WO2008102903A1 - Système de détection d'épissage par arn - Google Patents

Système de détection d'épissage par arn Download PDF

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WO2008102903A1
WO2008102903A1 PCT/JP2008/053130 JP2008053130W WO2008102903A1 WO 2008102903 A1 WO2008102903 A1 WO 2008102903A1 JP 2008053130 W JP2008053130 W JP 2008053130W WO 2008102903 A1 WO2008102903 A1 WO 2008102903A1
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fragment
vector
seq
exon
protein
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PCT/JP2008/053130
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Ohoshi Murayama
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School Corporation, Azabu Veterinary Medicine Educational Institution
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • 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 selective splicing in situ.
  • Non-patent document 1 Among the diseases known as genetic diseases, there are known ones caused by selective splicing abnormalities and those caused by mutations that cause abnormal alternative splicing.
  • Non-patent Document 2 In order to elucidate the mechanism of alternative splicing regulation in vivo, C. elegans is used to visualize cells and subsequent alternative splicing using fluorescent proteins as a repo overnight. A production method was developed (Non-patent Document 2). However, this method can only identify the splicing phenomenon of a specific gene disclosed in the literature, and can be used to analyze the alternative splicing control mechanism for other genes. As a typical analysis tool, it has the disadvantage that it cannot be used.
  • Non-Patent Document 1 Johnson J.M., et al., Science. 2003 302: 2141-2144
  • Non-patent Document 2 Kuroyanagi H., et al., Nat Methods. 2006 3: 909-915 Disclosure of the Invention
  • the present invention relates to a gene that defines a protein that is subject to alternative splicing. It is an object of the present invention to provide a DNA construct, such as a DNA fragment or an expression vector containing the DNA fragment, which can clarify how exon is selectively spliced in cells.
  • the inventors of the present invention have clarified that the above problem can be solved by inserting an intervening fragment to be subjected to selective splicing so as to cleave the cDNA of lipo-overnight protein.
  • This intervening fragment contains a cloning site into which an exon of a gene defining a protein can be inserted.
  • the inventors of the present invention provide a first fragment and a second fragment of a coding region of DNA encoding a reporter protein; 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; To do.
  • 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. .
  • 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 that has a splicing motif and includes a cloning site; an exogenous alternative splicing cassette inserted into said cloning site;
  • a eukaryotic expression vector prepared by inserting a DNA fragment into a cloning site of a vector for protein expression.
  • the present invention can also provide a kit for detecting alternative splicing of a desired exon exon comprising the above-described protein expression vector.
  • the inventors of the present invention provide a reporter protein.
  • An intervening fragment comprising: an exogenous alternative splice cinder cassette inserted into the cloning site; and created by inserting a DNA fragment into a cloning site of a protein expression vector, Transforming the eukaryotic expression vector into a eukaryotic cell; culturing the transfected eukaryotic cell; expressing the DNA fragment inserted into the expression vector in the cell; and As a result of alternative splicing.
  • Selective splicing in eukaryotic cells including in situ monitoring of functional report proteins generated by direct ligation of one fragment and a second fragment Provides a method for monitoring in situ.
  • FIG. 1 is a diagram showing a splicing mechanism using a lipo-overnight gene as an example.
  • FIG. 2 is a diagram showing a specific structure and mechanism of action of the DNA fragment of the present invention.
  • FIG. 3 shows the first step in the construction of the DNA fragment GFP int l.
  • FIG. 4 is a schematic diagram showing the preparation method of fragment B, which is the first step in the construction method of DNA fragment GFPintl.
  • 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 GFPintl.
  • FIG. 6 is a schematic diagram showing a method for preparing a DNA fragment GFPintl by linking fragments A to (:, which is the second step in the construction method of the DNA fragment GFPintl.
  • Figure 7 shows HEK293 cells transfected with either a pCI-neo vector, a pCI-GFP vector, a pCI-GFPintl vector, or a pCI-GFPint ⁇ TE10 vector. It is the fluorescence image and phase contrast image which show the result monitored in situ.
  • Figure 8 shows the results of in situ monitoring of SH-SY5Y cells transfected with either CI-GFP vector, CI-GFPintl vector, or pCI-GFPinU-TE10 vector. It is a fluorescence image and a phase contrast image.
  • Figure 9 shows the DNA fragment structure resulting from splicing in cells transfected with the expression vector pCI-GFPint ⁇ TE10 vector and the positional relationship of the DNA amplified by the primer set.
  • FIG. 10 shows the result of RT-PCR of total RNA expressed in HEK293 cells transfected with the expression vector pCI-GFPint ⁇ TE10 vector prepared in Example 1. It is a figure which shows an image.
  • FIG. 11 shows SH transfected with either pCI-GFPintl vector, pCI-GFPint ⁇ TE2 vector, pCI-GFPint ⁇ TE10 vector, GFP-neo vector, or pCI-neo vector.
  • pCI-GFPintl vector pCI-GFPint ⁇ TE2 vector
  • pCI-GFPint ⁇ TE10 vector GFP-neo vector
  • GFP-neo vector GFP-neo vector
  • pCI-neo vector pCI-neo vector
  • Figure 12 shows C0S1 cells transfected with either pCI-GFPintl vector, pCI-GFPint ⁇ TE2 vector, pCI-GFPinU-TElO vector, GFP-neo vector, or pCI-neo vector. This is a fluorescence image showing the result of in situ monitoring.
  • Figure 13 shows the structure of the DNA fragment resulting from splicing in cells transfected with the expression vector pCI-GFPint ⁇ TE2 vector and the positional relationship of the DNA amplified by the primer set.
  • FIG. 14 shows RT-total RNA expressed in C0S1 cells transfected with the expression vector pCI-GFPint ⁇ TE2 vector prepared in Example 4 or pCI-GFPint ⁇ TE10 vector. It is a figure which shows the electrophoresis image of the result of PCR.
  • 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 GL3intl.
  • 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 GL3intl.
  • FIG. 17 is a schematic diagram showing a method for preparing a DNA fragment GL3intl by combining fragments D to F, which is the second step in the construction method of the DNA fragment GL3intl.
