JP2016131516A - Linear double-stranded dna sequentially comprising terminator sequence and promoter sequence - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide linear double-stranded DNA capable of expressing RNA derived from a DNA sequence for RNA expression in a cell without connecting a promoter sequence or a terminator sequence to the DNA sequence for RNA expression in advance, and a method for expressing RNA derived from a DNA sequence for RNA expression using such a linear double-stranded DNA.SOLUTION: A linear double-stranded DNA that sequentially comprises a terminator sequence and a promoter sequence, in which a base number between a base at 3' terminal of the terminator sequence and 5' terminal of the promoter sequence is 0 to 3000 is fabricated. Further, by introducing the linear double-stranded DNA and a DNA sequence for RNA expression into a cell, RNA derived from the DNA sequence for RNA expression is expressed.SELECTED DRAWING: None

Description

  The present invention relates to a linear double-stranded DNA sequentially provided with a terminator sequence and a promoter sequence, and a method for expressing RNA derived from a DNA sequence for RNA expression using such a linear double-stranded DNA.

  In order to prepare a transformed cell and to express a target gene in the cell, a double-stranded DNA comprising a promoter sequence, a target gene and a terminator sequence is prepared in sequence, and the DNA construct is transferred to the cell via a vector. The method introduced into is common. When this method is used, it is necessary to link DNA so that a promoter sequence, a target gene, and a terminator sequence are sequentially provided. As described above, the most commonly used technique for ligating DNA is to bind DNA molecules having blunt ends or DNA molecules having complementary protruding ends using DNA ligase. is there. However, this method requires a complicated in vitro operation of restriction enzyme treatment and cleavage and ligation of the insert DNA and the vector by DNA ligase.

  In recent years, the present inventors have identified a promoter sequence and a target RNA expression DNA sequence prepared using a reverse primer for high expression containing a specific terminator sequence as a method for producing transformed cells without using a vector. A method has been proposed in which linear double-stranded DNA sequentially having a terminator sequence is introduced into animal cells (see Patent Document 1). In this method, the terminator sequence can be ligated downstream of the DNA sequence for RNA expression only by PCR without introducing restriction enzyme sites and annealing sequences, and without performing complicated circularization operations. By introducing double-stranded DNA into animal cells, it is possible to express RNA derived from the DNA sequence for RNA expression. However, when performing PCR, it was necessary to prepare in advance a template comprising a promoter sequence and a target RNA expression DNA sequence in sequence.

  In addition, the present inventors sequentially provided a linear double-stranded DNA for RNA expression comprising a terminator sequence, a promoter sequence, and a DNA sequence for RNA expression, or a DNA sequence for RNA expression, a terminator sequence, and a promoter sequence. A linear double-stranded DNA for RNA expression (see Patent Document 2) was proposed. If such a linear double-stranded DNA for RNA expression is used, the linear double-stranded DNA can be directly introduced into a cell and RNA derived from the DNA sequence for RNA expression can be easily expressed in the cell. It was necessary to connect a terminator sequence or promoter sequence in advance to the DNA sequence for expression.

  Furthermore, the present inventors introduced a linear double-stranded DNA comprising a promoter sequence, and a linear double-stranded DNA sequentially provided with a DNA sequence for RNA expression and a terminator sequence into the cell, thereby expressing the RNA expression. A method for producing a transformed cell that expresses RNA derived from a DNA sequence for use has been proposed (see Patent Document 3). In this method, it is necessary to prepare a linear double-stranded DNA that is sequentially provided with a DNA sequence for RNA expression and a terminator sequence in advance, and this is a complicated operation when analyzing the expression of various DNA sequences for RNA expression. I needed time.

International Publication No. 2012/147370 Pamphlet Japanese Patent Application No. 2013-213459 Japanese Patent Application No. 2013-258265

  An object of the present invention is to provide a linear double-stranded DNA capable of expressing RNA derived from an RNA expression DNA sequence in a cell without previously linking the promoter sequence or terminator sequence and the RNA expression DNA sequence. Another object of the present invention is to provide a method for expressing RNA derived from a DNA sequence for RNA expression using such a linear double-stranded DNA.

