JP5283044B2 - Vector and its use for highly amplifying target genes extrachromosomally - Google Patents

Abstract

It is intended to provide a vector DNA for amplification outside the chromosome of a host cell and a method for amplifying a target gene using the vector. The vector according to the invention comprises a mammalian replication initiation region which functions in a eukaryotic cell and a nuclear matrix association region and is characterized in that it is linear and at least one end is in the form of a hairpin structure. By using the vector of the invention, a target gene can be highly amplified in the DM form, i.e., extrachromosomally. Accordingly, it is considered that a level of a protein expressed from the target gene also increases in proportion to an increase in the number of amplified copies of the target gene without suffering repetitive sequence-dependent transcriptional repression.

Description

  The present invention provides a vector for highly amplifying a target gene in a mammalian cell outside the chromosome of the host cell, and a vector for highly amplifying the target gene outside the chromosome of the host cell using the vector. Regarding the method. More specifically, when a desired gene is amplified using the “advanced gene amplification system” developed by the present inventors, the vector can amplify the target gene without being incorporated outside the chromosome of the host cell. And a gene amplification method using the vector. Furthermore, this invention relates to the gene expression method using the vector concerning the said invention, and the kit used for the said gene expression method.

The inventor of the present invention uses a human-derived cancer cell (hereinafter referred to as “IR / MAR plasmid”) having a mammalian replication initiation region (IR) and a nuclear matrix binding region (MAR) as a matrix attachment region (MAR). COLO 320 colorectal cancer cell line and HeLa cell line) are introduced by lipofection method, and simply selected using a drug resistance gene (Blasticidine or Neomycin) present on the plasmid,
(1) The ability to amplify the number of intracellular copies of a gene encoding a protein to be expressed (hereinafter referred to as “target gene” as appropriate) to about 10,000 copies; and
(2) The target gene must be highly amplified, whether it is introduced as the same gene construct (cis) or a different gene construct (trans) into the IR / MAR plasmid. (See Patent Document 1 and Non-Patent Document 1). Then, based on this finding, the present inventor used an IR / MAR plasmid and a target gene for mammalian cells (for example, human-derived cancer cells (COLO320 colorectal cancer cell lines and HeLa cell lines), CHO cells, etc. ) And by using a drug resistance gene (Blasticidine or Neomycin) present on the plasmid, the target gene can be amplified to about 10,000 copies (hereinafter referred to as “advanced gene amplification system”). To complete).

  The amplified gene exists in the nucleus of the host cell in the form of DM (double-newt chromosome) which is a large extrachromosomal circular DNA or HSR (homogeneous staining region) which is a huge structure incorporated in the host cell chromosome. To do.

  In most mammalian cells, the target gene is amplified as HSR. However, HSR is a structure that is integrated into the host cell chromosome and, in addition, has a structure in which the target gene is highly repetitive, it undergoes repetitive sequence-dependent gene transcriptional repression. Therefore, there is a problem that the expression level of the protein from the target gene does not increase in proportion to the number of gene amplifications.

  In the method of amplifying the DHFR gene and the target gene in CHO cells, which is widely used as a protein mass production system, the amplification structure is HSR. Therefore, from the amplified copy number of the gene and the target gene, The above-mentioned problem that the amount of protein expression is not proportional has occurred.

On the other hand, when the target gene is amplified outside the chromosome of DM, that is, the host cell, or amplified after being incorporated into DM, the protein expression level from the target gene increases in proportion to the gene amplification copy number (Patent Document 2). reference). However, there is currently no method for controlling the amplified form of the gene DM or HSR.
JP 2003-245083 A (publication date: September 2, 2003) International Publication No. 2006/054561 Pamphlet (International Publication Date: May 26, 2006) Noriaki Shimizu, et al. (2001) Plasmids with a Mammalian Replication Origin and a Matrix Attachment Region Initiate the Event Similar to Gene Amplification.Cancer Research vol.61, no.19, p6987-6990.

  The advanced gene amplification system using the IR / MAR plasmid has made it very easy to amplify the number of target genes per cell in mammalian cells. However, from the viewpoint of protein expression level, as described above, when the vector containing the target gene is amplified in the form of HSR, transcription of the vector DNA is highly repetitive, resulting in transcriptional repression. There is a problem of not reaching.

  On the other hand, when the target gene is amplified in the form of DM, that is, outside the chromosome of the host cell, or incorporated into DM and amplified, the protein expression level from the target gene increases in proportion to the gene amplification copy number (Patent Literature). 2). Therefore, a method capable of amplifying the target gene outside the chromosome has been desired. However, a method for amplifying a vector containing the target gene in the form of DM has not been known to date.

  Therefore, the present invention aims to amplify a vector containing the target gene in the form of DM, that is, outside the chromosome of the host cell. More specifically, it is an object of the present invention to provide a vector DNA that can be amplified outside the chromosome of a host cell, and a method for amplifying a target gene using the vector. Furthermore, the present invention also provides a gene expression method using the vector according to the present invention and a kit used for the gene expression method.

