a specific structure and action mechanism of the DNA fragment of the present invention. FIG. 3 is a schematic diagram showing a method for preparing fragment A, which is the first step in the method for constructing DNA fragment GFPint1. FIG. 4 is a schematic diagram showing a method for preparing fragment B, which is the first step in the method for constructing DNA fragment GFPint1. FIG. 5 is a schematic diagram showing a method for preparing fragment C, which is the first step in the method for constructing DNA fragment GFPint1. FIG. 6 is a schematic diagram showing a method for preparing DNA fragment GFPint1 by linking fragments A to C, which is the second stage of the method for constructing DNA fragment GFPint1. FIG. 7 shows the fluorescence image and the results of in situ monitoring of HEK293 cells transfected with either pCI-neo vector, pCI-GFP vector, pCI-GFPint1 vector, or pCI-GFPint1-TE10 vector. It is a phase contrast image. FIG. 8 shows fluorescence images and phase contrast images showing the results of in situ monitoring of SH-SY5Y cells transfected with either pCI-GFP vector, pCI-GFPint1 vector, or pCI-GFPint1-TE10 vector. It is. FIG. 9 is a view showing the positional relationship between the structure of a DNA fragment resulting from splicing in a cell transfected with the expression vector pCI-GFPint1-TE10 vector and the DNA amplified by the primer set. FIG. 10 is a diagram showing an electrophoretic image of an RT-PCR result of total RNA expressed in HEK293 cells transfected with the expression vector pCI-GFPint1-TE10 vector prepared in Example 1. FIG. 11 shows SH-SY5Y cells transfected with either pCI-GFPint1 vector, pCI-GFPint1-TE2 vector, pCI-GFPint1-TE10 vector, GFP-neo vector, or pCI-neo vector in situ. It is a fluorescence image which shows the result monitored by. FIG. 12 shows in situ monitoring of COS1 cells transfected with either pCI-GFPint1 vector, pCI-GFPint1-TE2 vector, pCI-GFPint1-TE10 vector, GFP-neo vector, or pCI-neo vector. It is the fluorescence image which shows the result. FIG. 13 shows the positional relationship between the structure of a DNA fragment resulting from splicing in a cell transfected with the expression vector pCI-GFPint1-TE2 vector and the DNA amplified by the primer set. FIG. 14 shows an electrophoretic image of RT-PCR results of total RNA expressed in COS1 cells transfected with the expression vector pCI-GFPint1-TE2 vector or pCI-GFPint1-TE10 vector prepared in Example 4. FIG. FIG. 15 is a schematic diagram showing a method for preparing fragments D and E, which is the first step in the method for constructing DNA fragment GL3int1. FIG. 16 is a schematic diagram showing a method for preparing fragment F, which is the first step in the method for constructing DNA fragment GL3int1. FIG. 17 is a schematic diagram showing a method for preparing DNA fragment GL3int1 by linking fragments D to F, which is the second stage of the method for constructing DNA fragment GL3int1. FIG. 18 shows that when (A) SH-SY5Y cells or (B) HEmmmmmmK293 cells are transfected with the pGL3int1 vector, a splicing phenomenon occurs in which intervening fragments (corresponding to introns) are removed in the cells. FIG. 18 (FIG. 18A or B, respectively). FIG. 19 shows interstitial fragments (introns) in cells when COS1 cells were transfected with either pGL3int1 vector, pGL3int1-TE2 vector, pGL3int1-TE3 vector, pGL3int1-TE10 vector, or pCI-neo vector. It is a figure which shows that the splicing phenomenon from which an equivalent) is removed has arisen. FIG. 20 shows mRNA fragments generated as a result of splicing in cells transfected with the expression vector pGL3int1-TE2 vector (FIG. 20A), pGL3int1-TE3 vector (FIG. 20B), or pGL3int1-TE10 vector (FIG. 20C), respectively. It is a figure which shows the positional relationship of this structure, and mRNA amplified by a primer set. FIG. 21 shows an electrophoretic image of RT-PCR results of total RNA expressed in COS1 cells transfected with the expression vector pGL3int1-TE2 vector, pGL3int1-TE3 vector, or pGL3int1-TE10 vector prepared in Example 7. FIG.

Structure of DNA fragment An example of a general splicing mechanism that has been considered to date is shown in FIG. 1, taking a reporter gene as an example. In this figure, an intervening fragment (intron) is inserted between the first fragment and the second fragment of the reporter gene. In order for this intervening fragment to be removed by splicing, a sequence of GU (1) at the 5 ′ end side, a sequence of AG (2) at the 3 ′ end side, and a sequence of YYRAY inside the intervening fragment are included. This combination of sequences is known as a splicing motif (the splicing motif shown in FIG. 1 is an example of currently known splicing motifs). .
This splicing motif physically forms a loop structure between the GU sequence (1) on the 5 ′ end side and the YYRAY sequence inside the intervening fragment. As a result, the GU sequence (1) and 3 on the 5 ′ end side are formed. The intervening fragment is cleaved at the terminal AG sequence (2) and removed from the mRNA, whereby the first and second fragments of the reporter gene are directly ligated to form mature mRNA. It is thought that it is done. When such mature mRNA is formed, the target reporter protein is expressed as a functional reporter protein and plays a role as a reporter.
The present invention utilizes such a splicing control mechanism. First, in the present invention, a first fragment and a second fragment of a coding region of DNA encoding a reporter protein; an intervening fragment located between the first fragment and the second fragment, and splicing A DNA fragment is generated comprising an intervening fragment having a motif and comprising a cloning site; an exogenous alternative splicing cassette inserted into said cloning site. The specific structure and action mechanism of this DNA fragment is shown in FIG.
Reporter protein The reporter protein in the DNA fragment of the present invention may be any protein that is known to function as a reporter. For example, fluorescent protein, chromoprotein, enzyme protein, etc. Can be used. In the present invention, since it is preferable that splicing can be monitored in real time, it is preferable to use a fluorescent protein or a chromoprotein as a reporter protein. More specifically, as a reporter protein, a fluorescent protein such as green fluorescent protein (GFP), red fluorescent protein (RFP), luciferase (LF), DsRed, or phytochrome, blue light absorbing protein (NPL1) (Kagawa T. et al.). , Et al., Science, 2001, 291, 2138-2141).
When a fluorescent protein or a chromoprotein is used as a reporter protein, the generated fluorescence or color development or fluorescence quenching or light absorption can be monitored in real time while the cells remain alive. As an apparatus that can be used for real-time monitoring of such living cells, when using fluorescent protein, for example, fluorescent microscope, confocal microscope, CCD camera, etc., when using chromoprotein, A microscope, a CCD camera, and the like can be mentioned as examples, but the invention is not limited to these as long as changes in fluorescence or dye can be measured.
Intervening Fragment The intervening fragment present in the DNA fragment described above may be of any sequence as long as it has a splicing motif as described above. In the present invention, for example, a natural intron sequence contained in a genomic DNA sequence in a reporter gene can be used as an intervening fragment. For example, when the base sequence of the genomic DNA of a gene region encoding a reporter protein has a sequence structure consisting of exon 1, intron 1 and exon 2, the genomic DNA region can be used as a DNA fragment as it is. Alternatively, in the case of a reporter protein having three or more exons, any intron can be used as an intervening fragment, in which case the intron and the exons on both sides thereof are derived from the base sequence of genomic DNA, The other part can be a DNA fragment having a chimeric structure derived from the base sequence of cDNA.
In the present invention, intron sequences in other genes can also be used as intervening fragments. In this case, the DNA fragment of the present invention can be obtained by inserting an intron sequence derived from another gene into the nucleotide sequence of the gene encoding the reporter protein as an intervening fragment. In the present invention, an artificial intron can also be used as an intervening fragment. Examples of splicing using artificial introns have been reported in Umekage and Kikuchi (Nucleic Acids Sympo. Ser, (50): 323-324 (2006)), etc., and cloning using splicing It is used for the vector pDNR-Dual (clontech). Therefore, by inserting such an artificial intron as an intervening fragment into the cDNA base sequence of a gene encoding a reporter protein, the DNA fragment of the present invention can be obtained.
There may be one or more cloning sites present in the intervening fragment of the DNA fragment of the present invention. Such a cloning site may use, for example, a restriction enzyme site originally present in an intron when an intron is used as an intervening fragment, or a nucleic acid of a multiple cloning site of a cloning vector used in the art. The sequence may be cut out and diverted.
Alternative splicing phenomenon In order to utilize the above-described DNA fragment, an exogenous alternative splicing cassette is first inserted into the cloning site of the DNA fragment. This exogenous alternative splicing cassette is characterized in that it contains exons to be alternatively spliced among genes defining a protein together with sequences of intron portions upstream and downstream of the exons (FIG. 2). Middle).