  The inventors of the present invention conducted extensive studies to solve the above-mentioned problems, and prepared various linear double-stranded DNAs including promoter sequences and terminator sequences, and genes encoding proteins, As a result, a linear double-stranded DNA having a terminator sequence and a promoter sequence in sequence and a gene encoding a protein are unexpectedly introduced into the cell. In this case, it was found that the target protein was expressed in the cells, and the present invention was completed.

That is, the present invention is as follows.
(1) A linear double-stranded DNA sequentially having a terminator sequence and a promoter sequence, wherein the number of bases between the 3 ′ end base of the terminator sequence and the 5 ′ end of the promoter sequence is 0 to 3000. A linear double-stranded DNA characterized by the above.
(2) The linear double-stranded DNA according to (1) above, which does not contain a plasmid replication origin.
(3) The linear double-stranded DNA according to (1) or (2) above, wherein the terminator sequence is a sequence derived from SV40 terminator or rabbit β-globin terminator.
(4) The linear double-stranded DNA according to any one of (1) to (3) above, wherein the promoter sequence is a human cytomegalovirus (CMV) promoter sequence.
(5) The RNA derived from the DNA sequence for RNA expression, wherein the linear double-stranded DNA according to any one of (1) to (4) and the DNA sequence for RNA expression are introduced into cells. A method of expressing
(6) The method according to (5) above, wherein the cell is a mammalian cell.

  According to the present invention, when a gene or the like is expressed in a cell, a step of linking a promoter sequence and a sequence encoding a DNA sequence for RNA expression, or a sequence encoding a DNA sequence for RNA expression and a terminator sequence are linked. And a step of preparing a plasmid are not required. In particular, when performing expression analysis of various DNA sequences for RNA expression, the present invention can be used for analysis in a short time and at low cost.

It is a figure which shows the example of the method of producing linear double stranded DNA provided with a terminator arrangement | sequence and a promoter arrangement | sequence sequentially. (A) shows a case where double-stranded DNA containing a promoter sequence is used as a template, and (b) shows a case where double-stranded DNA containing a terminator sequence is used as a template. It is a figure which shows the result of the agarose electrophoresis of the produced linear double stranded DNA. (1) Lane is SVter-CMVp-EGFP, (2) Lane is SVter-CMVp, (3) Lane is βGlopAter-CMVp, and (4) Lane is βGlopAter-CMVp-EGFP. It is a figure which shows the map of plasmid pCMV-hGluc-SVter. It is a figure which shows the result of the agarose electrophoresis of the produced linear double strand DNA hGluc. It is a figure which shows the expression level of a luciferase at the time of using a human embryonic kidney cell (HEK293 cell) in Example 1. FIG. The horizontal axis represents the introduced linear double-stranded DNA, and the vertical axis represents the relative fluorescence intensity of luciferase (RLU (counts / μl / sec)). It is a figure which shows the expression level of a luciferase at the time of using the human cervix epithelioid cell (HeLa cell) in Example 1. FIG. The horizontal and vertical axes are the same as in FIG. It is a figure which shows the result of the agarose electrophoresis of the produced linear double stranded DNA. (1) Lane is EGFP, (2) Lane is CMVp-EGFP-SVter. In Example 2, it is a figure which shows the result observed with the fluorescence microscope at the time of introduce | transducing (b) (beta) GlopAter-CMVp and EGFP, when (a) SVter-CMVp and EGFP are introduce | transduced into HEK293 cell. Moreover, in (a) and (b), the left figure is a fluorescence microscope image, and the right figure is a differential interference microscope image (the same applies to FIGS. 9 to 13). In Example 2, it is a figure which shows the result observed with the fluorescence microscope at the time of introduce | transducing (a) SVter-CMVp-EGFP into HEK293 cell, (b) (beta) GlopAter-CMVp-EGFP. In Example 2, when (a) CMVp-EGFP-SVter is introduce | transduced into HEK293 cell, (b) It is a figure which shows the result observed with the fluorescence microscope at the time of introduce | transducing EGFP. In Example 2, it is a figure which shows the result observed with the fluorescence microscope at the time of introduce | transducing (a) SVter-CMVp and EGFP into HeLa cell, and (b) (beta) GlopAter-CMVp and EGFP. In Example 2, it is a figure which shows the result observed with the fluorescence microscope at the time of introduce | transducing (b) (beta) GlopAter-CMVp-EGFP when (a) SVter-CMVp-EGFP is introduce | transduced into HeLa cell. In Example 2, when (a) CMVp-EGFP-SVter is introduce | transduced into HeLa cell, it is a figure which shows the result observed with the fluorescence microscope at the time of introduce | transducing (b) EGFP.