  • Figure 18 shows the splicing phenomenon in which intervening fragments (corresponding to introns) are removed when (A) SH-SY5Y cells or (B) HEmmmmmmK293 cells are transfected with the pGL3intl vector. Figure 18 shows what has happened ( Figure 18A or B respectively).
  • Figure 19 shows C0S1 cells transfected with either pGL3intl vector, pGL3inU-TE2 vector, pGL3inU-TE3 vector, pGL3inU-TE10 vector, or pCI-neo vector. It is a figure which shows that the splicing phenomenon in which the intervening fragment (equivalent to intron) is removed in the cell has arisen.
  • Fig. 20 shows the expression vector pGL3inU-TE2 vector (Fig. 20A), pGL3intl-TE3 vector (Fig. 20B), or pGUint ⁇ TE10 vector (Fig. 20C), in cells transfected. It is a figure which shows the positional relationship of the structure of the mRNA fragment which arises in each result of splicing, and mRNA amplified by a primer set.
  • FIG. 21 shows the expression vector prepared in Example 7 transfected with pGL3intl-TE2 vector, pGL3intl-TE3 vector, or pGL3int ⁇ TE10 vector.
  • FIG. 1 Shows an electrophoretic image of RT-PCR results for total RNA expressed in C0S1 cells.
  • Figure 1 shows an example of a general splicing mechanism that has been conceived up to now, using a reporter gene as an example.
  • an intervening fragment intron
  • the sequence GU (1) at the 5 'end, the AG sequence (2) at the 3' end, and YYRAY inside the intervening fragment This combination of sequences is called a splicing motif (the splicing motif shown in Fig. 1 is an example of currently known splicing motifs). Is).
  • This splicing motif physically forms a loop structure between the 5 'end GU sequence (1) and the YYRAY sequence inside the intervening fragment.
  • the 5' end GU sequence (1) and 3 'By cutting the intervening fragment at the AG sequence (2) on the terminal side and removing it from the mRNA the first and second fragments of the Ribo Yuichi gene are directly ligated to mature It is thought that 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.
  • 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
  • a DNA fragment is made comprising an intervening fragment having a splicing motif and comprising a cloning site; an exogenous alternative splicing cassette inserted into said cloning site.
  • Figure 2 shows the specific structure and mechanism of action of this DNA fragment.
  • the reporter protein in the DNA fragment of the present invention is used as a reporter. Any protein that is known to perform its function can be used. For example, fluorescent proteins, chromoproteins, enzyme proteins, and the like 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 reporter proteins, fluorescent proteins such as green fluorescent protein (GFP), red fluorescent protein (RFP), luciferase (LF), and DsRed, or photochrome, blue light absorbing protein (NPL1) (Kagawa T , et a., Science, 2001, 291, 2138-2141).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • LF luciferase
  • NPL1 blue light absorbing protein
  • a fluorescent protein or chromoprotein When a fluorescent protein or 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 are alive.
  • chromoproteins are used for examples such as fluorescent microscopes, confocal microscopes, and CCD cameras. In some cases, a microscope, a CCD camera, and the like can be cited as examples, but the present invention is not limited to these as long as changes in fluorescence or dye can be measured.
  • 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.
  • a natural intron sequence contained in a genomic DNA sequence in a reporter gene can be used as an intervening fragment.
  • the base sequence of genomic DNA in a gene region encoding a certain Ribo Yuichi protein has a sequence structure consisting of exon 1, intron 1, and exon 2, that genomic DNA region should be used as a DNA fragment as it is. Can do.
  • either intron can be used as an intervening fragment, in which case the intron and the exons on both sides are derived from the genomic DNA base sequence.
  • the other part can be a chimeric DNA fragment derived from the cDNA base sequence.
  • intron sequences in other genes can also be used as intervening fragments.
  • the DNA fragment of the present invention can be obtained by inserting, as an intervening fragment, an intron sequence derived from another gene into the cDNA base sequence of the gene encoding the lipo-overnight protein.
  • an artificial intron can also be used as an intervening fragment. Examples of splicing using artificial introns have been reported in Umekage and K i kuch i (Nucleic Acids Sympo. Ser, (50): 323-324 (2006)).
  • DNA fragment of the present invention can be obtained by inserting such an artificial intron as an intervening fragment into the cDNA base sequence of the gene encoding Ribo Yuichi protein.
  • cloning sites present in the intervening fragment of the DNA fragment of the present invention.
  • a cloning site may be a restriction enzyme site originally present in the intron, for example, when an intron is used as an intervening fragment, or the multiple cloning site of a cloning vector used in the art.
  • the nucleic acid sequence may be excised and diverted.
  • an exogenous alternative splicing cassette is first inserted into the cloning site of the DNA fragment.
  • This exogenous alternative splice cinder cassette is characterized in that it contains an exon that is the target of alternative splicing among genes that define a protein, together with sequences of introns upstream and downstream of that exon. (Figure 2, middle).
  • an exon that is the target of alternative splicing of a gene defining a certain protein inserted into the cloning site of a DNA fragment as an alternative splicing cinder cassette is already known to cause alternative splicing. Even an exon that is expected to cause alternative splicing can be used. If you insert an exon known to cause alternative splicing and monitor the splicing phenomenon, Splicing trends can be detected.
  • exon 10 of the tau gene can be used as an exon known to cause alternative splicing.
  • a DNA region composed of exon 10 of tau gene and intron and downstream introns thereof can be used as an alternative splice cinder cassette.
  • 3Rt au having a structure consisting of a sequence not containing exon 10 (ie, exon 9 to exon 11), or from a sequence containing exon 10 4Rtau having the following structure (ie, exon 9 to exon 10 to exon 11) is produced.
  • Human 3Rt au and human 4Rt au are proteins related to the pathology of Alzheimer's disease.
  • Alzheimer's disease frontotemporal dementia with Parkinson's syndrome linked to chromosome 17 (FTDP-1 7), Pick's disease, progressive supranuclear palsy (PSP), and cortical basal Diseases with dementia, such as nuclear degeneration (CBD), characterized by abnormal accumulation of t au gene products in degenerative neurons are known.