  In order to solve the above problems, the present inventors have conducted extensive studies on the structure and shape of the IR / MAR vector. As a result, the IR / MAR vector capable of amplifying the target gene exclusively outside the chromosome of the host cell. The present inventors have succeeded in identifying the structure and completed the present invention.

  The present invention includes the following inventions in order to solve the above problems. The vector according to the present invention is a vector comprising a mammalian replication initiation region that functions in a eukaryotic cell, and a nuclear matrix binding region, and is linear and has a hairpin structure at least on one end. It is characterized by being.

  The vector according to the present invention may have a hairpin structure at both ends.

  In the vector according to the present invention, the mammalian replication initiation region is derived from any one of the replication initiation regions of the c-myc locus, the dihydrofolate reductase locus, and the β-globin locus. Is preferred.

  In the vector according to the present invention, the nuclear matrix-binding region is preferably derived from any one of the Igκ locus, the SV40 early region, and the nuclear matrix-binding region of the dihydrofolate reductase locus.

  On the other hand, a method according to the present invention is a method for amplifying a target gene in a mammalian cell, comprising the step of introducing the vector according to the present invention and the target gene into the mammalian cell. It is said.

  The method according to the present invention may be a method in which the target gene and the vector are arranged in cis and introduced into mammalian cells. Further, the method according to the present invention may be a method in which the target gene and the vector are arranged in trans and introduced into a mammalian cell.

  The transformed cell according to the present invention is a transformed cell in which the vector according to the present invention and the target gene are introduced into a mammalian cell.

  The target gene expression method according to the present invention includes a step of culturing the transformed cell according to the present invention.

  The target gene expression method according to the present invention includes a step of culturing the transformed cell according to the present invention in a medium containing any one or more of a histone deacetylase inhibitor and a DNA methylation inhibitor. The method of including may be sufficient.

  The kit according to the present invention is a kit for amplifying a target gene, and is characterized by including the vector according to the present invention.

  The present invention also includes a kit for expressing a target gene comprising the vector according to the present invention and any one or more of a histone deacetylase inhibitor and a DNA methylation inhibitor.

  According to the present invention, the target gene can be amplified in DM form, that is, extrachromosomally. Therefore, unlike the case of amplification in the form of HSR incorporated in a host cell, it is possible to enjoy the advantage that the amount of the target protein to be obtained increases as the target gene is amplified.

  Therefore, the present invention has an effect that it is possible to establish a mass production system of the target protein that overcomes the problems of the conventional advanced gene amplification system.

  Other objects, features, and advantages of the present invention will be fully understood from the following description. The advantages of the present invention will become apparent from the following description with reference to the accompanying drawings.

It is a structure schematic diagram of the vector of this invention, A is bilateral hairpin DNA, B is single-sided hairpin DNA. In the vector structure diagram, a black solid line indicates a single-stranded DNA, and a gray portion indicates a hydrogen bonding region existing between complementary DNA strands. 1 is a schematic diagram showing a method for constructing a vector used in Example 1. FIG. In the vector structure diagram, a black solid line indicates a single-stranded DNA, and a gray portion indicates a hydrogen bonding region existing between complementary DNA strands. It is the fluorescence-microscope image which detected the vector arrangement | sequence by the FISH method about the metaphase nuclear metaphase spreading sample of the COLO 320DM cell which transformed the vector of this invention and was selected with the blasticidin for 5 weeks. It is the fluorescence-microscope image which detected the vector arrangement | sequence by the FISH method about the metaphase nuclear metaphase spreading sample of the COLO 320DM cell which transformed the vector of this invention and selected with blasticidin for 5 weeks. Arrows represent representative examples of locations where vector sequences have been detected. A plasmid sequence signal found in interphase nuclei after introducing a one-sided hairpin DNA prepared based on a plasmid with IR and MAR (pSFV dhfr) or a plasmid without IR and MAR (pSFV-V) It is the graph which showed the frequency of. The amount of signal is shown by classifying from + to +++ in ascending order of signal. It is a graph which shows the result of having examined how the plasmid form (closed ring (supercoil), linear form, one-sided hairpin, both-sided hairpin) to introduce | transduces influences formation of an amplification structure. FIG. 3 is a schematic diagram showing a method for constructing an IR / MAR plasmid having a hairpin structure on both sides (referred to as “double-sided hairpin DNA”) used in Example 2. (A) is the figure which shows the result of Example 2, (A) is d2EGFP expression level (p (DELTA) BM-d2EGFp, and is described as "ccc" in a figure) of the transformed cell which introduce | transduced the closed circular plasmid, and double-sided hairpin DNA. It is a histogram which shows the d2EGFP expression level (it describes with "hairpin" in a figure) of the introduce | transduced transformed cell, (B) is a closed circular plasmid (p (DELTA) BM-d2EGFp, "ccc" in a figure) which is a comparison object. Shows the result of analyzing the expression level of d2EGFP with a cell sorter, and (C) shows the result of analysis of the expression level of d2EGFP with a cell sorter. Indicates. It is a figure which shows the result of Example 3, and is the transformed cell (it describes with "hairpin" in a figure) by which the double-sided hairpin DNA was introduce | transduced, and the transformed cell by which the closed circular plasmid which does not have a hairpin structure was introduce | transduced ( (pΔBM-d2EGFp, expressed as “ccc” in the figure) is a histogram showing the results of measuring the expression level of GFP using a cell sorter for the cells of the polyclonal cells that have passed 5 days after the addition of the inhibitor. In addition, the result in which the inhibitor is not added is described with "control" in the figure.