  The linear double-stranded DNA of the present invention is a linear double-stranded DNA that is sequentially provided with a terminator sequence and a promoter sequence, and is located between the base at the 3 ′ end of the terminator sequence and the 5 ′ end of the promoter sequence. The DNA is not particularly limited as long as it is a linear double-stranded DNA characterized in that the number of bases is 0 to 3000, and preferably contains no plasmid replication origin.

  In addition, the method for expressing RNA derived from the DNA sequence for RNA expression of the present invention is not particularly limited as long as it is a method for introducing the linear double-stranded DNA of the present invention and the DNA sequence for RNA expression into cells. However, by this method, RNA derived from the RNA expression DNA sequence can be expressed in the cell without previously linking the promoter sequence or terminator sequence and the RNA expression DNA sequence.

  In the present invention, the terminator sequence may be any sequence that controls the end of gene transcription and stabilizes the transcribed RNA. The purpose of RNA expression, the type of cell that performs RNA expression, the promoter The terminator sequence can be appropriately selected depending on the type and the type of DNA sequence for RNA expression, and may be a naturally occurring terminator sequence or an artificially synthesized terminator sequence based on a sequence on a database. In addition, as long as it has a function as a terminator, a partial sequence of a naturally occurring terminator sequence or a sequence containing a base substitution, deletion, or insertion in a naturally occurring terminator sequence is also an artificially synthesized terminator sequence. included.

  When using a naturally occurring terminator sequence, the terminator sequence may be a viral terminator sequence, a prokaryotic terminator sequence such as Escherichia coli or Bacillus subtilis, or a eukaryotic organism such as yeast, mouse or human. The terminator sequence may be, specifically, simian virus 40 (SV40) terminator sequence, Rabbit β-globin terminator sequence, bovine growth hormone (BGH) terminator sequence, HSV · TK terminator sequence. CYC1 terminator sequence, ADH terminator sequence, SPA terminator sequence, Agrobacterium tumefaciens nopaline synthase (NOS) gene terminator sequence, Cauliflower mosaic virus (CaMV) 35S gene terminator And corn-derived Zein gene terminator sequence, Rubisco small subunit (SSU) gene terminator sequence, subclover stunt virus (SCSV) gene terminator sequence, LacZ alpha terminator sequence, polyT terminator sequence, etc. From the viewpoint of increasing the expression level of RNA, an SV40 terminator sequence or a rabbit β-globin terminator sequence can be preferably exemplified.

  The artificially synthesized terminator sequence is preferably an SV40 terminator or a sequence derived from a rabbit β-globin terminator. For example, an artificially synthesized terminator sequence described in Japanese Patent Application No. 2013-213659, preferably SV40. Examples include the 130th to 208th base number sequence (SEQ ID NO: 1) of the terminator sequence and the 121st to 190th base sequence (SEQ ID NO: 2) of the rabbit β-globin terminator sequence.

  In the present invention, the promoter sequence refers to controlling the expression of RNA derived from the RNA expression DNA sequence arranged downstream by binding to RNA polymerase when arranged upstream of the RNA expression DNA sequence. Any base sequence can be used, and it can be appropriately selected depending on the purpose of RNA expression, the type of cell such as a mammal expressing RNA, the type of DNA sequence for RNA expression, the type of terminator sequence, etc. Even an existing promoter sequence may be a promoter sequence artificially synthesized based on a sequence on a database. In addition, as long as it has a function as a promoter, a partial sequence of a naturally occurring promoter sequence or a sequence containing a base substitution, deletion, or insertion in a naturally occurring promoter sequence is also an artificially synthesized promoter sequence. included.