  • FTDP-1 7 frontotemporal dementia with Parkinson's syndrome linked to chromosome 17
  • PSP progressive supranuclear palsy
  • CBD cortical basal Diseases with dementia, such as nuclear degeneration
  • Both 3Rt au and 4Rt au are recognized as major components of structures abnormally accumulated in degenerative neurons, but it depends on each disease whether 3Rt au or 4Rt au accumulates. For example, both 3Rt au and 4Rt au accumulate in Alzheimer's disease, whereas only 3Rt au accumulates in Pick's disease, while familial mutations, PSP, and FTDP-17 are accumulated.
  • the first fragment of the reporter that was originally present in the DNA fragment In addition to the splicing motif at the 3 ′ end (1) and the motif at the 5 ′ end of the second fragment (2), the splicing motif derived from the exogenous alternative splicing cinder cassette (3) and (4) Will be inserted (middle of Fig. 2).
  • a first fragment and a second fragment of the coding region of DNA encoding the above-mentioned lipo-evening protein located between the first fragment and the second fragment An intervening fragment having a splicing motif and containing a cloning site; an exogenous alternative splicing cinder cassette inserted into said cloning site; and selecting a DM fragment
  • a eukaryotic cell expression vector prepared by inserting the DNA fragment into a cloning site of a protein expression vector for the purpose of expression in cells that cause alternative splicing.
  • the “alternative splicing” phenomenon that occurs in the evening is a phenomenon peculiar to eukaryotic cells
  • 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 is a known eukaryotic cell expression vector known in the art, which is compatible with cells that transfer the expression vector into which the DNA fragment has been inserted. Anything is acceptable.
  • the cell into which the expression vector is transferred is an insect cell (eg, S f 9 cell, S f 21 cell, etc.)
  • the pIEx vector, baculovirus vector, etc. may be mentioned as examples.
  • the cell into which the expression vector is transfected is a mouse cell (eg, CH0 cell, HN2 cell, Neuro2a cell, mouse ES cell, etc.), take pCI-neo vector, adenovirus vector, etc. as examples
  • the cells that transfect the expression vector are human cells (eg HEK293 cells, SH-SY5Y cells, HeLa cells, etc.), mention pCI-neo vectors, adenovirus vectors, etc. Can do.
  • Insertion of the DNA fragment into the vector may be performed using any method known or well known in the art, for example, Sambrook and Russel (Molecular Library: A Laboratory Manua l, 3rd ed iti on (2001)), Masamura Muramatsu (Lab Manual Genetic Engineering, 3rd Edition, Maruzen) can be used.
  • one of the cloning sites in the vector is cleaved with a restriction enzyme, while both ends of the DNA fragment ⁇ are converted to a base sequence that can be bound to the end of the cloning site cleaved with the restriction enzyme. It can be inserted by arranging and ligating the DNA fragment into the cloning site.
  • the expression vector prepared in this manner When the expression vector prepared in this manner is expressed in a eukaryotic cell by transfer, selective splicing occurs in the selective cell.
  • the first fragment and the second fragment A functional reporter protein is defined, defined by the directly linked DNA, or by the DNA formed from the first fragment and the exon in the alternative splicer cassette and the second fragment A fusion protein that does not function as a reporter protein is generated.
  • One of the expression patterns is taken (Fig. 2 bottom).
  • in situ monitoring of alternative splicing in eukaryotic cells includes: transfecting an eukaryotic cell with the expression vector described above; culturing the transformed eukaryotic cell; The DNA fragment inserted into the expression vector is expressed in the cell; as a result of alternative splicing, the first and second fragments of the coding region of the DNA encoding the reporter protein are directly linked.
  • the functional report protein defined by the DNA produced by Such in situ monitoring can be performed in real time by selecting a reporter.
  • kits for detecting alternative splicing of a desired exogenous exon comprising the above-described DNA fragment and any eukaryotic expression vector; or the above-described DNA fragment is inserted.
  • a kit for detecting alternative splicing of a desired exogenous exon can also be provided, including a vector for protein expression.
  • a green fluorescent protein (Enhanced Green) is used as a reporter gene.
  • Acc # M62654 is the first sequence (SEQ ID NO: 1) corresponding to exon 2 including the start codon.
  • SEQ ID NO: 2 a sequence corresponding to a sequence obtained by combining exon 3 and part of exon 4 (from the 5 'end to the stop codon) (SEQ ID NO: 2) is used as the second fragment, respectively.
  • Aequorea victoria GFP GenBank Acc # M62654
  • Aequorea victoria genomic region (531 bp, SEQ ID NO: 3) having a nucleotide sequence homologous to intron 2 of GFP GenBank Acc # M62654 is used as an intervening fragment and selective splicing is performed.
  • the known exon is composed of exon 10 (SEQ ID NO: 4) of human tau gene and its upstream intron (SEQ ID NO: 5) and downstream intron (SEQ ID NO: 6).
  • a DNA fragment was constructed using the DNA region produced.
  • the sequence of EGFP contained in the repo overnight gene (first fragment and second fragment) and intervening fragment of the present invention was obtained from Clontech.
  • the GFP and intron sequences of Aequorea victoria contained in these were obtained from genomic DNA extracted from Aequorea victoria (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 2). 3)
  • the procedure was as follows. First, the EcoRI-Notl restriction enzyme fragment containing the EGFP cDNA region from the EGFP expression vector PEGFP-N3 vector (GenBank Acc #: 1157609, Clontech) was transformed into the eukaryotic expression vector pCI-neo Mammalian Expression.
  • EGFP expression vector constructed by ligation and insertion into the EcoRI-Notl site of the multiple cloning site of Vector (GenBank Acc #: U47120, Promega)
  • the pCI-GFP vector is used as a saddle type primer set (pCI-neo Fragment A was prepared by PCR using a PCIF primer that hybridizes to a vector-derived sequence (tttctctccacaggtgtccac; SEQ ID NO: 7) and an EGFPE1R primer that hybridizes to EGFP cDNA (tgcatcaccttcaccctcgccggacac; SEQ ID NO: 10).
  • This fragment A consists of 86 bp from the pCI-GFP vector, 5 'untranslated region of EGFP 461 ⁇ , 97 bp of nucleotide numbers 1 to 97 of EGFP cDNA, and EGFPE1R primer (SEQ ID NO: 10)
  • a pCI-GFP vector is used as a saddle, and a primer set (PCIR primer (ctagttgtggtttgtccaaactc; SEQ ID N0: 8) Fragment B was prepared by PCR using the EGFPE2F primer (agtgccatgcccgaaggttatgtccag; SEQ ID NO: 9) that hybridizes to EGFP cDNA (Fig. 4).