In the present invention, an exon that is the target of alternative splicing of a gene that defines a certain protein that is inserted into the cloning site of a DNA fragment as an alternative splicing cassette is an exon that is already known to cause alternative splicing. Even exons that are expected to undergo alternative splicing can be used. By inserting an exon that is already known to cause alternative splicing and monitoring the splicing phenomenon, it is possible to detect the trend of splicing in the cell.
In the present invention, for example, exon 10 of the tau gene can be used as an exon known to cause alternative splicing. Specifically, a DNA region composed of exon 10 of the tau gene and intron portions upstream and downstream thereof can be used as an alternative splicing cassette. When exon 10 of the tau gene is alternatively spliced, 3Rtau having a structure consisting of a sequence not containing exon 10 (ie, exon 9 to exon 11), or a structure consisting of a sequence containing exon 10 (ie, exon 10) 4Rtau with ~ exon 9 ~ exon 10 ~ exon 11 ~) is produced. Human 3Rtau and human 4Rtau are proteins related to the pathology of Alzheimer's disease. In humans, Alzheimer's disease, frontotemporal dementia with Parkinson's syndrome linked to chromosome 17 (FTDP-17), Pick's disease, progressive supranuclear palsy (PSP), and cortical basal ganglia degeneration Diseases associated with dementia, such as (CBD), characterized by abnormal accumulation of tau gene products in degenerative neurons are known. Both 3Rtau and 4Rtau are recognized as main components of structures abnormally accumulated in degenerative neurons, but which of 3Rtau and 4Rtau accumulates differs depending on each disease. For example, both 3Rtau and 4Rtau accumulate in Alzheimer's disease, whereas only 3Rtau accumulates in Pick's disease, whereas only 4Rtau in FTDP-17 is a familial mutation, PSP, and CBD. It is known to accumulate. For this reason, whether 3Rtau or 4Rtau is produced is strongly related to the pathology of a disease involving the tau gene. Therefore, when the alternative splicing is monitored using the DNA region composed of exon 10 of the tau gene and the intron part upstream and downstream of the exon as the target of alternative splicing, what kind of abnormality does the cell have? Thus, it is possible to predict what kind of disease can occur.
In addition, by inserting an exon that is expected to cause alternative splicing and monitoring the splicing phenomenon, it is possible to identify whether the target exon is actually involved in alternative splicing in the cell. Can do.
By inserting such an alternative splicing cassette into the cloning site, the splicing motif (1) present at the 3 ′ end of the first fragment of the reporter and the 5 ′ of the second fragment were originally present in the DNA fragment. In addition to the motif (2) present at the end, splicing motifs (3) and (4) derived from the exogenous alternative splicing cassette will be inserted (middle in FIG. 2).
When a DNA fragment having such a structure is incorporated into an expression vector and transfected into a cell, splicing in (1)-(2) occurs in the cell, or (1)-(3) and (4) )-(2), splicing occurs at two locations, and the function of alternative splicing can be mimicked (lower part of FIG. 2).
When splicing at (1)-(2) occurs in the cell, the first and second fragments of the coding region of the DNA encoding the reporter protein will be directly ligated and formed as a result. A mature reporter mRNA generates a functional reporter protein, and a reporter function (for example, generation of fluorescence, expression of pigment, etc.) occurs (lower left in FIG. 2).
On the other hand, when splicing at two locations (1)-(3) and (4)-(2) occurs in the cell, the first fragment and the second fragment of the coding region of the DNA encoding the reporter protein A fusion protein defined by DNA into which an exon from an alternative splicing cassette is inserted is generated. This fusion protein does not have a function as a reporter protein, and the reporter function does not occur in such cells (lower right in FIG. 2).
Expression vector In another embodiment of the present invention, a first fragment and a second fragment of the coding region of DNA encoding the above-mentioned reporter protein; an intervening fragment located between the first fragment and the second fragment A DNA fragment comprising an intervening fragment having a splicing motif and comprising a cloning site; an exogenous alternative splicing cassette inserted into said cloning site; For this purpose, a eukaryotic expression vector prepared by inserting the DNA fragment into a cloning site of a protein expression vector is provided. Since the “alternative splicing” phenomenon monitored in the present invention is a phenomenon peculiar to eukaryotic cells, the protein expression vectors that can be used in the present invention are limited to eukaryotic expression vectors.
The vector into which the DNA fragment of the present invention is inserted may be any eukaryotic expression vector known in the art as long as it is compatible with the cell into which the expression vector into which the DNA fragment has been inserted is transfected. It may be. For example, when the cell into which the expression vector is transfected is an insect cell (eg, Sf9 cell, Sf21 cell, etc.), pIEx vector, baculovirus vector, etc. can be mentioned as examples. In the case of mouse cells (for example, CHO cells, HN2 cells, Neuro2a cells, mouse ES cells, etc.), pCI-neo vectors, adenovirus vectors, etc. can be mentioned as examples, and cells transfected with expression vectors are human. When it is a cell (for example, HEK293 cell, SH-SY5Y cell, HeLa cell, etc.), a pCI-neo vector, an adenovirus vector, etc. can be mentioned as an example.
The DNA fragment may be inserted into the vector by any method known or well known in the art. For example, Sambrook and Russel (Molecular Cloning: A Laboratory Manual, 3rd edition (2001)), Masaaki Muramatsu A well-known method described in (Lab Manual Genetic Engineering, 3rd edition, Maruzen) can be used. Specifically, one of the cloning sites in the vector is cleaved with a restriction enzyme, while both ends of the DNA fragment are arranged in a base sequence that can be combined with the end of the cloning site cleaved with the above restriction enzyme, In addition, the DNA fragment can be inserted by ligation.
When the expression vector thus prepared is transfected into eukaryotic cells and expressed, selective splicing occurs in the selective cells, resulting in direct ligation of the first fragment and the second fragment. A functional reporter protein defined by the generated DNA, or a reporter defined by the DNA formed from the first fragment and the exon in the alternative splicing cassette and the second fragment Either expression mode is used to indicate whether a fusion protein that does not have a function as a protein is produced (lower part of FIG. 2).
In situ monitoring of alternative splicing using expression vectors In the present invention, alternative splicing resulting from expression based on such expression vectors is monitored in situ in cells by methods well known in the art. be able to. Specifically, in situ monitoring of alternative splicing in eukaryotic cells includes: transfecting a eukaryotic cell with the expression vector described above; culturing the transfected eukaryotic cell; Expressed by the DNA produced by direct ligation of the first and second fragments of the coding region of the DNA encoding the reporter protein as a result of alternative splicing Monitoring functional reporter protein in situ. Such in-situ monitoring can be performed in real time by selecting a reporter.
Other Embodiments In the present invention, a kit for detecting alternative splicing of a desired exogenous exon comprising the above-described DNA fragment and any eukaryotic expression vector, or a protein into which the above-described DNA fragment is inserted A kit for detecting alternative splicing of a desired exogenous exon comprising an expression vector can also be provided.

  In the remainder of the description, the invention will be described in more detail using exemplary sequences. However, the following examples are not intended to limit the invention to this embodiment.
  Example 1: Construction of DNA fragment and expression vector (1)
  In this example, a fluorescent protein of green fluorescent protein (Enhanced Green Fluorescent Protein: EGFP, Clontech) and jellyfish (Aequorea victoria) GFP is used as a reporter gene, and Aequorea victoria A26 A sequence corresponding to exon 2 including the start codon (SEQ ID NO: 1) corresponds to a sequence obtained by combining exon 3 and a part of exon 4 (from the 5 ′ end to the stop codon). The sequence (SEQ ID NO: 2) was used as the second fragment, respectively, and GFP (Gen) of Aequorea victoria an Aequorea victoria genome-derived region (531 bp, SEQ ID NO: 3) having a nucleotide sequence homologous to intron 2 of ank Acc # M62654) as an intervening fragment and as an exon known to cause alternative splicing , DNA fragment using exon 10 of human tau gene (SEQ ID NO: 4) and its upstream intron portion (SEQ ID NO: 5) and downstream intron portion (SEQ ID NO: 6) Built.
  The sequence of EGFP contained in the reporter gene of the present invention (first fragment and second fragment) and the intervening fragment was obtained from Clontech. The GFP and intron sequences of Aequorea victoria contained therein were obtained from genomic DNA extracted from Aequorea victoria (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3). ).
  Specifically, the procedure was as follows. First, an EcoRI-NotI restriction enzyme fragment containing an EGFP cDNA region from a pEGFP-N3 vector (GenBank Acc #: U57609, Clontech), which is an EGFP expression vector, was converted into a pCI-neo Mammalian Expression Vector (GenBank, an eukaryotic cell expression vector). Acc #: U47120, Promega) multi-cloning site EcoRI-NotI site constructed by ligation and insertion The EGFP expression vector pCI-GFP vector constructed as a template is hybridized to a primer set (sequence derived from pCI-neo vector) PCIF primer (ttttctccacaggtgtccac; SEQ ID NO: 7) and EGFP cDNA Fragment A was prepared by PCR using a hybridizing EGFPE1R primer (tgcatcacctcacccctcgccggacac; SEQ ID NO: 10) (FIG. 3). This fragment A is, in order from the 5 ′ side, 86 bp derived from the pCI-GFP vector, 5 bp untranslated region of EGFP 46 bp, 97 bp of nucleotide numbers 1 to 97 of EGFP cDNA, and EGFPPE1R primer (SEQ ID NO: 10) Is a nucleic acid having a length of 246 bp composed of 3 bp on the 3 ′ side (FIG. 3, SEQ ID NO: 13).