  When a naturally occurring promoter sequence is used, promoter sequences include human cytomegalovirus (CMV) promoter sequence, simian virus (SV40) promoter sequence, human elongation factor 1α (EF1α) promoter sequence, adenovirus major late (Adenovirus Major Late). AML), AML promoter sequence, SV40 and HTLV-1 LTR fusion promoter SRα promoter sequence, human ubiquitin C promoter sequence, α-actin promoter sequence, β-actin promoter sequence, U6 promoter sequence, H1 promoter sequence, tetracycline Tet-Off promoter sequence in which RNA expression is suppressed by Tet-On promoter, Tet-On promoter sequence in which RNA expression is induced by tetracycline, metal such as zinc, and a mechanism induced by various stimuli Loti Leone in promoter sequences, can be exemplified ARE promoter sequence induced by active oxygen, can be preferably exemplified a human cytomegalovirus (CMV) promoter sequence.

  The artificially synthesized promoter sequence is preferably a sequence derived from a human cytomegalovirus (CMV) promoter or a simian virus (SV40) promoter. For example, Japanese Patent Application No. 2014-123214 and Japanese Patent Application No. 2014-136929 Examples include sequences having the described promoter activity.

  The linear double-stranded DNA sequentially having the terminator sequence and the promoter sequence of the present invention is a linear double-stranded DNA in which the promoter sequence is operably linked downstream (3 ′ end side) of the terminator sequence. The number of bases between the 3 ′ terminal base of the terminator sequence and the 5 ′ terminal of the promoter sequence is 0 to 3000, preferably 0 to 1000, more preferably 0 to 500, still more preferably 0 to 100, and particularly preferably. Can be 0 to 50, most preferably 0.

  A method for producing a linear double-stranded DNA sequentially comprising a terminator sequence and a promoter sequence is not particularly limited. For example, PCR is performed using a double-stranded DNA containing a promoter sequence as a template and a primer containing a terminator sequence. A method of preparing by PCR, a method of preparing by PCR using a primer containing a promoter sequence using a double-stranded DNA containing a terminator sequence as a template, and a linear double-stranded DNA containing a terminator sequence and a promoter sequence Examples thereof include a method of ligating and producing linear double-stranded DNA by a known method such as a fusion PCR method (JP 2009-268360 A) or a method using DNA ligase.

  As a method of producing by PCR using a double-stranded DNA containing a promoter sequence as a template and using a primer containing a terminator sequence, as shown in FIG. 1 (a), using a double-stranded DNA containing a promoter sequence as a template, A method of performing PCR using a forward primer composed of a sequence including a terminator sequence and a sequence of the 5 ′ end region of the promoter sequence and a reverse primer composed of a complementary sequence of the 3 ′ end region of the promoter sequence can be exemplified. . As a terminator sequence in such a forward primer, an artificially synthesized terminator sequence described in the above-mentioned Japanese Patent Application No. 2013-213459, preferably the sequence of nucleotide numbers 130 to 208 (SEQ ID NO: 1) of the SV40 terminator sequence, It is preferable to use the base number 121-190th sequence (SEQ ID NO: 2) of the rabbit β-globin terminator sequence from the viewpoint of increasing the expression level of RNA and producing a primer at low cost.

  As a method for producing a double-stranded DNA containing a terminator sequence as a template and a PCR method using a primer containing a promoter sequence, a double-stranded DNA containing a terminator sequence as a template, as shown in FIG. Exemplifying a method of performing PCR using a forward primer composed of the sequence of the 5 ′ end region of the terminator sequence and a reverse primer composed of a complementary sequence of the sequence including the sequence of the 3 ′ end region of the terminator sequence and the promoter sequence in sequence. Can do. As a promoter sequence in such a reverse primer, an artificially synthesized sequence having promoter activity such as the sequence having promoter activity described in Japanese Patent Application No. 2014-123214 and Japanese Patent Application No. 2014-136929 described above may be used. From the viewpoint of increasing the expression level of RNA and producing a primer at low cost.