  • PCIR primer ctagttgtggttttgtccaaactc
  • Fragment B was prepared by PCR using the EGFPE2F primer (agtgccatgcccgaaggttatgtccag; SEQ ID NO: 9) that hybridizes to EGFP cDNA (Fig. 4).
  • This fragment B in order from the 5 'side, is 21 bp from the 5' side of the EGFPE2F primer (SEQ ID NO: 9), 441 bp from nucleotide numbers 280 to 720 of EGFP cDNA, 9 bp from the 3 'untranslated region of EGFP, And a nucleic acid having a length of 546 bp composed of 75 bp derived from the pCI-GFP vector (FIG. 4, SEQ ID NO: 14).
  • Genomic DNA extracted from Aequorea victoria is used as a saddle type primer set (GFPI1F primer that hybridizes to exon 2 of the GFP gene (tgtccggcgagggtgaaggtgatgcaacatacgg; SEQ ID NO: 11) and IGFP1 that hybridizes to GFP gene exon 3 Fragment C was prepared by PCR using a primer (ctggacataaccttcgggcatggcactcttg; SEQ ID NO: 12) (FIG. 5).
  • This fragment C is an onkage that is homologous to nucleotide numbers 95 to 206 of the GFP cDNA derived from the GFP gene exon 2 in the 5 'side of the GFPI1F primer (SEQ ID NO: 11), 9 bp from the 5' side.
  • Genomic DNA region 112 bp jellyfish homologous to GFP gene intron 1 ⁇ Genomic DNA region (531 bp), GFP gene derived from GFP gene exon 3 GFP cDNA homologous to nucleotide numbers 207-276 * Genomic DNA region 70 bp, And a 728 bp long nucleic acid composed of 6 bp from the GFPI1R primer (SEQ ID NO: 12) ( Figure 5, SEQ ID NO: 15).
  • the specific base sequence of the DNA fragment prepared by digesting this DNA fragment with the restriction enzymes EcoRI and Notl has the sequence consisting of nucleotide numbers 87 to 1392 of SEQ ID N0: 16, and SEQ ID NO : 1, SEQ ID NO: 3 and SEQ ID NO: 2 are included in this order.
  • This DNA fragment having the sequence consisting of nucleotide numbers 87 to 1392 of SEQ ID NO: 16 is referred to as GFPintl hereinafter. ( Figure 6, bottom).
  • alternative splice cinder cassettes containing exon 10 of the tau gene include exon 10 (SEQ ID NO: 4) of the tau gene and its upstream intron (SEQ ID NO: 5) and downstream intron ( A DNA region composed of a sequence in which SEQ ID NO: 6) is arranged in the order of SEQ ID NO: 5, SEQ ID NO :, and SEQ ID NO: 6 is used.
  • the desired sequence of the tau gene (SEQ IDNOs: 4-6) was obtained from genomic DNA isolated and purified from human blood using the Wizard® Genomic DNA Purification Kit.
  • the cloning site PmaCI site in the intervening fragment of the DNA fragment GFPintl inserted into the expression vector pCI-GFPintl vector was inserted into exon 10 (SEQ ID NO: 4) of the above-mentioned tau gene and its upstream and downstream introns (
  • SEQ ID NO: 3 In the sequence homologous to intron 2 (SEQ ID NO: 3) in the DNA fragment inserted into the expression vector, there is a restriction enzyme PmaCI site (CACGTG) at nucleotides 358 to 363. When the enzyme site is cleaved with an enzyme, a blunt end is formed.
  • 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.
  • vector pCI-GFPintl vector 50 ng of vector pCI-GFPintl vector and 60 ng of the above-described alternative splicer cassette were each treated with PmaCI at 37 for 17 hours to produce blunt ends.
  • the vector cleaved by this treatment was dephosphorylated with CIAP.
  • the expression vector pCI-GFPintl-TE10 vector prepared in Example 1 was used to transfect eukaryotic cells, and alternative splicing actually occurs for the gene exon 10. The purpose was to confirm whether or not.
  • HEK293 cells and SH-SY5Y cells were used to transfer the expression vector prepared in Example 1, pCI-GFPintl-TE10 vector.
  • These HEK293 cells and SH-SY5Y cells can be subcultured in DMEM medium (GIBC0) supplemented with fetal bovine serum to a final concentration of 10%.
  • the expression vector pCI-GFPinU-TE10 vector prepared in Example 1 0.8 / zg was subjected to a lance transfer using 1 ⁇ l of the cationic lipid aipofectamine 2000, Invitrogen).
  • ⁇ 293 cells and SH-SY5Y cells transfected with expression vectors express a green fluorescent protein that can function by splicing RNA synthesized from DNA fragments inserted with the target alternative splicing cassette. It was confirmed based on observation using a fluorescence microscope (Nikon) (Figs. 7 and 8).
  • pCI-neo vector which is a parent vector that does not contain the DNA fragment of the present invention at all
  • pCI-neo vector which contains EGFP cDNA in pCI-neo vector, but does not contain intron -GFP vector or DNA fragment of the present invention
  • pCI-GFPintl vector that incorporates GFPintl but does not contain alternative splicing cassettes HEK293 cells and SH-SY5Y cells transformed with one of the cells were used (Figs. 7 and 8).
  • 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-mentioned culture medium at 37 under humid conditions for 2 days. Thereafter, the cells were recovered, and total RNA expressed from within the cells was recovered using RNeasy Mini Kit (QIAGEN).
  • RT-PCR using OneStep5 RT-PCR kit (QIAGEN) was performed using the collected 50 ng of total RNA as a saddle type. In the RT-PCR reaction solution (total volume 25 zl), EGFP- 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).
  • F-65 primer (acgtaaacgg ccacaagt tc ag; SEQ ID NO: 17) and green fluorescent protein of the present invention (EGFP-GFP chimeric protein) EGFP-R-391 primer (gcgggtc t tg tagt tgcc; SEQ ID NO: 18) (10 pmol each) primer set that is hyper-hybridized to the sequence on the second fragment It was.