  Next, using the pCI-GFP vector as a template, a primer set (PCIR primer (ctagttgtgtgtgtccaactc; SEQ ID NO: 8) that hybridizes to a sequence derived from the pCI-neo vector and EGFPPE2F primer (agtgcccatgcccgagagtggtggtgagtggtggtgagtggtgagtggtggtgagtggtgagtgtag Fragment B was prepared by PCR using NO: 9)) (FIG. 4). This fragment B comprises, in order from the 5 ′ side, 21 bp of the EGFPPE2F primer (SEQ ID NO: 9), 441 bp of nucleotide numbers 280 to 720 of EGFP cDNA, 3 bp of EGFP 3 ′ untranslated region 9 bp, and pCI− It is a nucleic acid having a length of 546 bp composed of 75 bp derived from the GFP vector (FIG. 4, SEQ ID NO: 14).
  Using genomic DNA extracted from Aequorea victoria as a template, primer set (GFPI1F primer that hybridizes to exon 2 of the GFP gene (tgtccggggagggtgaaggtgtcacacatacgg; SEQ ID NO: 11) that hybridizes to exon 3 of the GFP gene Fragment C was prepared by PCR using ctggacataactttcggggcatggactctttg; SEQ ID NO: 12)) (FIG. 5). This fragment C is a jellyfish genomic DNA region homologous to nucleotide numbers 95 to 206 of the GFP cDNA derived from the 5 ′ side 9 bp of the GFPPI1F primer (SEQ ID NO: 11) and the GFP gene exon 2 in order from the 5 ′ side. 112 bp, Aequorea genomic DNA region homologous to GFP gene intron 2 (531 bp), Aequorea genus genomic DNA region 70 bp homologous to nucleotide numbers 207 to 276 of GFP cDNA derived from GFP gene exon 3, and GFPI1R primer (SEQ ID NO : 12) is a nucleic acid having a length of 728 bp composed of 6 bp derived from (FIG. 5, SEQ ID NO: 15).
  By performing PCR from a mixed solution of the three fragments A, B and C thus prepared, using a primer set (PCIF (SEQ ID NO: 7) and PCIR (SEQ ID NO: 8)) (FIG. 6, upper part), a DNA fragment containing fragments A, B and C was prepared (middle part of FIG. 6, SEQ ID NO: 16). This DNA fragment has a restriction enzyme EcoRI site on the 5 'end side and a restriction enzyme NotI site on the 3' end side.
  A specific base sequence of a DNA fragment prepared by digesting this DNA fragment with restriction enzymes EcoRI and NotI has a sequence consisting of nucleotide numbers 87 to 1392 of SEQ ID NO: 16, and SEQ ID NO: : 1, SEQ ID NO: 3 and SEQ ID NO: 2 are included in this order. This DNA fragment having a sequence consisting of nucleotide numbers 87 to 1392 of SEQ ID NO: 16 is referred to as GFPint1 in the following description of the present specification (lower part of FIG. 6).
  Using pCI-neo Mammalian Expression Vector (Promega) as a parent vector used for the construction of the expression vector, the restriction enzyme EcoRI is located between the EcoRI site and the NotI site in the multicloning site in the pCI-neo vector. The DNA fragment cleaved with NotI, GFPint1, was ligated and inserted to prepare a pCI-GFPint1 vector (lower panel in FIG. 6).
  In contrast, the alternative splicing cassette containing exon 10 of the tau gene includes exon 10 (SEQ ID NO: 4) of the tau gene and its upstream intron (SEQ ID NO: 5) and downstream intron (SEQ ID NO: 5). A DNA region composed of a sequence in which NO: 6) is arranged in the order of SEQ ID NO: 5, SEQ ID NO: 4 and SEQ ID NO: 6 is used. The desired sequence of the tau gene (SEQ ID NOs: 4-6) was obtained from genomic DNA isolated and purified from human blood using the Wizard® Genomic DNA Purification Kit.
  Next, exon 10 (SEQ ID NO: 4) of the tau gene described above and its upstream and downstream intron portions (SEQ SEQ ID NO: 4 respectively) were inserted into the cloning site PmaCI site in the intervening fragment of the DNA fragment GFPint1 inserted into the expression vector pCI-GFPint1 vector. An alternative splicing cassette consisting of ID NO: 5 and SEQ ID NO: 6) was inserted to create the pCI-GFPint1-TE10 vector. A restriction enzyme PmaCI site (CACGTG) exists in nucleotides 358 to 363 in the sequence homologous to intron 2 (SEQ ID NO: 3), which is an intervening fragment in the DNA fragment inserted into the expression vector, and this restriction is present. When the enzyme site is cleaved with an enzyme, a blunt end is formed. In the present invention, this restriction enzyme site was used as a cloning site to insert an alternative splicing cassette containing exon 10 of the tau gene described above.
  Specifically, 50 ng of the vector pCI-GFPint1 vector and 60 ng of the above-described alternative splicing cassette were each treated with PmaCI at 37 ° C. for 17 hours to prepare blunt ends. The vector cleaved by this treatment was dephosphorylated with CIAP. The samples were combined and reacted at 16 ° C. for 17 hours using a ligation kit (TaKaRa ligation kit ver 2.1) to incorporate the above-described alternative splicing cassette DNA fragment into the expression vector pCI-GFPint1 vector. It is.
  Using an expression vector pCI-GFPint1-TE10 vector extracted from E. coli JM109 transformed with the ligation reaction solution as a template, using a primer set (PCIF primer (SEQ ID NO: 7) and PCIR primer (SEQ ID NO: 8)) PCR and primer Confirmed by dideoxy method using BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied biosystems) using PCIF primer (SEQ ID NO: 7) or PCIR primer (SEQ ID NO: 8) However, a DNA fragment having a structure as shown in the middle of FIG. 2 is present in the expression vector pCI-GFPint1-TE10 vector. It is shown that is inserted expressible, a eukaryotic expression vector of interest was shown to have been constructed.
  Example 2 Alternative Splicing for Tau Gene Exon 10
  In this example, eukaryotic cells are transfected using the expression vector pCI-GFPint1-TE10 vector prepared in Example 1, and it is confirmed whether or not alternative splicing actually occurs for the tau gene exon 10. I went for that purpose.
  In this example, HEK293 cells and SH-SY5Y cells (both obtained from RIKEN) were used to transfect the expression vector prepared in Example 1, the pCI-GFPint1-TE10 vector. These HEK293 cells and SH-SY5Y cells can be subcultured in DMEM medium (GIBCO) supplemented with fetal bovine serum to a final concentration of 10%. Subcultured in this way 2 × 10⁴HEK293 cells and 5 × 10⁴Each SH-SY5Y cell was transfected with 0.8 μg of the expression vector pCI-GFPint1-TE10 vector prepared in Example 1 using 1 μl of cationic lipid (Lipofectamine 2000, Invitrogen). HEK293 cells and SH-SY5Y cells transfected with an expression vector express whether or not green fluorescent protein capable of functioning by splicing RNA synthesized from a DNA fragment into which the target alternative splicing cassette has been inserted is expressed. This was confirmed based on observation using a fluorescence microscope (Nikon) (FIGS. 7 and 8).
  As a comparative control, a pCI-neo vector which is a parent vector and a vector which does not contain the DNA fragment of the present invention at all, a pCI-GFP vector which is a vector in which EGFP cDNA is incorporated into a pCI-neo vector but does not contain an intron, or HEK293 and SH-SY5Y cells transfected with any of the pCI-GFPint1 vectors incorporating the DNA fragment GFPint1 of the present invention but without an alternative splicing cassette were used (FIGS. 7 and 8).
  As a result, both HEK293 cells and SH-SY5Y cells generate green fluorescence in some cells when transfected with the pCI-GFPint1 vector and when transfected with the pCI-GFPint1-TE10 vector. It became clear that This result shows that a splicing phenomenon has occurred in which the intervening fragment (corresponding to an intron) has been removed in cells transfected with the pCI-GFPint1 vector, and that tau in cells transfected with the pCI-GFPint1-TE10 vector. It shows that a splicing phenomenon has occurred in which intervening fragments (corresponding to introns) are removed so as not to include exon 10 of the gene.