  In the method for expressing RNA derived from the RNA expression DNA sequence of the present invention, the RNA expression DNA sequence may be a linear double-stranded DNA sequence capable of expressing RNA, in addition to a gene encoding a protein, Functional nucleic acids such as shRNA (small hairpin RNA) expression sequence, siRNA (short interfering RNA) expression sequence, miRNA (micro-RNA), nucleic acid aptamer expression sequence, degoi expression sequence, antisense oligonucleotide expression sequence, ribozyme expression sequence, etc. Examples of the DNA sequence to be expressed can be given.

  In the method of expressing RNA derived from an RNA expression DNA sequence of the present invention, when a protein is expressed in a cell into which the RNA expression DNA sequence has been introduced, a gene encoding the protein can be selected as the RNA expression DNA sequence. The gene encoding the protein may be a full-length gene or a part thereof depending on the application. The origin may be a gene isolated from any organism or an artificial gene produced by genetic engineering.

  In the method of expressing RNA derived from an RNA expression DNA sequence of the present invention, when knocking down the expression of a protein in a cell into which the RNA expression DNA sequence has been introduced, the RNA expression DNA sequence may be a gene encoding the protein. ShRNA expression DNA sequence, siRNA expression DNA sequence, and antisense oligonucleotide expression DNA sequence that can suppress the expression of mRNA of RNA can be selected, and the activity of intracellular proteins into which the RNA expression DNA sequence has been introduced In the case of inhibiting or suppressing the activation, a DNA sequence for expressing a nucleic acid aptamer that can inhibit or suppress the activation of protein or a DNA sequence for expressing a ribozyme can be selected as the DNA sequence for RNA expression. , Each target RNA sequence and its transcription factor bind It can be designed by a conventional method based on the sequence information of that.

  In the method for expressing RNA derived from an RNA expression DNA sequence of the present invention, a linear double-stranded DNA having a terminator sequence and a promoter sequence in sequence, and a cell into which the RNA expression DNA sequence is introduced may be a mammal. Examples include cells, plant cells, and insect cells. Among them, mammalian cells can be preferably exemplified. Such mammalian cells may be cells derived from mammals such as humans, monkeys, mice, rats, hamsters, rabbits, goats, sheep, horses, pigs, dogs and the like. Specifically, HEK293 cells, HeLa cells, African green monkey kidney-derived cells (COS-7) cells, NIH-derived mouse embryonic fibroblasts (NIH-3T3 cells), human osteosarcoma cells (HOS cells), cultured human osteoblasts (SaM-1 cells), human Leukocyte T cells (jurkat cells), human breast cancer established cells (MCF-7 cells), human liver cancer-derived cells (HepG2 cells), human colon cancer-derived cells (CaCO-2 cells), human osteoblasts (SaOS cells) Human chronic myeloid leukemia cells (K562 cells), monkey kidney-derived cells (CV-1 cells), African green monkey kidney cells (COS-1 cells) Mouse fibroblasts (L929 cells), mouse teratoma cells (F9 cells), mouse osteoblast-like cells (MC-3T3-E1 cells), rat adrenal medulla melanoma (PC-12 cells), rat osteoblasts Like cells (ROS17 / 2.8 cells), Chinese hamster ovary-derived cells (CHO-K1 cells), hamster kidney cells (BHK-21 cells), etc., among which HEK293 cells or HeLa cells are preferred. The mammalian cells include embryonic stem cells (ES cells) such as EB3 cells and primary cultured cells collected from tissues.

  As a method for introducing linear double-stranded DNA comprising a promoter sequence and linear double-stranded DNA sequentially comprising an RNA expression DNA sequence and a terminator sequence into the cell, the DNA is introduced into a cell such as a mammal. Any method may be used as long as it is a general method to be introduced into a liposome, lipofection method, microinjection method, DEAE dextran method, calcium phosphate method, electroporation method, etc., and Lipofectin Reagent (registered trademark), Lipofectamine ( (Registered trademark), Lipofectamine (registered trademark) 2000 Reagent (manufactured by Invitrogen), SuperFect (registered trademark) Transfection Reagent (manufactured by Qiagen), FuGENE (registered trademark) HD Tra sfection Reagent, can be used FuGENE (TM) Transfection Reagent technique widely used in commercial transfection reagent and the art, such as (manufactured by Roche Diagnostics).