  • This primer set is used when mRNA encoding the green fluorescent protein of the present invention (EGFP-GFP chimeric protein) is formed in the cell (that is, when the intervening sequence is completely spliced out with exon).
  • TEX10-F primer gtgcagataa t taataagaa get; SEQ ID NO: 19
  • TEXIO-R primer actgccgcct cccggg; SEQ ID NO: 20
  • RNA samples obtained from HEK293 cells transfected with either the pCI-neo vector, pCI-GFP vector, pCI-GFPint l vector, or pCI-GFPint l-TE10 vector the EGFP-F-65 primer (SEQ ID N0: 17) and EGFP-R-391 primer (SEQ ID N0: 18) PCR sample and TEX10-F primer (SEQ ID NO: 19) and TEX10-R primer (SEQ ID NO Figure 20 (A) and (B) show the results of electrophoresis for the samples subjected to PCR using the primer set of 20).
  • Lane 1 is the pCI-neo vector 1
  • Lane 2 is the pCI-GFP vector
  • Lane 3 is the pCI-GFPint l vector
  • Lane 4 is the pCI-GFPint l-TE10 vector.
  • the short mRNA is revealed to be a 266 bp DNA from which exon 10 of the tau gene has been removed as a result of alternative splicing, ie, the sequence corresponding to exon 2 of GFP.
  • the first fragment (SEQ ID NO: 1) is directly ligated to the second fragment (SEQ ID NO: 2) consisting of the sequence corresponding to the sequence of GFP exons 3 and 4. It was revealed that this is a nucleic acid sequence that defines GFP as a functional reporter protein (Fig. 10 (A), lane 4).
  • PCR was performed using a primer set of TEX10-F primer (SEQ ID NO: 19) and TEX10-R primer (SEQ ID NO: 20). It was revealed that 93 bp of exon 10 of the tau gene was amplified (Fig. 10 (B)).
  • the pCI-GFPint vector prepared in Example 1 is used, and exon known to cause alternative splicing is exon 2 of human tau gene and its upstream and downstream introns.
  • a DNA fragment was constructed using a DNA region composed of a portion.
  • Alternative splice cinder cassettes containing tau exon 2 include tau exon 2 (SEQ ID NO: 21) and its upstream intron (SEQ ID NO: 22) and downstream intron (SEQ ID NO: 23) is used as a DNA region composed of sequences arranged in the order of SEQ ID NO: 22, SEQ ID NO: 21 and SEQ ID NO: 23.
  • the 5 desired sequences of the tau gene (SEQ IDNOs: 21-23) were obtained from genomic DNA isolated and purified from human blood using the Wizard® Genomic DNA Purification Kit.
  • An alternative splice cinder cassette consisting of 10 23) was inserted to prepare a pCI-GFPinU-TE2 vector.
  • a sequence homologous to intron 2 (SEQ ID NO: 3), which is an intervening fragment in the DNA fragment inserted into the expression vector, contains a restriction enzyme PmaCI site (CACGTG) at nucleotides 358 to 363.
  • PmaCI site CACGTG
  • this restriction enzyme site is used as a cloning site.
  • the expression vector pCI-GFPinU-TE2 vector extracted from E. coli JM109 transformed with the ligation reaction mixture was used as a primer type (PCIF (SEQ ID NO: 7) and
  • the expression vector pCI-GFPint ⁇ TE2 vector prepared in Example 3 and the expression vector pCI-GFPinU-TElO vector prepared in Example 1 were used to transfect eukaryotic cells.
  • the purpose of this study was to confirm whether or not selective splicing actually occurs for tau gene exon 2.
  • SH- SY5Y cells and C0S1 cells were used.
  • SH-SY5Y cells and C0S1 cells can be subcultured in DMEM medium supplemented with fetal bovine serum to a final concentration of 10%.
  • Example 1 prepared in Example 3
  • the prepared expression vector pCI-GFPintl-TE10 vector was transfected using a solution in which a cationic lipid (Lipofectamine 2000, Invitrogen) 11 was mixed.
  • SH-SY5Y and C0S1 cells transfected with the expression vector have a green fluorescent protein that can function by splicing RNA synthesized from a DNA fragment inserted with the target alternative splicer cassette. Whether it was expressed or not was confirmed based on observation using a fluorescence microscope (Nikon) (FIGS. 11 and 12).
  • SH-SY5Y cells and C0S1 cells in which the expression vectors pCI-GFPinU-TE2 and pCI-GFPint1-TE10 vectors were expressed in the cells in this way were added to the culture medium described above. Then, after culturing at 37 under humid conditions for 2 days, 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 saddle.
  • 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 gcgggtcttg tagttgcc
  • EGFP-GFP chimeric protein SEQ ID NO: 18m
  • This primer set is used when mRNA encoding the green fluorescent protein of the present invention (EGFP-GFP chimeric protein) is formed in the cell (that is, when the intervening sequence is completely spliced out with exon).
  • a 266 bp PCR product is generated, and 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.
  • mRNA consisting of the sequence is generated, a PCR product of 353 b or 359 bp is generated, respectively.
  • This RT-PCR reaction solution at 50 for 30 minutes (reverse transcription), 95 for 15 minutes, followed by PCR (94 for 30 seconds-59.6 for 30 seconds-72 for 1 minute, 30 cycles), Furthermore, reaction was performed at 72 for 5 minutes (Fig. 13).
  • TEX2-F primers (aatctcccctgcaga; SEQ ID NO: 24) and TEX2-R primers (cttccgctgttggagtgct; SEQ ID NO: 25) that hybridize to exon 2 of the human tau gene are used as primer sets.
  • mRNA consisting of 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 primer set of TEX2-F primer (SEQ ID NO: 24) and TEX2-R primer— (SEQ ID NO: 25) produces a 61 bp PCR product (FIG. 13).
  • EGFP Figure 14 shows the results of electrophoresis of a sample that had been subjected to PCR using the primer set of -F-65 primer (SEQ ID 0: 17) and EGFP-R-391 primer (SEQ ID NO: 18).
  • lane 1 is the pCI-GFPintl vector
  • lane 2 is the pCI-GFPint ⁇ TE2 vector
  • lane 3 is the pCI-GFPintl-TE10 vector
  • lane 4 is the pCI-GFP vector
  • lane 5 is the pCI-neo Total RNA collected from C0S1 cells transfected with each vector was used as a sample.