  Hereinafter, the present invention will be described in detail. However, the scope of the present invention is not limited to these descriptions, and modifications other than the following examples can be made as appropriate without departing from the spirit of the present invention. Moreover, all the well-known literatures described in this specification are used as reference in this specification.

<1. Gene amplification method of the present invention>
One embodiment of the present invention relates to a method for amplifying a gene of interest. The above method is referred to as “the gene amplification method of the present invention”.

  Here, the gene amplification method of the present invention comprises a mammalian replication initiation region that functions in a eukaryotic cell, and a nuclear matrix binding region, the shape of which is linear, and either or both of its ends. In which a vector having a hairpin structure (hereinafter referred to as “the vector of the present invention”) and a target gene encoding a useful protein are introduced into mammalian cells (hereinafter referred to as “introduction step”). is there.

  Here, the “target gene” means a gene encoding a protein to be expressed. The target gene is not particularly limited, and a polynucleotide encoding a desired protein may be appropriately selected and employed. The polynucleotide which is the target gene may be obtained using a known technique such as PCR based on the base sequence information.

  By carrying out the gene amplification method of the present invention, the target gene is amplified on an extrachromosomal double-new chromosome (hereinafter referred to as “DM” where appropriate) and / or in the form of DM, that is, extrachromosomally. That is, if an amplification structure is formed, it can be determined that the target gene is amplified. The method for detecting whether or not the amplified structure is formed in the clone of the transformed cell is not particularly limited. For example, a known FISH method (fluorescence in situ hybridization) is performed on chromosomes at mitosis, It can be determined by detecting a gene introduced into a mammalian cell. Those skilled in the art can easily make the above determination. A specific method for carrying out the FISH method is not particularly limited, and a conventionally known method may be appropriately selected and adopted.

  In addition to the introduction step, the gene amplification method of the present invention includes a step of separating mammalian cells into which a target gene and a vector have been introduced (hereinafter referred to as “selection step”), and a selection step. In addition, a culture step for culturing mammalian cells (ie, transformed cells) (hereinafter referred to as “culture step”) may be included.

  Moreover, the target gene can be expressed by culturing the transformant. That is, it can be said that the present invention includes a method of expressing a target gene.

  In addition, a method for producing a protein encoded by a target gene is constructed by combining a method for purifying the target protein produced by the above culture step (hereinafter referred to as “purification step”) with the gene amplification method of the present invention. Can do. Hereinafter, the method according to the present invention will be described step by step.

[1-1. Introduction process)
The introduction step in the gene amplification method of the present invention is a step of introducing the vector of the present invention described later and the target gene into mammalian cells.

  The mammalian replication initiation region and mammalian nuclear matrix binding region contained in the vector of the present invention are particularly limited as long as they are replication initiation regions and nuclear matrix binding regions that function in eukaryotic cells including mammals. It is not a thing. Examples of the mammalian replication initiation region include replication initiation regions derived from the c-myc gene locus, the dihydrofolate reductase (DHFR) gene locus, the β-globin gene locus, and the like. The replication initiation region derived from the c-myc locus (hereinafter referred to as “c-myc locus-derived replication initiation region” as appropriate, “McWhinney, C. et al., Nucleic Acids Res. Vol. 18, p1233-1242”). (1990) "and the replication initiation region of the dihydrofolate reductase locus is referred to" Dijkwel, PA et al., Mol. Cell. Biol. Vol. 8, p5398-5409 (1988) ". For the replication initiation region of the β-globin locus, see “Aladjem, M. et al., Science vol. 281, p1005-1009 (1998)”.

  Examples of the nuclear matrix-binding region include polynucleotides derived from a nuclear matrix-binding region such as Igκ locus, SV40 early region, dihydrofolate reductase locus, and the like. Regarding the nuclear matrix-binding region of the Igκ locus, see “Tsutsui, K. et al., J. Biol. Chem. Vol. 268, p12886-12894 (1993)”. See also “Pommier, Y. et al., J. Virol., Vol 64, p419-423 (1990)” for the nuclear matrix binding region of the SV40 initial region. See also “Shimizu N. et al., Cancer Res. Vol. 61, p6987-6990” for the nuclear matrix-binding region of the dihydrofolate reductase locus.

