JP2006055175A - Method for highly amplifying target gene in mammalian cells and vector for performing the method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for mass production of protein products by amplifying a target gene in mammalian cells irrespective of its chain length or structure, wherein the method is available to any mammalian cells and convenient. <P>SOLUTION: A vector having a mammalian replication point and a nuclear matrix binding region is used. The target gene desired to be amplified is arranged in a cis (an identical structure with the vector) or trans (a different structure from the vector) conformation to such vector and introduced into the mammalian cells. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for highly amplifying a target gene in mammalian cells, and a vector used for carrying out the method.

  The only known method for increasing the intracellular copy number of a transgene in a mammalian cell is to use a Chinese hamster ovary (CHO) cell or the like as a host, and the target gene is dihydrofolate reductase (DHFR). A dihydrofolate reductase) gene, which is selected by adding methotrexate (MTx), an inhibitor of the dihydrofolate reductase gene product, to the culture medium. This method is now widely used for mass production of useful substances such as pharmaceuticals and has become an important technique.

  However, this method has a problem that the applicable host cells are limited to CHO cells and the like and lacks versatility. In addition, it is necessary to select for a long period of time using methotrexate, which is a selective drug, and it is necessary to consider increasing the concentration gradually while considering the cytotoxic effect of methotrexate. And skill is also required. Further, this method is applicable only to a relatively short DNA as a target gene, and there is a problem that the application range is narrow.

  In general, proteins are often subjected to various modifications such as phosphorylation and acetylation depending on the type of the protein, and even with the same protein, the type of modification and the site to be modified depending on conditions such as the cell in which it is expressed, the time of expression, and stimulation of the cell. Are known to be different. Therefore, when the gene encoding the target protein to be expressed is to be expressed ectopically and / or chronologically in a host cell derived from a tissue where the protein is not originally expressed, There is no guarantee that it will accurately reflect the nature of the. In general, specific modifications are often required for the functional expression of proteins. Therefore, it is preferable that the host cell for expressing the target protein can be appropriately selected according to the protein gene to be introduced. Therefore, as in the prior art described above, the fact that applicable cells are limited to CHO cells or the like is a great obstacle to producing useful proteins such as pharmaceuticals in mammalian cells.

  The present invention has been made in view of the above circumstances, and the problem to be solved is that a wide range of mammalian cells can be used as a host, and experience and skill are not required. Regardless, it is a simple procedure to amplify.

  The present invention provides a vector useful for achieving this object and a method for amplifying a target gene in a mammalian host cell using the vector.

  According to one aspect of the present invention, a vector for highly amplifying and / or expressing a gene of interest in mammalian cells, comprising a replication origin that functions in eukaryotic cells and a nuclear matrix-binding region. A vector is provided that is capable of autonomous replication in transfected cells and stably distributed to daughter cells.

  According to another aspect of the present invention, there is provided a method for highly amplifying and / or expressing a target gene in a mammalian cell, wherein the target gene is placed in cis with respect to the vector of the present invention. There is provided a method characterized in that it is transfected into a suitable mammalian host cell for amplification and / or expression.

  According to another aspect of the present invention, there is provided a method for highly amplifying and / or expressing a target gene in mammalian cells, wherein the target gene is placed in trans with respect to the vector of the present invention, There is provided a method characterized in that this is transfected into a suitable mammalian host cell together with said vector for amplification and / or expression.

  In the present invention, “arranging the target gene in cis with respect to the vector” means that the target gene is disposed in the vector. Also, “arranging the target gene in trans with respect to the vector” means that the target gene is disposed in a different structure from the vector.

  The method according to the present invention can be applied to any mammalian cell, and the target gene can be amplified from several thousand to 10,000 copies or more in the cell by a very simple operation. Furthermore, since the method according to the present invention can amplify a gene of any length in principle, a wide range of target gene regions on the genome can be amplified.

  Hereinafter, the present invention will be described in detail. In the following description, various known documents are referred to by numbers in parentheses. Bibliographic items of these documents are listed at the end of the detailed description section.

  The present invention relies on the findings observed in gene amplification phenomena in tumor cells and the like. Gene amplification is a major mechanism by which tumor cells acquire unlimited growth or drug resistance (1). Cytogenetically speaking, amplified genes are extrachromosomal double chromosomal chromosomes (DM) in vivo. ) Most frequently detected (but long-term in vitro passage usually leads to a dominant growth of cells with a homogeneously stained region (HSR) of chromosomes). Previous studies have shown that removal of amplified genes on DM from tumor cells reverts from a state exhibiting tumor phenotype and cell differentiation to a normal state (2-4). This type of removal process is mediated by selective uptake of DM into micronuclei released from dividing cells (3, 5, 6). It is understood that such a micronucleation process is closely related to the intracellular behavior of DM during the cell cycle (7). DM is composed of acrocentric circular DNAs of various sizes (8) and, despite being acentromeric, adheres to mitotic chromosomes and is stably separated into daughter cells ( 7-9). Several recent important findings include several viral nuclear plasmids, including bovine papillomavirus (10), EB virus (11), Kaposi's sarcoma-associated herpesvirus (12), and SV40 (13). However, it has been shown to utilize similar mechanisms during cell division. Furthermore, recent interesting studies have shown that plasmids carrying the EB virus replicon can be integrated into DM in tumor cells (14). In addition, the establishment of the attachment process in the plasmid having the SV40 origin of replication requires the expression of the viral large T antigen, but the large T gene encoded by the plasmid is a cell-derived nuclear matrix-binding region (MAR; It has been shown that separation can be achieved if a matrix attachment region) is substituted (13). Indeed, nuclear matrix-binding regions are frequently found near the origin of replication in the mammalian genome, suggesting a role for matrix attachment in genome replication (15).