  • the mainly amplified DNA in lane 2 is obtained by PCR using the primer set of TEX2-F primer (SEQ ID N0: 24) and TEX2-R primer (SEQ ID NO: 25). It was revealed that 93 bp was amplified (data not shown).
  • photoprotein protein luciferase (pGL3-controll: GenBank Acc # U47296, Clontech) is used as a reporter gene
  • Photinus pyral is (firefly) luciferase (GenBank Acc # Ml 5077) exon 1 to 3 combined nucleic acid (668 base pairs, SEQ ID NO: 26) corresponding to the sequence (668 base pairs), the first fragment is exon 4 to 6 and exon 7 Sequence that joins up to the stop codon Nucleic acid (985 base pairs, SEQ ID NO: 27) linked to a sequence equivalent to (985 base pairs) was used as the second fragment, respectively, and a GFP (GenBank Acc # M62654) intron of Aequorea victoria 5 'and 6' bases of the Aequorea victoria genome-derived region (SEQ IDN0: 3) having a nucleotide sequence homologous to 2 Photinus pyralis (Firefly) Luciferase
  • Exons 1 to 3 and exons 4 to 6 and 7 of the luciferase gene of the present invention were obtained from Clontech, respectively (SEQ ID NO: 26 and SEQ ID NO: 27, respectively).
  • Intron C sequence was obtained from GenBank (GenBank Acc # Ml 5077).
  • the pGL3-control vector (GenBank Acc #: U47296, Clontech), a Luciferase expression vector, is used as a saddle type primer set (PGL3-387F primer (gcagcctacc gtggtgttcg tttcc; SEQ ID NO: 29) and PGL3-int5R primer ( Fragment D was prepared by PCR using gagtataaag tgcacatacc tggcatgcga gaatc; SEQ ID NO: 34)).
  • Fragment D consists of 325 bp consisting of 306 bp from nucleotide numbers 363 to 668 of pGL3-controI luciferase cDNA and 19 bp from 3 ′ side of PGL3-int5R primer in order from 5 ′. It is a nucleic acid having a length (FIG. 15, SEQ ID 0: 35). Of these, the 5 '13 bp of the PGL3-int5R primer (SEQ ID NO: 34) is derived from the 5' end (nucleotide number? -19) of the intervening fragment (SEQ ID NO: 3) of the pCI-GFPintl vector. The 6 bp next to it is a sequence (gtatgt) derived from intron C of firefly luciferase (GenBank Acc # M15077).
  • pGL3-control vector GenBank Acc #: U47296, Clontech
  • a Luci f erase expression vector is used as a primer
  • primer set PGL3-int3F primer (ggaattacgt tgtctcttgt agagatccta tttttggcaa tc; SEQ ID NO: 31)
  • PGL3- Fragment E was prepared by PCR using 1249R primer (cccagtaagc tatgtctcca gaatgtagcca; SEQ ID NO: 30).
  • This fragment E is the PGL3-int3F braid in order from the 5 'side. 5'-side 22 bp, pGL3-control luciferase cDNA nucleotide number 669 ⁇
  • the 5 '16 bp of the PGL3-int3F primer (SEQ ID NO: 31) is derived from the 3' end (nucleotide numbers 510-525) of the intervening fragment of the pCI-GFPintl vector (SEQ IDN0: 3),
  • the 6 bp next to it is a sequence (ttgtag) derived from in ⁇ ron C of firefly luciferase (GenBank Acc # M15077).
  • Fragment by PCR using (SEQ ID NO: 29) and PGL3-int3R primer (SEQ ID NO: 32))! A fragment G (SEQ ID N0: 38) having a length of 857 bp containing F was prepared (see the second row from the top in FIG. 17).
  • PCR was performed using a primer set (PGL3-387F primer (SEQ ID NO: 29) and PGL3-1249R primer (SEQ ID NO: 30)).
  • Fragment H (SEQ ID NO: 39) was prepared (see the third row from the top in FIG. 17).
  • the specific base sequence of fragment H prepared in this way is as shown in SQ ID NO: 39, which 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 is the parent vector used for the construction of the expression vector.
  • the expression vector pGL3intl vector was constructed by ligation and insertion of the recipient H.
  • This expression vector pGL3intl vector includes SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO:
  • GL3intl a DNA fragment containing NO: 27 in this order.
  • This DNA fragment is referred to as GL3intl in the rest of this specification ( Figure 17, bottom row).
  • the intervening fragment (SEQ ID NO: 28) in the DNA fragment GL3intl inserted into the expression vector pGL3intl vector contains a restriction enzyme PmaCI site (CACGTG) at nucleotides 358 to 363.
  • PmaCI site CACGTG
  • Primer sets (PGL3-387F primer (SEQ ID NO: 29) and PGL3-1249R primer (SEQ ID NO: 30)) were expressed in the shape of an expression vector extracted from E. coli JM109 transformed with the ligation reaction solution. PCR performed using and primers PGL3- 387F primer (SEQ ID NO: 29), PGL3-1249R primer (SEQ ID NO: 30) and PGL3-int5F primer (SEQ ID NO: 33) When confirmed by the dideoxy method using BigDye (registered trademark) Terminater v3.1 Cycle Sequencing Kit (Applied biosystems), a DNA fragment having the structure shown in the upper part of Fig. 2 was inserted into the vector for expression. It was shown that the target eukaryotic cell expression vector was constructed.
  • Example 6 Splicing in an expression vector using luciferase
  • Example 5 the expression vector prepared in Example 5 was used to transfection into eukaryotic cells to confirm whether splicing actually occurs in the luciferase gene into which the intervening fragment has been inserted. Done as a purpose.
  • the pGL3intl vector prepared in Example 5 produces splicing in the cells
  • the pGL3intl vector was transfected into SH-SY5Y cells and HEK293 cells (RIKEN) and spliced in the cells. Whether or not occurred was detected by detecting chemiluminescence.
  • SH-SY5Y cells and HEK293 cells (RIKEN) can be subcultured in DMEM medium supplemented with fetal bovine serum to a final concentration of 10%.