  The hairpin structure (also referred to as “hairpin loop structure”) present at the end of the vector of the present invention may be achieved by any means. For example, it may be achieved by ligating an oligonucleotide capable of taking a hairpin structure to the end of the vector of the present invention. The base sequence constituting the hairpin structure is not particularly limited as long as it has a base sequence capable of forming a hairpin structure.

  In the present specification, unless otherwise specified, the mammalian replication initiation region is denoted as “IR”, the nuclear matrix binding region as “MAR”, and the oligonucleotide having a hairpin structure as “hairpin DNA”. FIG. 1 shows a schematic diagram of the structure of the vector of the present invention. FIG. 1A shows double-sided hairpin DNA, and B shows single-sided hairpin DNA. In the vector structure diagram, a black solid line indicates a single-stranded DNA, and a gray portion indicates a hydrogen bonding region existing between complementary DNA strands. The vector shown in FIG. 1 may contain a drug resistance gene, a target gene, a signal sequence, and the like.

  The gene amplification method of the present invention is characterized by using a vector having IR, MAR, and hairpin DNA ends. Here, the length of the hairpin DNA is not particularly limited, but is preferably at least 30 bp or more at the single-stranded stage. In addition, the single-stranded DNA is preferably bent by generating a hydrogen bond between complementary sequences and has a double-stranded hairpin structure of 15 bp or more.

  The vector of the present invention used in the introduction step of the gene amplification method according to the present invention may be any vector that has the aforementioned IR and MAR and has a hairpin DNA at the end of the linear DNA. Includes sequences necessary for cloning in E. coli, drug resistance genes (blasticidin resistance gene, neomycin resistance gene, hygromycin resistance gene, etc.) or green fluorescent protein gene as a selection marker (marker protein) In addition, a target gene encoding a useful protein may be included. By using these selection markers as indices, mammalian cells into which the vector of the present invention has been introduced can be selected.

  Moreover, it is preferable that the target gene introduced in the introduction step of the gene amplification method according to the present invention is controllably linked to a promoter. The promoter is not particularly limited as long as it functions in a mammalian cell to be introduced. A promoter (this book) whose transcription activity is activated or inactivated by a predetermined operation using a transcription factor or the like. It may be a “transcriptional activity-regulated promoter” in the specification, or a constitutive promoter in which transcriptional activity is constantly activated. The “transcriptional activity-regulated promoter” is not particularly limited as long as it has the above-mentioned characteristics. For example, commercially available products such as TRE promoter (Clontech) and T-REX promoter (Invitrogen) are available. It can be used in the method according to the invention. As the constitutive promoter, CMV promoter, SV40 promoter, SRalpha promoter, LTR promoter, MMTV promoter and the like can be used.

  In the introduction step of the gene amplification method according to the present invention, the vector of the present invention and the target gene are simultaneously introduced into mammalian cells. By doing so, the target gene is highly amplified in mammalian cells.

  The mammalian cells are particularly preferably human colon cancer COLO 320DM cells (obtained from, for example, ATCC CCL-220). However, it is not particularly limited, and examples thereof include various tumor cells such as CHO-K1 cells (source: for example, ATCC CCL-61, RIKEN RCB0285, RIKEN RCB0403, etc.). However, as the mammalian cells, tumor cells having infinite proliferation ability are particularly preferable. Examples of the tumor cells include HeLa cells (source: eg, ATCC CCL-2, ATCC CCL-2.2, RIKEN RCB0007, RIKEN RCB0191, etc.), human colon cancer COLO 320HSR cells (source: eg, ATCC). CCL-220.1), NS0 cells (source: RIKEN RCB0213) and the like. For human colon cancer COLO 320DM cells, see "Shimizu, N. et al. Nat. Genet., 12: 65-71, 1996."

  The introduction of the vector of the present invention and the target gene into mammalian cells is not particularly limited as long as both are introduced into mammalian cells at the same time. It may be introduced as a construct or may be introduced as a separate gene construct. Here, the former is referred to as “arranging the vector of the present invention and the target gene in cis”, and the latter is referred to as “arranging the vector of the present invention and the target gene in trans”. In the former case, one gene construct may be introduced into a mammalian cell, and there is an advantage that the operation is easy. On the other hand, in the latter case, since the size of each gene construct can be reduced, there is an advantage that high gene transfer efficiency can be obtained.

  The form of the gene construct may be a plasmid or a cosmid. In addition, the method of introducing the vector of the present invention and the target gene into mammalian cells is not particularly limited, and a known method such as lipofection, electroporation, or particle gun may be appropriately selected and employed. .