  On the other hand, it was clarified that the number of cells emitting green fluorescence decreased when transfected with the pCI-GFPint1-TE10 vector, compared with the case where transfected with the pCI-GFPint1 vector. This phenomenon is caused by splicing in which mRNA comprising a sequence encoding the green fluorescent protein of the present invention (EGFP-GFP chimeric protein) is produced when transfected with the pCI-GFPint1-TE10 vector, and the green fluorescent protein of the present invention. The cause is that splicing in which mRNA consisting of a sequence in which exon 10 of the tau gene is inserted between the first fragment and the second fragment of (EGFP-GFP chimeric protein) is generated occurs. it was thought. This indicates that alternative splicing is carried out in cells transfected with the pCI-GFPint1-TE10 vector.
  Next, each of the four types of HEK293 cells and SH-SY5Y cells in which the respective expression vectors were expressed in the cells in this manner were cultured in the above-described culture solution for 2 days at 37 ° C. under humid conditions. Thereafter, the cells were collected, and total RNA expressed from the cells was collected using RNeasy Mini Kit (QIAGEN). RT-PCR using OneStep RT-PCR kit (QIAGEN) was performed using the collected 50 ng of total RNA as a template. In the RT-PCR reaction solution (total volume 25 μl), EGFP-F- hybridizes to the sequence on the first fragment (SEQ ID NO: 1) of the green fluorescent protein of the present invention (EGFP-GFP chimeric protein). EGFP-R-391 primer (gcgggtctttg tagttgcc; SEQ) hybridizing to a sequence on the second fragment of the 65 primer (acgtaaacgg cccagattc ag; SEQ ID NO: 17) and the green fluorescent protein of the present invention (EGFP-GFP chimeric protein) ID NO: 18) (each 10 pmol) contained a primer set. This primer set is a 266 bp PCR when mRNA encoding the green fluorescent protein of the present invention (EGFP-GFP chimeric protein) is formed in the cell (ie, when the intervening sequence is completely spliced out with the exon). A product is generated, and mRNA consisting of a sequence in which exon 10 of the tau gene is inserted between the first fragment and the second fragment of the green fluorescent protein of the present invention (EGFP-GFP chimeric protein) is generated in the cell. A 359 bp PCR product is generated. This RT-PCR reaction solution was subjected to PCR at 30 ° C. for 30 minutes (reverse transcription reaction) and at 95 ° C. for 15 minutes, followed by PCR (94 ° C. for 30 seconds to 59.6 ° C. for 30 seconds to 72 ° C. Min, 30 cycles), and further reacted at 72 ° C. for 5 min (FIG. 9).
  Separately from the above, TEX10-F primer (gtgcagataata tataataga gct; SEQ ID NO: 19) and TEX10-R primer (actgccgcct cccggg; SEQ ID NO: 20) that hybridize to exon 10 of the human tau gene are added as a primer set. The RT-PCR reaction solution was added at 50 ° C. for 30 minutes (reverse transcription reaction), 95 ° C. for 15 minutes, followed by PCR (94 ° C. for 30 seconds−52.3 ° C. for 30 seconds at −72 ° C. Second, 30 cycles), and further reacted at 72 ° C. in 1 minute (FIG. 9).
  For a total RNA sample from HEK293 cells transfected with either pCI-neo vector, pCI-GFP vector, pCI-GFPint1 vector, or pCI-GFPint1-TE10 vector, EGFP-F-65 primer (SEQ ID NO: 17) and a sample subjected to PCR using a primer set of EGFP-R-391 primer (SEQ ID NO: 18) and of TEX10-F primer (SEQ ID NO: 19) and TEX10-R primer (SEQ ID NO: 20). The results of electrophoresis of the samples subjected to PCR using the primer set are shown in FIGS. 10A and 10B, respectively. In this figure, lane 1 is a pCI-neo vector, lane 2 is a pCI-GFP vector, lane 3 is a pCI-GFPint1 vector, and lane 4 is a total collected from HEK293 cells transfected with a pCI-GFPint1-TE10 vector. RNA was used as a sample.
  As a result of RT-PCR, it was revealed that two mRNAs of 359 bp and 266 bp were produced from the cells transfected with the pCI-GFPint1-TE10 vector. The base sequence was confirmed by the dideoxy method using the BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied biosystems) using the amplified fragment prepared by RT-PCR as a template. 359 bp DNA incorporating exon 10 of the tau gene, that is, a first fragment consisting of a sequence corresponding to exon 2 of GFP (SEQ ID NO: 1), exon 10 of the tau gene (SEQ ID NO: 4), And a second fragment (SEQ ID NO: 2) consisting of a sequence corresponding to a sequence obtained by binding a part of exons 3 and 4 of GFP and a nucleic acid sequence encoding a non-functional fusion protein Is On the other hand, the short mRNA is a 266 bp DNA from which exon 10 of the tau gene has been removed as a result of alternative splicing, ie, a first fragment consisting of a sequence corresponding to exon 2 of GFP (SEQ ID NO: 1 ) And a second fragment (SEQ ID NO: 2) consisting of a sequence corresponding to a sequence obtained by binding a part of exons 3 and 4 of GFP, and a nucleic acid sequence defining GFP as a functional reporter protein (FIG. 10A, lane 4). In the sample of lane 4 in which two bands were amplified, the tau gene exon was obtained by PCR using a primer set of TEX10-F primer (SEQ ID NO: 19) and TEX10-R primer (SEQ ID NO: 20). It was revealed that 10 93 bp were amplified (FIG. 10B).
  On the other hand, when the pCI-GFP vector and the pCI-GFP int1 vector were transfected, only the 266 bp fragment was specifically amplified. Therefore, in these cells, EGFP and the green fluorescent protein of the present invention, respectively, It was shown that mRNA of (EGFP-GFP chimeric protein) was expressed (FIG. 10 (A), lanes 2 and 3 respectively). As a result of sequence analysis of these amplification products, it was confirmed that in both lanes 2 and 3, the amplified sequences were part of the 266 bp green fluorescent protein of the present invention (EGFP-GFP chimeric protein).
  Example 3: Construction of DNA fragments and expression vectors (2)
  In this example, the exon 2 of the human tau gene and its upstream and downstream intron parts are used as exons that use the pCI-GFPint1 vector prepared in Example 1 and are known to cause alternative splicing. A DNA fragment was constructed using the DNA region composed of
  Alternative splicing cassettes containing exon 2 of the tau gene include exon 2 (SEQ ID NO: 21) of the tau gene and its upstream intron part (SEQ ID NO: 22) and downstream intron part (SEQ ID NO: 23). , SEQ ID NO: 22, SEQ ID NO: 21, and SEQ ID NO: 23, and a DNA region composed of a sequence arranged in this order is used. The desired sequence of the tau gene (SEQ ID NOs: 21-23) was obtained from genomic DNA isolated and purified from human blood using the Wizard® Genomic DNA Purification Kit.
  In the cloning site in the intervening fragment of the DNA fragment GFPint1 inserted into the expression vector pCI-GFPint1 vector, exon 2 (SEQ ID NO: 21) of the tau gene described above and its upstream and downstream intron parts (SEQ ID NO: 22 respectively) And an alternative splicing cassette consisting of SEQ ID NO: 23) was inserted to create the pCI-GFPint1-TE2 vector. A restriction enzyme PmaCI site (CACGTG) exists in nucleotides 358 to 363 in the sequence homologous to intron 2 (SEQ ID NO: 3), which is an intervening fragment in the DNA fragment inserted into the expression vector, and this restriction is present. When the enzyme site is cleaved with an enzyme, a blunt end is formed. In the present invention, this restriction enzyme site was used as a cloning site to insert an alternative splicing cassette containing exon 2 of the tau gene described above.
  Specifically, 50 ng of the expression vector pCI-GFPint1 vector and 60 ng of the above-mentioned alternative splicing cassette were each treated with PmaCI at 37 ° C. for 17 hours to prepare blunt ends. The vector cleaved by this treatment was dephosphorylated with CIAP. The samples were combined and reacted at 16 ° C. for 17 hours using a ligation kit (TaKaRa ligation kit ver 2.1) to incorporate the above-described alternative splicing cassette DNA fragment into the expression vector pCI-GFPint1 vector. It is.
  PCR performed using primer set (PCIF (SEQ ID NO: 7) and PCIR (SEQ ID NO: 8)) using the expression vector pCI-GFPint1-TE2 vector extracted from E. coli JM109 transformed with the ligation reaction solution as a template 2 and the dideoxy method using BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied biosystems) performed using primer PCIF (SEQ ID NO: 7) or PCIR (SEQ ID NO: 8). It was shown that the DNA fragment having the structure as shown in the middle row of FIG. 2 is inserted into the expression vector pCI-GFPint1-TE2 vector so that it can be expressed. Eukaryotic cell expression vectors have been shown to have been constructed.
  Example 4: Alternative Splicing for Tau Gene Exon 2
  In this example, eukaryotic cells were transfected using the expression vector pCI-GFPint1-TE2 vector prepared in Example 3 and the expression vector pCI-GFPint1-TE10 vector prepared in Example 1, and a tau gene exon was used. The purpose was to confirm whether alternative splicing actually occurred for 2.