  • Expression vector SH-SY5Y cells and HEK293 cells transfected with pGL3intl vectors express functional photoproteins by splicing RNA synthesized from a DNA fragment containing the luciferase gene with an intervening sequence inserted. This was confirmed based on measurements using Dim® Glo luci ferase 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 medium at 37 for 1 day under humid conditions, and then the medium is removed.
  • the sample was dissolved with the luci ferase Reagent of Dua® Glo luciferase Assay System (Promega), and the luminescence was measured with a luminescence sensor (a I ⁇ 1).
  • a luminescence sensor a I ⁇ 1.
  • luminescence by water luciferase was measured, and luminescence by Renilla luciferase expressed by pRL-CMV was measured by treatment with Stop & GloReagent . Record the integrated value of each luminescence and divide it by the amount of luminescence by renilla luciferase to correct the error in transfer efficiency, and then compare the amount of luminescence by water luciferase ( Figures 18A and B).
  • the SH-SY5Y cells in which the expression vector is expressed in the cells in this way are cultured in the above-mentioned culture medium at 37 under humid conditions for 1 day, and then the cells are collected, Using the RNeasy Mini Kit (QIAGEN), total RNA expressed from the cells was recovered.
  • RT-PCR using OneStep RT-PCR kit (QIAGEN) was performed using the collected lng total RNA as a saddle type.
  • primer sets EGFP-F-65 and EGFP-R-391 each final concentration 0.6 M
  • This RT-PCR reaction solution is 50 to 30 minutes (reverse transcription), 95 to 15 minutes, followed by PCR (30 seconds at 94 "-30 seconds at 50-90 seconds at 72, 35 Cycle), and further reacted at 72 for 10 minutes.
  • RT-PCR reaction solution containing TEX10-F and TEX10-R as a primer set at 50 for 30 minutes (reverse transcription), 95 for 15 minutes, followed by PCR (30 for 94 The reaction was carried out at 72-5 for 30 seconds-72 for 30 seconds, 30 cycles), and 72 for 1 minute.
  • the base sequence was confirmed by the dideoxy method using BigDye (registered trademark) Terminater v3.1 Cycle Sequencing Kit (Applied biosystems) with the amplified fragment prepared by RT-PCR as a saddle type.
  • Example 5 the pGL3intl vector prepared in Example 5 is used, and exons that are known to cause alternative splicing are exon 1 of the human tau gene and its upstream and downstream intron portions.
  • a DNA fragment was constructed by a method substantially similar to the method described in Example 3 using the DNA region composed of
  • Alternative splicing cassettes containing exon 2 of the tau gene include exon 2 of the tau gene (SEQ ID NO: 21) and its upstream intron (SEQ ID NO: 22) and A DNA region composed of a sequence in which the downstream intron portion (SEQ ID NO: 23) is arranged in the order of SEQ ID NO: 11, SEQ ID NO: 21 and SEQ ID NO: 23 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.
  • Alternative splicing cassettes that include exon 3 of the tau gene include exon 3 of the tau gene (SEQ ID NO: 40) and its upstream intron (SEQ ID NO: 41) and downstream intron (SEQ ID NO: 42). ) Is used in a DNA region composed of sequences arranged in the order of SEQ ID NO: 41, SEQ ID NO: 40 and SEQ ID NO: 42.
  • the desired sequence of the tau gene was obtained from genomic DNA isolated and purified from human blood using Wizard® Genomic DNA Purification Kit.
  • Alternative splicing cassettes containing tau gene exon 10 include tau gene exon 10 (SEQ ID NO: 4) and its upstream intron (SEQ ID NO: 5) and downstream intron (SEQ ID NO: 6). ) Using a DNA region composed of sequences arranged in the order of SEQ ID NO: 5, SEQ ID NO: 4 and SEQ ID NO: 6. 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.
  • Expression vector Intervening fragment inserted into the pGL3intl vector In the cloning site in GL3intl, the exon 2 alternative splice cinder cassette, exon 3 alternative splice cinder cassette, or exon 10 alternative splice cinder of the tau gene described above
  • a pGL3int ⁇ ⁇ ⁇ TE2 vector, a pGL3int ⁇ TE3 vector, or a pGL3inU-TE10 vector was prepared by inserting one of the sets.
  • the intervening fragment (SEQ ID NO: 28) in the DNA fragment inserted into the expression vector pGL3int vector contains a restriction enzyme PmaCI site (CACGTG) at nucleotides 358 to 363. When cut, a blunt end is formed.
  • this restriction enzyme site as a cloning site, an alternative splice cinder cassette containing any exon of the tau gene described above was inserted.
  • Expression vectors extracted from Escherichia coli JM109 transformed with the ligation reaction solution pGL3intl-TE2 vector, pGL3intl-TE3 vector, or pGL3inU-TE10 vector are used as primers, and each primer set (PCIF (SEQ ID NO: 7 ) And PCIR (SEQ ID NO: 8)) and PCR and primers PCIF (SEQ ID NO: 7) or PCIR (SEQ ID NO: 8) and BigDye® Terminater v3.1
  • the DNA fragment having the structure shown in the middle of Figure 2 was found to be the expression vector pCI-GFPintl-TE2 vector, pGL3inU-TE3 vector, or pGL3inU- It was shown that it was inserted into any of the TE10 vectors so as to allow expression, and that the target eukaryotic expression vector was constructed.
  • the three expression vectors pGL3int ⁇ TE2 vector, pGL3int ⁇ TE3 vector, or pGL3int ⁇ TE10 vector prepared in Example 7 were used to transfect eukaryotic cells, and tau gene exon 2 The purpose of this study was to confirm whether alternative splicing actually occurred for Exon 3 or Exon 10.
  • C0S1 cells RIKEN were used to transfect the pGL3int ⁇ TE2 vector, the pGL3int ⁇ TE3 vector, or the pGL3int ⁇ TE10 vector.
  • the C0S1 cells can be subcultured in DMEM medium supplemented with fetal bovine serum to a final concentration of 10%.
  • DMEM medium supplemented with fetal bovine serum
  • GL3intl-TE2 vector GL3intl-TE3 vector
  • pGL3int ⁇ TE10 vector 0.8 / xg was added to cationic lipid (Lipofectamine2000, Invitrogen) 1 I. Transfer was performed using the mixed solution.