  When the vector of the present invention and the target gene are introduced as separate gene constructs, it is preferable that each gene construct contains a gene encoding a selection marker. This is because it is possible to select mammalian cells into which the polynucleotide has been introduced. At this time, it goes without saying that the selection marker contained in the vector of the present invention is preferably different from the selection marker contained in the gene construct containing the target gene.

[1-2. Selection process)
The “selection step” in the gene amplification method of the present invention is a step of separating mammalian cells into which a target gene and a vector have been introduced. More specifically, this step involves the latter cell from a polyclonal population of cells that contain mammalian cells into which the gene of interest and vector have not been introduced, and cells into which the gene of interest has been introduced and vector. It is a process of selecting. In the case where this step is performed using drug resistance as an index, a step of culturing mammalian cells in a medium may be included, but in this step, mammalian cells into which the target gene and vector have not been introduced, and In contrast to culturing a mixture of cells containing mammalian cells into which the target gene and vector have been introduced, cells that have already been selected as mammalian cells into which the target gene and vector described below have been introduced However, the two processes are clearly different. By such a selection step, a mammalian cell in which the target gene introduced into the mammalian cell is highly amplified can be selected.

  The specific method of the selection step is not particularly limited. For example, when a drug construct is included in a gene construct used when introducing a target gene and a vector into a mammalian cell, the drug What is necessary is just to select the mammalian cell which introduce | transduced the target gene and the vector using resistance.

  The selection step in the gene amplification method of the present invention can also be performed by detecting a target gene or vector or a nucleotide fragment thereof contained in a mammalian cell by PCR or Southern blotting. Moreover, the specific method of the said drug resistance, PCR method, and Southern blot method is not specifically limited, A well-known method can be utilized suitably.

[1-3. (Culture process)
The “culturing step” in the gene amplification method of the present invention is a step of culturing mammalian cells (ie, “transformed cells”) that have already been selected by the above selection step. By such a culture step, a mammalian cell into which the target gene has been introduced and highly amplified can be grown, and the target gene is encoded by expressing the target gene by a predetermined operation (transcription induction operation or the like). Protein can be produced.

  The specific method of the culture step is not particularly limited, and may be appropriately adopted after examining optimum conditions for mammalian cells to be cultured.

  In particular, it is possible to further improve the expression level of the target gene by including at least one of histone deacetylase inhibitor and DNA methylation inhibitor in the medium used in the culture process. We have found that (see Example 3). Therefore, in order to further enhance the expression of the target gene by culturing the transformed cells, it can be said that it is preferable to include one or more of a histone deacetylase inhibitor and a DNA methylation inhibitor in the medium. . Histone deacetylase inhibitors increase the histone acetylation level, and DNA methylation inhibitors decrease the DNA methylation level to release the epigenetic expression suppression of the target gene. Thus, the target gene can be highly expressed.

  Histone deacetylase inhibitors and DNA methylation inhibitors are not particularly limited, but as histone deacetylase inhibitors, butyrate, Trichostatin A (TSA), MS-275, Oxamflatin, DMSO, etc. Examples of the DNA methylation inhibitor include 5-aza-2'-deoxycytidine, 5-aza-2'-cytidine and the like.

  The amount of each inhibitor added to the medium may be adopted after examining the amount of addition that improves the expression level of the target gene within a range that does not affect the growth of the transformed cells to be cultured.

[1-4. Purification process]
The “purification step” in the present invention is a method for purifying the target protein produced by the culture step.

  As a specific method of protein purification in this purification step, for example, mammalian cells are suspended in a buffer solution such as PBS (Phosphate Buffered Saline), then the cells are disrupted with a homogenizer or an ultrasonic wave, and centrifuged. And collect the supernatant. A surfactant for promoting protein solubilization, a reducing agent for stabilizing the three-dimensional structure of the protein, and a protease inhibitor for preventing protein degradation can be appropriately added to the buffer solution. . As the surfactant, CHAPS (3-[(3-cholaminopropyl) -dimethylamino-1-propanesulfonate], Triton X-100, Nikkol, n-octyl glycoside, etc. can be used. Can use DTT (dithiothreitol), DET (dithioerythritol), etc. Moreover, aprotinin and leupeptin can be used as the protease inhibitor.

  The protein encoded by the target gene (“target protein”) can be purified from the supernatant using column chromatography such as affinity chromatography, ion exchange chromatography, filtration chromatography or the like. Further, unnecessary salts can be removed by dialyzing the purified protein solution against an appropriate buffer. The protein purification step is preferably performed under low temperature conditions in order to suppress protein degradation. In particular, it is preferable that the purification step is performed at 4 ° C.

In addition, the specific method of the said refinement | purification process is not this limitation, A well-known method can be utilized suitably.
<2. Vector and Kit of the Present Invention>
The present invention includes the above-described vector of the present invention, a gene amplification kit comprising the vector (hereinafter referred to as “gene amplification kit of the present invention”), and a gene comprising the vector. An expression kit (hereinafter referred to as “gene expression kit of the present invention”) is also included.