  In this example, in order to transfect the expression vector prepared in Example 3, the pCI-GFPint1-TE2 vector and the expression vector prepared in Example 1, the pCI-GFPint1-TE10 vector, SH-SY5Y cells and COS1 Cells (RIKEN) were used. The SH-SY5Y cells and COS1 cells (RIKEN) can be subcultured in DMEM medium supplemented with fetal bovine serum to a final concentration of 10%. Subcultured in this way, 5 × 10⁴SH-SY5Y cells and 1 × 10⁴For each COS1 cell, 0.8 μg of the expression vector pCI-GFPint1-TE2 vector prepared in Example 3 or the expression vector pCI-GFPint1-TE10 vector prepared in Example 1 was added to a cationic lipid (Lipofectamine 2000, Invitrogen). ) Transfected with 1 μl mixed solution. Whether SH-SY5Y cells and COS1 cells transfected with the expression vector express a functional green fluorescent protein by splicing RNA synthesized from a DNA fragment into which an alternative splicing cassette of interest has been inserted. It confirmed based on the observation using a fluorescence microscope (Nikon) (FIG. 11 and FIG. 12).
  As a comparative control, transfected with either the pCI-GFP vector, which is a vector that incorporates EGFP cDNA but does not contain introns, or the pCI-GFPint1 vector that incorporates the DNA fragment GFPint1 of the present invention but does not contain an alternative splicing cassette. SH-SY5Y cells and COS1 cells were used (FIGS. 11 and 12).
  As a result, both SH-SY5Y cells and COS1 cells generate green fluorescence in some cells when transfected with the pCI-GFPint1 vector and when transfected with the pCI-GFPint1-TE10 vector. It became clear that This result shows that a splicing phenomenon has occurred in which the intervening fragment (corresponding to an intron) has been removed in cells transfected with the pCI-GFPint1 vector, and that tau in cells transfected with the pCI-GFPint1-TE10 vector. It shows that a splicing phenomenon has occurred in which intervening fragments (corresponding to introns) are removed so as not to include exon 10 of the gene (FIGS. 11 and 12).
  On the other hand, when transfected with the pCI-GFPint1-TE2 vector, it was revealed that the number of cells emitting green fluorescence was significantly smaller than that of the pCI-GFPint1-TE10 vector. This result shows that, in the cells transfected with the pCI-GFPint1-TE2 vector, splicing occurred mainly at the two ends of the tau gene exon 2, so that exon 2 remains between the two reporter fragments. It is shown that many splicing phenomena in which the intervening fragments (corresponding to introns) on both sides of A are removed (FIGS. 11 and 12). Next, SH-SY5Y cells and COS1 cells in which the expression vectors pCI-GFPint1-TE2 vector and pCI-GFPint1-TE10 vector were expressed in the cells in this manner were cultured in the above-mentioned culture medium at 37 ° C. under humid conditions. After culturing under 2 days, the cells were recovered, and total RNA expressed from within the cells was recovered using RNeasy Mini Kit (QIAGEN). RT-PCR using OneStep RT-PCR kit (QIAGEN) was performed using the collected 50 ng of total RNA as a template. In the RT-PCR reaction solution (total volume 25 μl), EGFP-F- hybridizes to the sequence on the first fragment (SEQ ID NO: 1) of the green fluorescent protein of the present invention (EGFP-GFP chimeric protein). EGFP-R-391 primer (gcgggtctttg tagttgcc; SEQ) hybridizing to a sequence on the second fragment of the 65 primer (acgtaaacgg cccagattc ag; SEQ ID NO: 17) and the green fluorescent protein of the present invention (EGFP-GFP chimeric protein) ID NO: 18m) (10 pmol each) primer set was included. This primer set is a 266 bp PCR when mRNA encoding the green fluorescent protein of the present invention (EGFP-GFP chimeric protein) is formed in the cell (ie, when the intervening sequence is completely spliced out with the exon). MRNA comprising a sequence in which exon 2 or exon 10 of the tau gene is inserted between the first fragment and the second fragment of the green fluorescent protein of the present invention (EGFP-GFP chimeric protein) in the cell Is generated, a 353 bp or 359 bp PCR product is generated, respectively. This RT-PCR reaction solution was subjected to PCR at 30 ° C. for 30 minutes (reverse transcription reaction) and at 95 ° C. for 15 minutes, followed by PCR (94 ° C. for 30 seconds to 59.6 ° C. for 30 seconds to 72 ° C. Min, 30 cycles), and further reacted at 72 ° C. for 5 min (FIG. 13).
  Separately from the above, RT- with a TEX2-F primer (aatctccccctgcaga; SEQ ID NO: 24) and a TEX2-R primer (cttccgctgtgtggagtgct; SEQ ID NO: 25) hybridizing to exon 2 of the human tau gene as a primer set PCR reaction solution was 30 minutes at 50 ° C. (reverse transcription reaction), 15 minutes at 95 ° C., followed by PCR (94 ° C. for 30 seconds−52.3 ° C. for 30 seconds−72 ° C. for 30 seconds, 30 Cycle) and further reacted at 72 ° C. for 1 minute (FIG. 13). When mRNA comprising a sequence in which exon 2 of the tau gene is inserted between the first fragment and the second fragment of the green fluorescent protein of the present invention (EGFP-GFP chimeric protein) is generated in the cell, A 61 bp PCR product is generated by the primer set of the TEX2-F primer (SEQ ID NO: 24) and the TEX2-R primer (SEQ ID NO: 25) (FIG. 13).
  For total RNA samples obtained from COS1 cells transfected with either pCI-neo vector, pCI-GFP vector, pCI-GFPint1 vector, pCI-GFPint1-TE2 vector or pCI-GFPint1-TE10 vector, EGFP-F-65 FIG. 14 shows the result of electrophoresis performed on a sample subjected to PCR using a primer set of a primer (SEQ ID NO: 17) and an EGFP-R-391 primer (SEQ ID NO: 18). In this figure, lane 1 is a pCI-GFPint1 vector, lane 2 is a pCI-GFPint1-TE2 vector, lane 3 is a pCI-GFPint1-TE10 vector, lane 4 is a pCI-GFP vector, and lane 5 is a pCI-neo vector, Total RNA collected from each transfected COS1 cell was used as a sample.
  In contrast to cells transfected with the pCI-GFPint1-TE10 vector in which two mRNAs of 359 bp and 266 bp were produced as a result of RT-PCR (FIG. 14, lane 3), pCI-GFPint1 -266 bp mRNA expression was found to be very weak in cells transfected with the TE2 vector (Figure 14, lane 2).
  Using BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied biosystems) with the amplified fragment amplified and prepared by RT-PCR method based on mRNA of cells transfected with pCI-GFPint1-TE2 vector as a template When the base sequence was confirmed by the dideoxy method, as a result of alternative splicing, 353 bp DNA incorporating exon 2 of the tau gene, that is, the first fragment consisting of a sequence corresponding to exon 2 of GFP (SEQ ID NO: 1 ), Exon 2 of the tau gene (SEQ ID NO: 21), and a second fragment consisting of a sequence corresponding to a sequence in which a part of exons 3 and 4 of GFP are combined (SEQ ID N : 2) linked to the non-functional fusion protein-encoding nucleic acid sequence was found to be mainly produced, while the slightly amplified mRNA product was the result of alternative splicing. 266 bp DNA from which exon 2 of the tau gene was removed, that is, the first fragment consisting of a sequence corresponding to exon 2 of GFP (SEQ ID NO: 1) and a part of exons 3 and 4 of GFP were combined. A second fragment (SEQ ID NO: 2) consisting of a sequence corresponding to the sequence was directly ligated to reveal a nucleic acid sequence defining GFP as a functional reporter protein (FIG. 14, lane 2). .
  In addition, the DNA amplified mainly in Lane 2 has 93 bp of exon 10 of the tau gene by PCR using a primer set of TEX2-F primer (SEQ ID NO: 24) and TEX2-R primer (SEQ ID NO: 25). Amplification was found (data not shown).
  On the other hand, when the pCI-GFPint1-TE10 vector was used, it was revealed that exon 10 of the tau gene was removed as a result of alternative splicing and 266 bp DNA was expressed (FIG. 14, lane 3).
  In lanes 2 and 3, a band due to non-specific amplification appeared in the vicinity of 450 bp, but these sequences are completely unrelated to the sequence encoding the green fluorescent protein (EGFP-GFP chimeric protein) of the present invention. (Data not shown).