  In addition, the linear double-stranded DNA sequentially having a terminator sequence and a promoter sequence to be introduced into the cell and the DNA sequence for RNA expression may be introduced into the cell at the same time, and either one is introduced into the cell first. After the introduction, the other may be introduced into the cell, but it is preferable to introduce into the cell at the same time. The concentration of the linear double-stranded DNA sequentially having a terminator sequence and a promoter sequence and the DNA sequence for RNA expression are not particularly limited as long as the RNA of the DNA sequence for RNA expression can be expressed in the transformed cells. Illustrate that the concentration of linear double-stranded DNA comprising a terminator sequence and a promoter sequence in sequence is 0.2-5 times, preferably 0.5-2 times the concentration of the DNA sequence for expression. Can do.

[Expression of luciferase]
(Production of linear double-stranded DNA having SV40 terminator sequence and CMV promoter sequence in sequence)
Using pEGFP-C1 (Clontech) as a template and using a forward primer (SV40pA (130-208) -eCMV-600) shown in SEQ ID NO: 1 and a reverse primer (CMVpc) shown in SEQ ID NO: 3, an SV40 terminator A linear double-stranded DNA (SVter-CMVp) was prepared which was sequentially provided with the sequence of nucleotide numbers 130 to 208 of the sequence and the full length sequence of the CMV promoter. PCR was performed according to the recommended protocol using GXL polymerase (manufactured by Takara Bio Inc.). Each final concentration was adjusted to 10 pg of template (1 μl of 100 pg / μl), 0.3 μM of forward primer and 0.3 μM of reverse primer, and 98 ° C. for 10 seconds at 60 ° C. using iCycler thermal cycler (manufactured by BIO-RAD). A cycle of 15 ° C. for 15 seconds and 68 ° C. for 1 minute was performed 40 times. The results of agarose electrophoresis of the prepared linear double-stranded DNA are shown in FIG. 2 (2) lane.

(Production of linear double-stranded DNA having a rabbit β-globin terminator sequence and a CMV promoter sequence in sequence)
Using pEGFP-C1 (Clontech) as a template, β-globin using a forward primer (βGlopA (121-190) -eCMV-600) shown in SEQ ID NO: 2 and a reverse primer (CMVpc) shown in SEQ ID NO: 3 A linear double-stranded DNA (βGlopAter-CMVp) having a sequence of the 121st to 190th base numbers of the terminator sequence and the full length sequence of the CMV promoter was prepared. In the PCR reaction, a cycle of 98 ° C. for 10 seconds, 60 ° C. for 15 seconds and 68 ° C. for 1 minute was performed 40 times with the same reaction solution composition as described above. The results of agarose electrophoresis of the prepared linear double-stranded DNA are shown in FIG. 2 (3) lane.

(Production of linear double-stranded DNA having a sequence encoding luciferase (hGluc) gene)
Using pCMV-Gluc (manufactured by New England Biolabs), according to the method described in Japanese Patent Application No. 2013-213659, the full-length sequence of the CMV promoter, the sequence encoding the luciferase gene, and the base number 121 of the SV40 terminator A plasmid DNA (pCMV-hGluc-SVter: FIG. 3) having a sequence of ˜220th sequence was prepared. Using this pCMV-hGluc-SVter as a template, a forward primer (hGluc + 1) shown in SEQ ID NO: 4 and a reverse primer (hGluc + 558taaC) shown in SEQ ID NO: 5 were used to provide a sequence encoding the hGluc gene. A linear double-stranded DNA (hGluc) was prepared. In the PCR reaction, a cycle of 98 ° C. for 10 seconds, 60 ° C. for 15 seconds and 68 ° C. for 1 minute was performed 40 times with the same reaction solution composition as described above. The results of agarose electrophoresis of the prepared linear double-stranded DNA are shown in FIG.