  For the description of the vector of the present invention, the description of the vector of the present invention in the section <1. Gene amplification method of the present invention> is incorporated.

  The gene amplification kit and gene expression kit of the present invention are characterized by including the vector of the present invention. The kit of the present invention is not limited to the above configuration as long as the gene amplification method according to the present invention can be carried out, and may include other configurations. For example, a buffer necessary for performing transformation, a restriction enzyme for inserting a target gene into a vector, vials, tubes, and the like can be included in the kit of the present invention. The gene expression kit of the present invention further comprises any one or more of a histone deacetylase inhibitor and a DNA methylation inhibitor, thereby constructing a kit capable of further expressing the gene expression level. be able to.

  Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited thereto.

<Example 1>
[Vector used in Example 1]
The vector of the present invention used in this example was constructed based on a plasmid (pSFV dhfr) having IR and MAR derived from the DHFR locus. A control plasmid (pSFV-V) without IR and MAR was also constructed in the same manner. These plasmids are the plasmid DNAs already described in “N. Shimizu, et al. (2001) Cancer Research, vol. 61, p6987-6990”. A specific method for constructing the vector of the present invention is described below.

  First, linear DNA was obtained by simultaneously cleaving the above two plasmid DNAs with two restriction enzymes, Ase I and Mlu I, respectively. The linear DNA was mixed with a synthetic oligonucleotide having a hairpin structure having a large excess of moles having an Ase I cleavage site, and ligation was performed using Ligation high Kit (Toyobo Co., Ltd.). This ligation product was referred to as “bilateral hairpin DNA”, and the DNA further digested with Mlu I was referred to as “single hairpin DNA”. An order for the synthesis of an oligonucleotide having a hairpin structure was received from a DNA synthesis contractor. The sequence of the hairpin oligonucleotide used in this example is shown in SEQ ID NO: 1 (taatatgctgcactgacgtcagtgcagcatat). See Figure 1 for the structures of these plasmids and Figure 2 for their construction.

〔experimental method〕
The following experiment was performed using the plasmid prepared above.

  In this example and reference examples (referred to as “Examples”), the above plasmid was introduced into a cell, a transformed cell transformed with the plasmid was selected, and the transformed cell was transformed into the transformed cell using the FISH method. The form of the amplified structure was examined.

  The gene introduction method used in this example and the like is as follows. First, “bilateral hairpin DNA” and “single hairpin DNA” constructed by the above method were introduced into cells.

  The cell into which the gene is introduced is COLO 320DM, which is a human colon cancer cell line. The cell line was obtained from the site described in “N. Shimizu, et al. (2001) Cancer Research, vol. 61, p6987-6990” and cultured under the same conditions as described above. In COLO 320DM, many endogenous DMs are generated by amplification of the c-myc gene.

  The transformed cells were selected by culturing the cells in a selective medium supplemented with blasticidin to a final concentration of 5 μg / ml two days after gene transfer. As for the selective medium, half of the selective medium in culture was replaced with the freshly prepared selective medium every 3 to 5 days.

  The FISH method, the preparation of a probe for detecting a transgene used in the FISH method, and the metaphase spreading sample collect a part of cells in culture 5 weeks after the gene transfer. And according to the method described in “N. Shimizu, et al. (2001) Cancer Research, vol. 61, p6987-6990”. The probe is biotinylated and can be detected by streptavidin bound with FITC (fluorescein isothiocyanate) that emits green fluorescence. In addition, DNA was counterstained with PI (propidium iodide) that emits red fluorescence. Inverted fluorescence microscope (ECLIPSE) equipped with a filter set appropriate for detecting fluorescent dyes and a 100 × objective lens (Nikon Plan Fluor, NA 1.30 oil) were placed on the slide glass fluorescently labeled by the FISH method. Using the Fuji FinePix S1Pro digital camera (Fuji Film Co. Tokyo), which was observed with TE2000-U, Nikon), and connected with the microscope, a picture of the transgene and DNA in the cell as a digital image I took a picture. Each of the obtained images was synthesized using image analysis software Adobe (registered trademark) Photoshop (registered trademark) version 4.0J (Adobe Systems Inc).

  After introducing the constructed vector “bilateral hairpin DNA” and “single hairpin DNA” of the present invention into COLO320 DM cells, a metaphase spreading sample was prepared for a group of cells selected by blasticidin for 5 weeks, and this was performed by FISH method. The amplification site of the vector of the invention was visualized (see FIGS. 3 and 4).

  FIG. 3 shows an image of the interphase nucleus. A very small signal (the part that appears white) is very numerous in almost all interphase nuclei. In order to know the amplification form of the vector of the present invention in the host cell in more detail, a chromosomal image in which the vector sequence of the present invention is detected in dividing cells is shown in FIG. As indicated by the arrows in FIG. 4, the signal of the vector of the present invention exists outside the chromosome, and many plasmid signals are attached to the large DM originally present in the cell. Existed.