  Example 5: Construction of DNA fragment and expression vector (3)
  In this example, a photoprotein firefly luciferase (pGL3-control: GenBank Acc # U47296, Clontech) was used as a reporter gene, and exons 1 to 3 of Phototinus pyralis (firefly) luciferase (GenBank Acc # M15077) were combined. A nucleic acid having a sequence corresponding to the sequence (668 base pairs) (668 base pairs, SEQ ID NO: 26) is used as a first fragment, and the sequence (985) is bound to the codons in exons 4 to 6 and exon 7. A nucleic acid (985 base pairs, SEQ ID NO: 27) to which a sequence corresponding to (base pairs) is bound is used as the second fragment, respectively, and GFP (Ge) of Aequorea victoria Bank Acc # M62654) Aequoreavicoria genome-derived region (SEQ ID NO: 3) having a nucleotide sequence homologous to intron 2 contains 5'-end 6 bases and 3'-end 6 bases, respectively, with Photinus pyralis (firefly) luciferase (GenBank Acc # M15077) a nucleic acid (531 base pairs, SEQ ID) having a sequence substituted with 5 ′ terminal 6 bases (gtatgt) and 3 ′ terminal 6 bases (ttgttag) of intron C (48 base pairs) sandwiched between exons 3 and 4 of M15077) NO: 28) was used as an intervening fragment to construct a DNA fragment, GL3int1, for inserting the splicing cassette.
  Exons 1 to 3 and exons 4 to 6 and 7 of the luciferase gene of the present invention were obtained from Clontech (SEQ ID NO: 26 and SEQ ID NO: 27, respectively). Intron C sequence was obtained from GenBank (GenBank Acc # M15077).
  Using Luciferase expression vector pGL3-control vector (GenBank Acc #: U47296, Clontech) as a template, primer set (PGL3-387F primer (gcacccctaccgtgggtgtcgtgtgtgtgtcgtgtgtgcc) Fragment D was prepared by PCR using ID NO: 34)). This fragment D is a nucleic acid having a length of 325 bp composed of 306 bp derived from nucleotide numbers 363 to 668 of pGL3-control luciferase cDNA and 19 bp on the 3 ′ side of the PGL3-int5R primer in this order from the 5 ′ side. Yes (FIG. 15, SEQ ID NO: 35). Among these, the 5 ′ side 13 bp of the PGL3-int5R primer (SEQ ID NO: 34) is derived from the 5 ′ end (nucleotide numbers 7 to 19) of the intervening fragment (SEQ ID NO: 3) of the pCI-GFPint1 vector, and The 6 bp next to it is a sequence (gtatgt) derived from intron C of firefly luciferase (GenBank Acc # M15077).
  On the other hand, using the Luciferase expression vector pGL3-control vector (GenBank Acc #: U47296, Clontech) as a template, a primer set (PGL3-int3F primer (ggaattacgtttgttgttgtgtgatcctgtcgttcttgtcgttcgttcgttcgttcgttgtcgttcgttcgttgtcgtcgttcgttgtcgttcgtgtt Fragment E was prepared by PCR using tatgtctcca gaatgttagcc a; SEQ ID NO: 30)). This fragment E is a nucleic acid having a length of 633 bp composed of 611 bp derived from nucleotide numbers 669 to 1279 of the 5 ′ side 22 bp of the PGL3-int3F primer and the luciferase cDNA of pGL3-control in order from the 5 ′ side. (FIG. 15, SEQ ID NO: 36). Of these, the 5 ′ 16 bp of the PGL3-int3F primer (SEQ ID NO: 31) is derived from the 3 ′ end (nucleotide numbers 510 to 525) of the intervening fragment (SEQ ID NO: 3) of the pCI-GFPint1 vector, and The 6 bp next to it is a sequence (ttgttag) derived from intron C of firefly luciferase (GenBank Acc # M15077).
  Using the pCI-GFPint1 vector prepared in Example 1 as a template, a primer set (PGL3-int5F primer (gattctcgca tgcctagt gtgcacttta tactc; SEQ ID NO: 33) and PGL3-int3catat catatatatc ) Was used to prepare fragment F having a length of 567 bp by PCR (FIG. 16, SEQ ID NO: 37).
  Using a primer set (PGL3-387F primer (SEQ ID NO: 29) and PGL3-int3R primer (SEQ ID NO: 32)) from a mixed solution of fragments D and F, 857 bp containing fragments D and F by PCR A fragment G (SEQ ID NO: 38) having a length was prepared (see the second row from the top in FIG. 17). Next, from the mixed solution of fragments G and E, using a primer set (PGL3-387F primer (SEQ ID NO: 29) and PGL3-1249R primer (SEQ ID NO: 30)), a length of 1448 bp was obtained by PCR. Fragment H (SEQ ID NO: 39) was prepared (see the third row from the top in FIG. 17).
  The specific base sequence of the fragment H thus prepared is as shown in SEQ ID NO: 39, and has a restriction enzyme BsrGI site on the 5 'end side and a restriction enzyme PpuMI site on the 3' end side.
  The pGL3-control vector (Clontech) was used as the parent vector used for the construction of the expression vector. Ligation of fragment H cleaved with restriction enzymes BsrGI and PpuMI between the BsrGI site (TGTACA: 491 to 497 nucleotides) and the PpuMI site (AGGTCCCT: 1179 to 1185 nucleotides) in the luciferase cDNA of the pGL3-control vector To construct an expression vector pGL3int1 vector. This expression vector pGL3int1 vector contains a DNA fragment in which SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 27 are arranged in this order. This DNA fragment is referred to as GL3int1 in the rest of this specification (FIG. 17, bottom row).
  An intervening fragment (SEQ ID NO: 28) in the DNA fragment GL3int1 inserted into the expression vector pGL3int1 vector has a restriction enzyme PmaCI site (CACGTG) at nucleotides 358 to 363. When cut, a blunt end is formed. In the present invention, this restriction enzyme site was used as a cloning site to insert an alternative splicing cassette containing exon 10 of the tau gene described above.
  PCR performed using a primer set (PGL3-387F primer (SEQ ID NO: 29) and PGL3-1249R primer (SEQ ID NO: 30)) using an expression vector extracted from E. coli JM109 transformed with the ligation reaction solution as a template And BigDye® Terminator v3 performed with primer PGL3-387F primer (SEQ ID NO: 29), PGL3-1249R primer (SEQ ID NO: 30) and PGL3-int5F primer (SEQ ID NO: 33) .1 DNA having the structure shown in the upper part of FIG. 2 was confirmed by the dideoxy method using Cycle Sequencing Kit (Applied biosystems). Ragumento is, it is shown that is inserted expressible manner into the vector, a eukaryotic expression vector of interest was shown to have been constructed.
  Example 6: Splicing in an expression vector using luciferase
  In this example, for the purpose of confirming whether or not splicing actually occurs in the luciferase gene into which the intervening fragment was inserted by transfecting eukaryotic cells using the expression vector prepared in Example 5. went.
  In order to examine whether the expression vector pGL3int1 vector prepared in Example 5 causes splicing in cells, is the pGL3int1 vector transfected into SH-SY5Y cells and HEK293 cells (RIKEN) and splicing occurs in the cells? It was confirmed by detecting chemiluminescence. The SH-SY5Y cells and HEK293 cells (RIKEN) can be subcultured in DMEM medium supplemented with fetal bovine serum to a final concentration of 10%. Subcultured in this way, 3.1 × 10⁴SH-SY5Y cells and 3.1 × 10⁴For each HEK293 cell, 0.27 μg of the expression vector pGL3int1 vector prepared in Example 5 and 20 ng of a co-reporter vectorpRL-CMV vector (GenBank Acc # AF025843, Clontech) for correcting transfection efficiency, It was transfected using a solution mixed with 0.34 μl of lipid (Lipofectamine 2000, Invitrogen).
  SH-SY5Y cells and HEK293 cells transfected with the expression vector pGL3int1 vector indicate whether or not a functional photoprotein is expressed by splicing RNA synthesized from a DNA fragment containing a luciferase gene into which an intervening sequence has been inserted. It confirmed based on the measurement using Dual-Glo luciferase Assay System (Promega). That is, SH-SY5Y cells and HEK293 cells in which the expression vector is expressed in the cells in this way are cultured in the above-mentioned culture solution for 1 day at 37 ° C. under humid conditions, and after the medium is removed, Dual -It melt | dissolved in Luciferase Reagent of Gloluciferase Assay System (Promega), and light emission was measured with the luminescence sensor (Ato Co., Ltd.). After treatment of the cells with Luciferase Reagent of Dual-Glo luciferase Assay System (Promega), luminescence by firefly luciferase was measured, and luminescence by Renilla luciferase expressed by pRL-CMV was measured by Stop & Glo Reagent. The integrated value of each luminescence was recorded and divided by the amount of luminescence by Renilla luciferase to correct the error in transfection efficiency, and then the amount of luminescence by firefly luciferase was compared (FIGS. 18A and B).
  Chemiluminescence was similarly detected in SH-SY5Y cells and HEK293 cells transfected with pGL3-control vector containing luciferase gene cDNA as a positive control and pGL3-basic vector without luciferase gene cDNA as a negative control. did. The results are shown in FIGS. 18A and B.