(Introduction to cells)
HEK293 cells and HeLa cells were seeded on a 96-well plate at 4000 cells / well and cultured for 23 hours. (I) SVter-CMVp, (II) βGlopAter-CMVp, (III) hGluc, (IV) SVter-CMVp And hGluc (SVter-CMVp + hGluc), (V) βGlopAter-CMVp and hGluc (βGlopAter-CMVp + hGluc) linear double-stranded DNA 20 ng, 5% (W / V) PEG3350, 1.25 μg tRNA, Prepare 20 μl of a solution containing 2 μl of FuGENE (registered trademark) HD Transfection Reagent reagent and distilled water, and use this solution to transfer the linear double-stranded DNAs (I) to (V) to HEK293 cells and HeLa cells. Introduced. Next, the expression level of secretory luciferase contained in the culture supernatant collected after 25 hours was measured using a final concentration of 10 μM CTZ substrate and Centro LB960 (Berthold Technology). FIG. 5 shows the expression level of luciferase when HEK293 cells are used, and FIG. 6 shows the expression level of luciferase when HeLa cells are used.

(result)
As shown in FIGS. 5 and 6, even though SVter-CMVp, βGlopAter-CMVp, or hGluc alone was introduced, luciferase was not expressed in the cells, but SVter-CMVp and hGluc, or βGlopAter-CMVp and It was revealed that luciferase was expressed in cells when hGluc was introduced.

[Expression of EGFP]
(Preparation of linear double-stranded DNA having a sequence encoding the EGFP gene)
Using pEGFP-C1 (Clontech) as a template, a forward primer (pEGFP + 1) shown in SEQ ID NO: 6 and a reverse primer shown in SEQ ID NO: 7 (pEGFP + 720c), a sequence encoding the EGFP gene A linear double-stranded DNA (EGFP) provided with was prepared. In the PCR reaction, the same reaction solution composition as described in Example 1 was used, and a cycle of 98 ° C. for 10 seconds, 60 ° C. for 15 seconds, and 68 ° C. for 1 minute was performed 40 times. The results of agarose electrophoresis of the prepared linear double-stranded DNA are shown in FIG. 7 (1) lane.

(Production of linear double-stranded DNA sequentially comprising SV40 terminator sequence, CMV promoter sequence, and sequence encoding EGFP gene)
Using pEGFP-C1 (Clontech) as a template and using the forward primer (SV40pA (130-208) -eCMV-600) shown in SEQ ID NO: 1 and the reverse primer (pEGPF + 720c) shown in SEQ ID NO: 7, SV40 A linear double-stranded DNA (SVter-CMVp-EGFP) having a sequence of base numbers 130 to 208 of the terminator sequence, a full-length sequence of the CMV promoter, and a sequence encoding the EGFP gene was prepared. In the PCR reaction, the same reaction solution composition as described in Example 1 was used, and a cycle of 98 ° C. for 10 seconds, 60 ° C. for 15 seconds, and 68 ° C. for 1 minute was performed 40 times. The results of agarose electrophoresis of the prepared linear double-stranded DNA are shown in FIG. 2 (1) lane.

(Production of linear double-stranded DNA sequentially comprising β-globin terminator sequence, CMV promoter sequence, and sequence encoding EGFP gene)
Using pEGFP-C1 (manufactured by Clontech) as a template, a forward primer (βGlopA (121-190) -eCMV-600) shown in SEQ ID NO: 2 and a reverse primer (pEGPF + 720c) shown in SEQ ID NO: 7 -A linear double-stranded DNA (βGlopAter-CMVp-EGFP) having a sequence of base numbers 121 to 190 of the globin terminator sequence, a full-length sequence of the CMV promoter, and a sequence encoding the EGFP gene was prepared. In the PCR reaction, the same reaction solution composition as described in Example 1 was used, and a cycle of 98 ° C. for 10 seconds, 60 ° C. for 15 seconds, and 68 ° C. for 1 minute was performed 40 times. The results of agarose electrophoresis of the prepared linear double-stranded DNA are shown in FIG. 2 (4) lane.