  • C0S1 cells are DNA fragments containing the desired alternative splice cinder cassette. Whether or not a photoprotein (Luc i ferase) capable of functioning by splicing RNA synthesized from sputum was expressed based on observations using a chemiluminescence analyzer (Ascent fl uoroscan CF) (Fig. 19). ).
  • the positive control vector pGL3-cont rol prepared in Example 5 or the pGL3 in tl vector incorporating the DNA fragment GL3int l of the present invention but not containing the alternative splicer cassette, or the negative control C0S1 cells transfected with any of the pCI-neo vectors were used (FIG. 19).
  • exon 2 There are many splicing events that remove intervening fragments (corresponding to introns) on both sides of Exon 2 so that they remain between two reporter fragments; in C0S1 cells transfected with pCI-GFPint ⁇ 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, and the intervening fragment does not contain exon 3 of the tau gene.
  • a splicing phenomenon occurs in which (equivalent to an intron) is removed; and
  • C0S1 cells expressing the expression vectors pGL3intl-TE2 vector, pGL3intl-TE3 vector and pGL3intl-TE10 vector in the cells in this manner were cultured in the above-mentioned culture medium at 37 "C, wet. After culturing under conditions for 2 days, cells are collected and RNeasyMini
  • RNA expressed from the cells was recovered.
  • RT-PCR using OneStep RT-PCR kit (QIAGEN) was performed using the collected 50 ng of total RNA as a saddle.
  • a PGL3-520F primer (cgtcacatct catctacctc; SEQ IDN) that hybridizes to the sequence on the first fragment (SEQ IDN0: 26) of the luciferase protein of the present invention.
  • a PGL3-820R primer (taatcctgaa ggctcctcag; SEQ ID NO: 44) (10 pmol each) primer set that hybridizes to the sequence on the second fragment of the luciferase protein of the present invention was included.
  • This primer set generates a 339 bp PCR product when the mRNA encoding the luciferase zeta protein of the present invention is formed in the cell (ie, when the intervening sequence is completely spliced out with exon).
  • 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 in a cell
  • PCR products of 426 bp, 426 bp or 432 bp are generated, respectively.
  • This RT-PCR reaction solution is 50 minutes for 30 minutes (reverse transcription), then 95 for 15 minutes, followed by PCR (30 seconds for 94-30 seconds for 59.6-1 minute for 72 seconds, 30 cycles) Further, the reaction was performed at 72T: for 5 minutes (Fig. 20).
  • 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 (cttccgctgttggagtgct; SEQ ID NO: 25)), a primer pair that hybridizes to exon 3 (ie TEX3-F primer (atgtgacagc acccttagt; SEQ ID NO: 45) and TEX3-R primer (ctgtggttcc ttctggga; SEQ ID NO: 46)), or exon 10 RT- with either of a pair of primers that hybridize to (ie, TEX10-F primer (gtgcagataa ttaataagaa get; SEQ ID NO: 19) and TEX10-R primer (actgccgcct ccggg;
  • the pCI-neo vector, the pGL3control vector, the pGL3intl vector, the pGL3inU- TE2 vector, the pGL3int ⁇ TE3 vector, or the pGL3int ⁇ TE10 vector For total RNA samples obtained from transfected C0S1 cells, PCR was performed using a primer set of PGL3-520F primer (SEQ ID NO: 43) and PGL3-820R primer (SEQ ID NO: 44). The results of electrophoresis are shown in Figure 21.
  • lane 1 is the pGUinil vector
  • lane 1 is the pGL3int ⁇ TE2 vector
  • lane 3 is the pGL3int ⁇ TE3 vector
  • lane 4 is the pGL3int ⁇ TE10 vector
  • lane 5 is the pGL3control vector
  • lane 6 is the pCI-neo vector

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Abstract

L'invention vise à proposer un produit de construction d'ADN tel qu'un fragment d'ADN ou un vecteur d'expression contenant le fragment d'ADN capable d'élucider la manière dont un épissage alternatif d'un exon d'un gène spécifiant une protéine devant subir un épissage alternatif est régulé dans les cellules. Les présents inventeurs ont découvert que l'objectif ci-dessus peut être atteint par l'insertion d'un fragment intermédiaire contenant une région de clonage capable d'insérer un exon d'un gène spécifiant une protéine devant subir un épissage alternatif de telle sorte que l'ADNc pour une protéine reporter est divisé.
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Publication number Priority date Publication date Assignee Title
CN113817772A (zh) * 2021-09-08 2021-12-21 浙江赛微思生物科技有限公司 一种用于验证人类基因内含子变异对基因剪接影响的质粒及其构建方法和应用
CN114934058A (zh) * 2022-05-31 2022-08-23 北京海创科业生物科技有限责任公司 一种pMini-CopGFP质粒载体的构建方法及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005023255A2 (fr) * 2003-09-04 2005-03-17 Centre National De La Recherche Scientifique (Cnrs) Derives d’indole pour le traitement de maladies liees au processus d’epissage

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005023255A2 (fr) * 2003-09-04 2005-03-17 Centre National De La Recherche Scientifique (Cnrs) Derives d’indole pour le traitement de maladies liees au processus d’epissage

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GAO L. ET AL.: "SR PROTEIN 9G8 MODULATES SPLICING OF TAU EXON 10 VIA ITS PROXIMAL DOWNSTREAM INTRON, A CLUSTERING REGION FOR FRONTOTEMPORAL DEMENTIA MUTATIONS", MOLECULAR AND CELLULAR NEUROSCIENCE, vol. 34, no. 1, January 2007 (2007-01-01), pages 48 - 58, XP005751264 *
NASIM M.T. AND EPERON C.: "A DOUBLE-REPORTER SPLICING ASSAY FOR DETERMINING SPLICING EFFICIENCY IN MAMMALIAN CELLS", NATURE PROTOCOLS, vol. 1, no. 2, 2006, pages 1022 - 1028 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113817772A (zh) * 2021-09-08 2021-12-21 浙江赛微思生物科技有限公司 一种用于验证人类基因内含子变异对基因剪接影响的质粒及其构建方法和应用
CN114934058A (zh) * 2022-05-31 2022-08-23 北京海创科业生物科技有限责任公司 一种pMini-CopGFP质粒载体的构建方法及其应用

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