  As shown in this figure, almost no chemiluminescence was detected in SH-SY5Y cells transfected with the pGL3-basic vector, whereas strong chemiluminescence was observed in SH-SY5Y cells transfected with the pGL3-control vector. was detected. In contrast, it became clear that SH-SY5Y cells transfected with the pGL3int1 vector produced chemiluminescence that was weaker than the positive control but distinct. Therefore, it was shown that, in SH-SY5Y cells, the pGL3int1 vector caused a splicing phenomenon in which an intervening fragment (corresponding to an intron) was removed in the transfected cells, and could express a functional luciferase.
  On the other hand, it was revealed that substantially only luciferase activity occurs in HEK293 cells transfected with the pGL3int1 vector. Therefore, splicing between exon 3 and exon 4 of luciferase does not occur in HEK293 cells, and as a result, pGL3int1 vector has a splicing phenomenon in which intervening fragments (corresponding to introns) are removed in the transfected cells. It was shown not to occur.
  Next, the SH-SY5Y cells in which the expression vector is expressed in the cells in this way are cultured in the above-described culture medium for 1 day at 37 ° C. under humid conditions, and then the cells are collected and RNeasy Mini. Using Kit (QIAGEN), total RNA expressed from inside the cells was recovered. RT-PCR using OneStep RT-PCR kit (QIAGEN) was performed using the recovered 1 ng of total RNA as a template. Primer sets EGFP-F-65 and EGFP-R-391 (each final concentration 0.6 μM) were contained in the RT-PCR reaction solution (total volume 10 μl). This RT-PCR reaction solution was subjected to PCR at 30 ° C. for 30 minutes (reverse transcription reaction), 95 ° C. for 15 minutes, followed by PCR (94 ° C. for 30 seconds−50 ° C. for 30 seconds−72 ° C. for 90 seconds, 35 cycles), and further reacted at 72 ° C. for 10 minutes.
  Separately from the above, the RT-PCR reaction solution with TEX10-F and TEX10-R added as a primer set for 30 minutes at 50 ° C. (reverse transcription reaction) and 15 minutes at 95 ° C. 30 seconds at 52.3 ° C. for 30 seconds at 72 ° C. for 30 seconds and 30 cycles), and further at 72 ° C. for 1 minute.
The nucleotide sequence was confirmed by the dideoxy method using BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied biosystems) using the amplified fragment prepared by the RT-PCR method as a template.
  Example 7: Construction of DNA fragment and expression vector (4)
  In this example, the pGL3int1 vector prepared in Example 5 is used, and the exon known to cause alternative splicing is composed of exon 2 of the human tau gene and its upstream and downstream intron parts. A DNA fragment was constructed by a method substantially similar to the method described in Example 3 using the DNA region to be prepared.
  Alternative splicing cassettes containing exon 2 of the tau gene include exon 2 (SEQ ID NO: 21) of the tau gene and its upstream intron part (SEQ ID NO: 22) and downstream intron part (SEQ ID NO: 23). , SEQ ID NO: 22, SEQ ID NO: 21, and SEQ ID NO: 23, and a DNA region composed of a sequence arranged in this order is used. The desired sequence of the tau gene (SEQ ID NOs: 21-23) was obtained from genomic DNA isolated and purified from human blood using the Wizard® Genomic DNA Purification Kit.
  As an alternative splicing cassette containing exon 3 of the tau gene, exon 3 of the tau gene (SEQ ID NO: 40) and its upstream intron portion (SEQ ID NO: 41) and downstream intron portion (SEQ ID NO: 42) are used. , SEQ ID NO: 41, SEQ ID NO: 40, and SEQ ID NO: 42, a DNA region composed of a sequence arranged in this order is used. The desired sequence of the tau gene (SEQ ID NOs: 40-42) was obtained from genomic DNA isolated and purified from human blood using the Wizard® Genomic DNA Purification Kit.
  Alternative splicing cassettes containing exon 10 of the tau gene include exon 10 (SEQ ID NO: 4) of the tau gene and its upstream intron part (SEQ ID NO: 5) and downstream intron part (SEQ ID NO: 6). , SEQ ID NO: 5, SEQ ID NO: 4, and SEQ ID NO: 6 are used to make use of a DNA region composed of sequences arranged in this order. The desired sequence of the tau gene (SEQ ID NOs: 4-6) was obtained from genomic DNA isolated and purified from human blood using the Wizard® Genomic DNA Purification Kit.
  Either the exon 2 alternative splicing cassette, the exon 3 alternative splicing cassette or the exon 10 alternative splicing cassette of the tau gene described above is inserted into the cloning site in the intervening fragment GL3int1 inserted into the expression vector pGL3int1 vector. As a result, a pGL3int1-TE2 vector, a pGL3int1-TE3 vector, or a pGL3int1-TE10 vector was prepared. In the intervening fragment (SEQ ID NO: 28) in the DNA fragment inserted into the expression vector pGL3int1 vector, the restriction enzyme PmaCI site (CACGTG) is present at nucleotides 358 to 363, and this restriction enzyme site is cleaved with an enzyme. A blunt end is then formed. In the present invention, using this restriction enzyme site as a cloning site, an alternative splicing cassette containing any exon of the tau gene described above was inserted.
  Specifically, 50 ng of expression vector pGL3int1 vector and 60 ng of any of the above-mentioned alternative splicing cassettes were each treated with PmaCI at 37 ° C. for 17 hours to prepare blunt ends. The vector cleaved by this treatment was dephosphorylated with CIAP. The samples were combined and reacted at 16 ° C. for 17 hours using a ligation kit (TaKaRa ligation kit ver2.1) to incorporate a DNA fragment in which the above-described alternative splicing cassette was inserted into the expression vector pGL3int1 vector.
  Using an expression vector pGL3int1-TE2 vector, pGL3int1-TE3 vector, or pGL3int1-TE10 vector extracted from E. coli JM109 transformed with the ligation reaction solution as a template, primer sets (PCIF (SEQ ID NO: 7) and PCIR (SEQ TD No. 8)) PCR and primer PCIF (SEQ ID NO: 7) or BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied biosystems) performed using PCIR (SEQ ID NO: 8) or PCIR (SEQ ID NO: 8) ) By the dideoxy method, the DNA fragment having the structure shown in the middle of FIG. -GFPint1-TE2 vector, pGL3int1-TE3 vector, or pGL3int1-TE10 vector is shown to be inserted in an expressible manner, indicating that the desired eukaryotic expression vector has been constructed. It was done.
  Example 8: Alternative Splicing for Tau Gene Exon 2, Exon 3, and Exon 10
  In this example, eukaryotic cells were transfected using the three expression vectors pGL3int1-TE2 vector, pGL3int1-TE3 vector, or pGL3int1-TE10 vector prepared in Example 7, and tau gene exon 2 and exon were used. The purpose of this study was to confirm whether alternative splicing actually occurs for 3 or exon 10.
  In this example, COS1 cells (RIKEN) were used to transfect the pGL3int1-TE2 vector, pGL3int1-TE3 vector, or pGL3int1-TE10 vector. The COS1 cells (RIKEN) can be subcultured in DMEM medium supplemented with fetal bovine serum to a final concentration of 10%. Subcultured in this way 2 × 10³Each COS1 cell was transfected with 0.8 μg of pGL3int1-TE2 vector, pGL3int1-TE3 vector, or pGL3int1-TE10 vector using a solution mixed with 1 μl of cationic lipid (Lipofectamine 2000, Invitrogen). The COS1 cells transfected with the expression vector are chemiluminescent whether or not a photoprotein (Luciferase) that can function by splicing RNA synthesized from a DNA fragment into which the target alternative splicing cassette is inserted is expressed. It confirmed based on the observation using a measuring apparatus (Ascent fluoroscan CF) (FIG. 19).
  As a comparative control, either the positive control vector pGL3-control produced in Example 5, or the pGL3int1 vector that incorporates the DNA fragment GL3int1 of the present invention but does not contain an alternative splicing cassette, or the negative control pCI-neo vector Transfected COS1 cells (FIG. 19).
  The detailed results of this experiment are shown in Table 1 below. As a result of this experiment, COS1 cells have slightly lower luciferase activity when transfected with the pGL3int1-TE10 vector, and when transfected with the pGL3int1-TE3 vector. It was shown to have significantly higher luciferase activity. In contrast, when COS1 cells were transfected with the pGL3int1-TE2 vector, almost no luciferase activity was shown (FIG. 19).