(Production of a linear double-stranded DNA comprising a CMV promoter sequence, a sequence encoding an EGFP gene, and an SV40 terminator sequence in sequence)
Using pEGFP-C1 (manufactured by Clontech) as a template, using the forward primer (pEGFP-600) shown in SEQ ID NO: 8 and the reverse primer (pEGPF + 1018c) shown in SEQ ID NO: 9, the full-length sequence of the CMV promoter and EGFP A linear double-stranded DNA (CMVp-EGFP-SVter) having a gene-coding sequence and an SV40 terminator sequence in sequence was prepared. In the PCR reaction, the same reaction solution composition as described in Example 1 was used, and a cycle of 98 ° C. for 10 seconds, 60 ° C. for 15 seconds, and 68 ° C. for 1 minute was performed 40 times. The results of agarose electrophoresis of the prepared linear double-stranded DNA are shown in FIG. 7 (2) lane.

(Introduction to cells)
HEK293 cells and HeLa cells were seeded on a 96-well plate at 4000 cells / well and cultured for 23 hours, then (I) SVter-CMVp and EGFP (SVter-CMVp + EGFP), (II) βGlopAter-CMVp and EGFP (βGlopAter− CMVp + EGFP), (III) SVter-CMVp-EGFP, (IV) βGlopAter-CMVp-EGFP, (V) CMVp-EGFP-SVter, and (VI) EGFP each linear double-stranded DNA (20 ng) in the same manner as in Example 1. (Trademark registration) It introduced into HEK293 cell and HeLa cell using the solution containing HD Transfection Reagent reagent, and it observed with the fluorescence microscope 26 hours afterward. Observation results when HEK293 cells are used are shown in FIGS. 8 to 10, and observation results when HeLa cells are used are shown in FIGS.

(result)
As shown in FIGS. 8 to 10, when HEK293 cells are used, expression of EGFP is not observed in cells even when EGFP is introduced alone, but SVter-CMVp and EGFP, or βGlopAter-CMVp and EGFP It was revealed that EGFP was expressed in the cells. Similarly, as shown in FIGS. 11 to 13, when HeLa cells are used, EGFP expression is not observed in cells even when EGFP is introduced alone, but SVter-CMVp and EGFP, or βGlopAter- When CMVp and EGFP were introduced, it was revealed that EGFP was expressed in the cells. SVter-CMVp and EGFP, although the expression level is inferior compared to the case where SVter-CMVp-EGFP, βGlopAter-CMVp-EGFP, CMVp-EGFP-SVter in which EGFP is previously bound to the promoter sequence and / or terminator sequence are introduced, or When βGlopAter-CMVp and EGFP are introduced, EGFP expression is expressed at a level that can be analyzed by fluorescence observation, and the use of the linear double-stranded DNA of the present invention requires complicated work and time. It became clear that the expression analysis of the gene introduced into the cell became possible.

  According to the present invention, when a gene or the like is expressed in a cell, a step of linking a promoter sequence and a sequence encoding a gene, a step of linking a sequence encoding a gene and a terminator sequence, a plasmid A manufacturing process is not necessary. Therefore, it can be used for analysis of genes, promoters and terminators.

Claims (6)

A linear double-stranded DNA comprising a terminator sequence and a promoter sequence in sequence, wherein the number of bases between the 3 ′ end base of the terminator sequence and the 5 ′ end of the promoter sequence is 0 to 3000 A linear double-stranded DNA. The linear double-stranded DNA according to claim 1, which does not contain a plasmid replication origin. The linear double-stranded DNA according to claim 1 or 2, wherein the terminator sequence is a sequence derived from SV40 terminator or rabbit β-globin terminator. The linear double-stranded DNA according to any one of claims 1 to 3, wherein the promoter sequence is a human cytomegalovirus (CMV) promoter sequence. A method of expressing RNA derived from the DNA sequence for RNA expression, wherein the linear double-stranded DNA according to any one of claims 1 to 4 and the DNA sequence for RNA expression are introduced into a cell. 6. The method of claim 5, wherein the cell is a mammalian cell.