  The frequency of cells in which the vector of the present invention was highly amplified extrachromosomally was graphed based on data from two experiments (see FIGS. 5 and 6). FIG. 5 shows the result of introducing a single-sided hairpin DNA prepared based on a plasmid having IR and MAR (pSFV dhfr) and a plasmid having no IR and MAR (pSFV-V) into cells. Based on the approximate number of plasmid sequence signals found in interphase nuclei, they were classified into + to +++ in ascending order of signal, and the frequency was shown in a graph. As a result, extrachromosomal signals were detected in a very high proportion of cells, but the frequency was significantly higher in the plasmid with IR and MAR than in the plasmid without IR and MAR. FIG. 6 shows the results of examining how the form of the plasmid vector DNA to be introduced (closed circle (supercoil), linear, single-sided hairpin, double-sided hairpin) affects the formation of the amplified structure. As a result, in the closed ring (supercoiled) vector, the formation of HSR was recognized as in the results of the experiments conducted by the inventors so far. It was shown that extrachromosomal DM was formed very efficiently by adopting the structure.

<Example 2>
Example 2 was performed using the same method as Example 1 unless otherwise stated.

[Vector used in Example 2]
The vector used in this example has IR and MAR derived from the human DHFR locus, and cleaves a plasmid (pΔBM-d2EGFP) having a GFP gene with Mlu I under the control of a constitutive promoter (CMV promoter). Subsequently, an IR / MAR plasmid having a hairpin structure on both sides (referred to as “bilateral hairpin DNA”) was constructed by ligating synthetic DNA having a hairpin structure at two cleavage ends (see FIG. 7). ). The sequence of the synthetic DNA having the hairpin structure was cgcgatatgctgcactgacgtcagtgcagcatat (34 bp, the base sequence is shown in SEQ ID NO: 2).

  The method for producing the above pΔBM-d2EGFP is as follows. The above pΔBM-d2EGFP is pSFV dhfr (Nonaki Shimizu, et al. (2001) Plasmids with a Mammalian Replication Origin and a Matrix Attachment Region Initiate the Event Similar to Gene Amplification. Cancer Research vol.61, no. 19, p6987-6990.]). First, pSFV dhfr was cleaved simultaneously with two types of restriction enzymes, BamH I and Mlu I. A linear synthetic oligonucleotide having BamH I and Mlu I cleavage sites at both ends and having a multiple cloning site was inserted into the cleavage site by ligation using a Ligation high Kit (Toyobo Co., Ltd.). The linear synthetic oligonucleotide was obtained by annealing a synthetic nucleotide consisting of the base sequences shown in SEQ ID NOs: 3 and 4. This plasmid was named pΔBM-linker. The multicloning site has Kpn I, Asc I, Sal I, Swa I, AsiS I, Sbf I, and BamH I cleavable sites in the direction from the BamH I cleavage terminal to the Mlu I cleavage terminal. However, in the ligation step, the Mlu I cleavage site at the adhesion site is restored, but the BamH I cleavage site is not restored. Next, pΔBM-d2EGFP was prepared by incorporating the d2EGFP gene controlled by the CMV promoter between the AsiS I and Asc I sites of pΔBM-linker.

〔experimental method〕
The bilateral hairpin DNA constructed by the above steps was introduced into COLO320DM cells by the lipofection method. From 2 days after gene introduction, 5 μg / ml blasticidin (manufactured by Funakoshi) corresponding to the blasticidin resistance gene on the plasmid was selected, and stable transformed cells of the polyclonal cells 75 days after the start of the culture were The expression level of GFP was measured using a cell sorter. For comparison, a closed circular plasmid (pΔBM-d2EGFp) was also tested in the same manner.

〔result〕
FIG. 8 shows the result. FIG. 8 (B) shows the result of analyzing the expression level of d2EGFP with a cell sorter for the transformed cells into which the closed circular plasmid (pΔBM-d2EGFp, referred to as “ccc” in the figure) to be compared was introduced. FIG. 8C shows the result of analyzing the expression level of d2EGFP with a cell sorter for the transformed cells into which the double-sided hairpin DNA was introduced. FIG. 8A shows the d2EGFP expression level (pΔBM-d2EGFp, expressed as “ccc” in the figure) of the transformed cells into which the closed circular plasmid was introduced, and d2EGFP of the transformed cells into which the bilateral hairpin DNA was introduced. It is a histogram which shows expression level (it describes with "hairpin" in a figure).

  According to FIG. 8, the cells into which the double-sided hairpin DNA was introduced clearly showed a marked increase in the expression level of the d2EGFP gene as compared with the IR / MAR plasmid having no hairpin structure (about 4 times).