  From these results,
  splicing phenomenon in which intervening fragments (corresponding to introns) are removed in COS1 cells transfected with the pGL3int1 vector;
  In COS1 cells transfected with the pGL3int1-TE2 vector, splicing occurs mainly at both ends of the tau gene exon 2, so that exon 2 intervening fragments on both sides of exon 2 so as to remain between the two reporter fragments. There are many splicing phenomena that remove (equivalent to introns);
  In COS1 cells transfected with the pCI-GFPint1-TE3 vector, a splicing phenomenon occurs in which intervening fragments (corresponding to introns) on both sides of exon 3 are removed so that exon 3 remains between the two reporter fragments, a splicing phenomenon has occurred in which the intervening fragment (corresponding to an intron) is removed so as not to contain exon 3 of the tau gene; and
  In COS1 cells transfected with the pCI-GFPint1-TE10 vector, a splicing phenomenon occurs in which intervening fragments (corresponding to introns) on both sides of exon 10 are removed so that exon 10 remains between the two reporter fragments, splicing phenomenon in which intervening fragments (corresponding to introns) are removed so as not to include exon 10 of the tau gene;
Became clear.
  Next, COS1 cells in which the expression vectors pGL3int1-TE2 vector, pGL3int1-TE3 vector, and pGL3int1-TE10 vector are expressed in the cells in this manner in the above-described culture medium at 37 ° C. under humid conditions. After culturing for a day, the cells were collected, and total RNA expressed from the cells was collected using RNeasy Mini Kit (QIAGEN). RT-PCR using OneStep RT-PCR kit (QIAGEN) was performed using the collected 50 ng of total RNA as a template. In the RT-PCR reaction solution (total volume 25 μl), PGL3-520F primer (cgtcacatct catactactc; SEQ ID NO :) that hybridizes to the sequence on the first fragment of the luciferase protein of the present invention (SEQ ID NO: 26). 43) and a PGL3-820R primer (taatcctgaa ggctcctcag; SEQ ID NO: 44) (each 10 pmol) primer set that hybridizes to the sequence on the second fragment of the luciferase protein of the present invention. This primer set generates a 339 bp PCR product when the mRNA encoding the luciferase protein of the present invention is formed in the cell (ie, when the intervening sequence is completely spliced out with the exon). When mRNAs having sequences in which exon 2, exon 3, or exon 10 of the tau gene is inserted between the first fragment and the second fragment of the luciferase protein of the present invention are generated, 426 bp and 426 bp, respectively. Alternatively, a 432 bp PCR product is generated. This RT-PCR reaction solution was subjected to PCR at 30 ° C. for 30 minutes (reverse transcription reaction) and at 95 ° C. for 15 minutes, followed by PCR (94 ° C. for 30 seconds to 59.6 ° C. for 30 seconds to 72 ° C. Min, 30 cycles), and further reacted at 72 ° C. for 5 min (FIG. 20).
  Separately from the above, a primer pair that hybridizes to exon 2 of the human tau gene as a primer set (ie, TEX2-F primer (aatctcccctgcaga; SEQ ID NO: 24) and TEX2-R primer (cttccgctgtttggagtgct; SEQ ID NO: 25) ), A primer pair that hybridizes to exon 3 (ie, TEX3-F primer (atgtgacaccacccttatag; SEQ ID NO: 45) and TEX3-R primer (ctgtggtttcc tttctgga; SEQ ID NO: 46)) or exon 10 Primer pair (ie, TEX10-F primer (gtgcagataa tataataga g ct; SEQ ID NO: 19) and TEX10-R primer (actgccgcct cccggg; SEQ ID NO: 20)) were added to the RT-PCR reaction solution at 50 ° C. for 30 minutes (reverse transcription reaction), 95 PCR was continued at 15 ° C. for 15 minutes (94 ° C. for 30 seconds−52.3 ° C. for 30 seconds−72 ° C. for 30 seconds, 30 cycles), and further reacted at 72 ° C. for 1 minute (FIG. 20). ). When mRNA comprising a sequence in which exon 2 of the tau gene is inserted between the first fragment and the second fragment of the luciferase protein of the present invention is generated in the cell, the TEX2-F primer (SEQ ID NO. : 24) and a primer set of TEX2-R primer (SEQ ID NO: 25) to generate a 87 bp PCR product of exon 2 of the tau gene (FIG. 20A); the tau gene between the first fragment and the second fragment When an mRNA having a sequence in which exon 3 is inserted is generated, a primer set of a TEX3-F primer (SEQ ID NO: 45) and a TEX3-R primer (SEQ ID NO: 46) is used. The 87 bp PCR product of exon 3 (FIG. 2 0B); and when a mRNA comprising a sequence in which exon 10 of the tau gene is inserted between the first fragment and the second fragment is generated, TEX10-F primer (SEQ ID NO: 19) and TEX10 A 93 bp PCR product of exon 10 of the tau gene is generated by PCR using a primer set of -R primer (SEQ ID NO: 20) (FIG. 20C), respectively.
  For a total RNA sample obtained from COS1 cells transfected with any of the pCI-neo vector, pGL3control vector, pGL3int1 vector, pGL3int1-TE2 vector, pGL3int1-TE3 vector, or pGL3int1-TE10 vector, PGL3-520F primer (SEQ ID FIG. 21 shows the result of electrophoresis performed on a sample subjected to PCR using a primer set of NO: 43) and PGL3-820R primer (SEQ ID NO: 44). In this figure, lane 1 is a pGL3int1 vector, lane 2 is a pGL3int1-TE2 vector, lane 3 is a pGL3int1-TE3 vector, lane 4 is a pGL3int1-TE10 vector, lane 5 is a pGL3control vector, and lane 6 is a pCI-neo vector, Total RNA collected from each transfected COS1 cell was used as a sample.
  As a result of RT-PCR, when the pGL3int1 vector (lane 1) and the pGL3control vector (lane 5) were transfected, only a 339 bp mRNA product was obtained, whereas the pGL3int1-TE2 vector ( Lane 2) contains only 426 bp mRNA, pGL3int1-TE3 vector (lane 3) essentially only 339 bp mRNA, and pGL3int1-TE10 vector (lane 4) contains two 339 bp and 432 bp mRNAs, respectively. It was confirmed that it was produced (FIG. 21).

Whether or not alternative splicing occurs in a target exon in a cell by transfecting a eukaryotic cell using a DNA fragment having the above-described sequence structure inserted into an expression vector and expressing the DNA fragment Can be monitored in real time in situ. This monitoring system can be applied to any organism by changing the vector, and can be used for both in vivo experiments and in vitro experiments. In addition, since reporter gene expression can be detected using any of the conventional detection methods, whether or not alternative splicing occurs in real time is monitored in situ without preparing a new device. be able to.
[Sequence Listing]

Claims (13)

A first fragment and a second fragment of the coding region of the DNA encoding the reporter protein;
An intervening fragment located between the first fragment and the second fragment, having a splicing motif and comprising a cloning site;
An exogenous alternative splicing cassette inserted into the cloning site;
A DNA fragment comprising   The DNA fragment according to claim 1, wherein the reporter protein is a fluorescent protein or a chromoprotein.   The DNA fragment according to claim 1, wherein the reporter protein is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), and luciferase (LF).   The DNA encoding the reporter protein is formed when the first fragment and the second fragment of the coding region of the DNA encoding the reporter protein are directly ligated. DNA fragment.   The DNA fragment according to any one of claims 1 to 4, wherein a plurality of cloning sites are present, and an exogenous alternative splicing cassette is inserted into any one of the cloning sites.   The alternative splicing cassette is composed of an exon that is subject to alternative splicing among genes known or predicted to cause alternative splicing and intron portions upstream and downstream thereof. The DNA fragment according to any one of 1 to 5.   The DNA fragment according to any one of claims 1 to 5, wherein the alternative splicing cassette is composed of exon 10 of the tau gene and intron portions upstream and downstream thereof.   A eukaryotic cell expression vector prepared by inserting the DNA fragment according to any one of claims 1 to 7 into a cloning site of a protein expression vector.   The protein expression vector is a eukaryotic cell expression vector selected from the group consisting of a pCI-neo vector (GenBank Acc #: U47120) and a pGL3-Control vector (GenBank Acc #: U47296). Expression vector.   When spliced into eukaryotic cells and expressed, alternative splicing occurs in the selected cells, resulting in the direct ligation of the first and second fragments to produce a functional reporter protein 10. A fusion protein is produced that does not function as a reporter protein, or is formed from the first fragment and an exon in the alternative splicing cassette and a second fragment. The expression vector described in 1. A eukaryotic cell is transfected with the expression vector according to any one of claims 8 to 10,
Culturing the transfected eukaryotic cells,
Expressing a DNA fragment inserted into an expression vector in a cell;
Monitoring in situ the functional reporter protein produced by direct ligation of the first and second fragments of the coding region of the DNA encoding the reporter protein as a result of alternative splicing;
A method for monitoring in situ alternative splicing in eukaryotic cells. The DNA fragment according to any one of claims 1 to 7;
Any eukaryotic expression vector;
A kit for detecting alternative splicing of a desired exogenous exon.   A kit for detecting alternative splicing of a desired exogenous exon, comprising the eukaryotic cell expression vector according to any one of claims 8 to 10.