<Example 3>
Example 3 was performed using the same method as Examples 1 and 2, unless otherwise specified.

〔experimental method〕
The bilateral hairpin DNA constructed in Example 2 was introduced into COLO320DM cells by the lipofection method. From 2 days after gene introduction, 5 μg / ml blasticidin (manufactured by Funakoshi) corresponding to the blasticidin resistance gene on the plasmid was selected, and stable transformed cells of the polyclonal cells after 70 days from the start of culture, Various histone deacetylase inhibitors (HDAC inhibitors: butyrate, Trichostatin A (TSA), MS-275, Oxamflatin, DMSO) or DNA methylation inhibitors (5-aza-2'-deoxycytidine: hereinafter referred to as “5-aza Added). Each inhibitor has a final concentration of 2 mM for butyrate (Sigma), 100 nM for TSA (Sigma), 3 μM for MS-275 (Alexis Biochemicals), and 0 μm for Oxamflatin (Calbiochem). 1 μg / ml, DMSO (manufactured by Nacalai Tesque) was added at 1.6% by volume, and 5-aza (manufactured by Sigma) was added at 3 μM. The expression level of GFP was measured using a cell sorter for the cells of the polyclonal cells after 5 days from the addition of the inhibitor. For comparison, a closed circular plasmid (pΔBM-d2EGFp) was also tested in the same manner.

〔result〕
FIG. 9 shows the result. FIG. 9 shows a transformed cell into which double-sided hairpin DNA was introduced (indicated as “hairpin” in the figure) and a transformed cell into which a closed circular plasmid having no hairpin structure was introduced (pΔBM-d2EGFp, in the figure). Shows the results of adding various inhibitors to the experimental system and the results of not adding them (designated as “control” in the figure).

  According to the results of FIG. 9, it is possible to further increase the gene expression level by treating the transformed cells in which the gene expression level is increased by the hairpin DNA on both sides with an HDAC inhibitor or a DNA methylation inhibitor. It was confirmed.

  As described above, according to the method of the present invention, the target gene can be amplified in the form of DM, that is, extrachromosomally. Therefore, the expression amount of the target protein from the target gene increases with the amplification of the target gene. You can enjoy the benefits of Therefore, it is possible to establish a mass production system of the target protein that overcomes the problems of the conventional advanced gene amplification system. Therefore, there is an effect that a desired protein (for example, useful protein) can be produced in a larger amount than the conventional method.

  Therefore, the present invention can be used in various industries that produce proteins, such as pharmaceuticals, chemistry, foods, cosmetics, and fibers.

Claims (11)

A vector for amplifying a gene of interest outside the chromosome of a mammalian cell that is a CHO cell or a tumor cell having infinite proliferation ability,
The vector and the vector capable of reducing the ratio of mammalian cells forming an amplification structure of the target gene in the chromosome relative to the mammalian cell into which the target gene has been introduced, to less than 1%,
A mammalian replication initiation region that functions in eukaryotic cells, and a nuclear matrix binding region;
A vector which is linear and has a hairpin structure at least on one end, and the hairpin structure is a synthetic oligonucleotide.   The vector according to claim 1, wherein both ends have a hairpin structure.   The vector according to claim 1 or 2, wherein the mammalian replication initiation region is derived from any one of a replication initiation region of a c-myc locus, a dihydrofolate reductase locus, and a β-globin locus.   The vector according to any one of claims 1 to 3, wherein the nuclear matrix-binding region is derived from any one of an Igκ locus, an SV40 early region, and a nuclear matrix-binding region of a dihydrofolate reductase locus. A method for amplifying a gene of interest outside a chromosome of a mammalian cell which is a CHO cell or a tumor cell having infinite growth ability,
A method comprising introducing the vector according to any one of claims 1 to 4 and a target gene into a mammalian cell.   The method according to claim 5, wherein the target gene and the vector are arranged in cis and introduced into a mammalian cell. The vector according to any one of claims 1 to 4 and a target gene are introduced into a mammalian cell which is a CHO cell or a tumor cell having infinite proliferation ability,
A transformed cell that amplifies a target gene outside the chromosome of a mammalian cell.   A method for expressing a target gene, comprising a step of culturing the transformed cell according to claim 8.   A method for expressing a target gene, comprising a step of culturing the transformed cell according to claim 8 in a medium containing any one or more of a histone deacetylase inhibitor and a DNA methylation inhibitor.   A kit for amplifying a target gene outside the chromosome of a mammalian cell which is a CHO cell or a tumor cell having infinite proliferation ability, comprising the vector according to any one of claims 1 to 4. A featured kit. A kit for amplifying and expressing a gene of interest outside the chromosome of a mammalian cell which is a CHO cell or a tumor cell having infinite growth ability,
A vector according to any one of claims 1 to 4;
A kit comprising one or more of a histone deacetylase inhibitor and a DNA methylation inhibitor.