  In the present invention, based on the basic knowledge recognized in the gene amplification phenomenon described above, the vector into which the target gene has been introduced by using a vector having a mammalian replication origin and a nuclear matrix binding region (MAR) De novo formation of DM was achieved. The vector of the present invention amplifies a long chain target gene in mammalian cells, and enables separation of the vector having the amplified gene into stable daughter cells, and as a result, accompanied with gene amplification. The effect of increasing protein production can be obtained.

  The present invention has overcome the problems such as versatility, restriction of the nucleic acid chain length of the transgene, and ease of implementation by newly applying a vector having the above characteristics. That is, the vector of the present invention is characterized by having a mammalian replication origin and a nuclear matrix binding region (MAR).

  The mammalian origin of replication and the nuclear matrix binding region may be any combination as long as the target gene introduced into the vector in the mammalian cell is amplified and separated and distributed to the daughter cells. EBV latent origin, c-myc locus, dihydrofolate reductase locus, β-globin locus, etc., and Igκ locus, SV40 early region, dihydrofolate, respectively It has a sequence derived from a nuclear matrix binding region such as a reductase locus.

  In addition, the vector may be a sequence necessary for cloning in Escherichia coli according to the purpose, or a drug resistance gene (blasticidine resistance, neomycin resistance, etc.) or a green fluorescent protein gene as a marker protein. It may have a gene for selecting the transformed cells. Cells transformed with a vector into which these marker proteins have been introduced can be selected by selecting or separating marker protein-expressing cells using drug selection based on drug resistance genes or a cell sorter.

  The vector may be a plasmid or a cosmid as long as it has a mammalian origin of replication and a nuclear matrix binding region.

  The host cell into which the vector is introduced may be a mammalian cell, but preferably a cell line such as COLO 320 cell or Hela cell is used.

  In the present invention, a target gene is amplified in a mammalian host cell by using the above vector. In the first embodiment, the gene of interest is directly integrated into the vector under the control of an appropriate promoter sequence and introduced into a mammalian cell. That is, in this embodiment, the target gene is arranged in cis (the same structure as the vector).

  In the second aspect of gene amplification according to the present invention, a target gene is incorporated together with a suitable promoter into a nucleic acid such as lambda phage, cosmid, etc., which has a sticky end and can be self-circulated, and this is a vector capable of autonomous replication. Mixed with a plasmid or the like) and transfected into a mammalian host cell for introduction. That is, in this embodiment, the target gene is arranged in trans (a structure different from the vector). In this embodiment, two types of nucleic acids may be mixed and transfected together, but the weight ratio of the two is most preferably 1: 1. In this second aspect, the target gene having a longer chain length can be amplified compared to the first aspect.

  Introduction of a vector into cells can be performed by methods well known to those skilled in the art, such as electroporation and lipofection.

  Proteins transcribed and translated from the amplified target gene can be prepared by various methods depending on the purpose of use. For example, the cells may be crushed in an appropriate buffer after recovery, extracted and used as a crude extract, or may be used after purification by various known chromatograph methods.

  Hereinafter, embodiments of the present invention will be described in detail. The following examples do not limit the present invention.

<Materials and methods>
[Plasmid]
The plasmid construct used in FIG. 1 is described. Plasmids pEPBG (11.0 kbp) and pSFVdhfr (11.0 kbp) were a gift from Dr. John Kolman and Dr. Geoffrey M. Wahl (The Salk Institute, San Diego, Calif.). The former plasmid contains a fusion gene of EBV latent origin of replication (OriP) and EBNA-1, plus a green fluorescent protein and a G-associated polypeptide (GFP-GAP). The latter plasmid has a 4.6 kbp fragment containing Oriβ derived from a region 3′-downstream of dihydrofolate reductase (16). The pSFV-V plasmid (6.4 kbp) lacking the origin of replication was constructed from pSFVdhfr by deleting all dihydrofolate reductase derived sequences with NotI digestion. The pNeo.Myc-2.4 plasmid (9.0 kbp; reference 17) was provided by Dr. Michael Leffak (Wright State University, Dayton, OH). This plasmid contains a 2.4 kbp HindIII / XhoI fragment derived from the promoter region of c-myc, and digested with NotI / HindIII to delete almost all the sequence derived from c-myc (pNeo-V (with the origin of replication). Used to build). The pNeo.MycΔSV plasmid (which lacks the nuclear matrix binding region) was constructed by deleting most of the SV40-derived sequences exhibiting nuclear matrix binding activity (18) by BamHI / BsmI digestion. The intronic enhancer nuclear matrix binding region (Intronic enhancer MAR) of the Igκ gene was amplified by PCR using the pG19 / 45 plasmid as a template, and a pAR1 plasmid was constructed from the amplified product (19). The pNeo.MycΔSV AR1 plasmid was prepared by inserting the nuclear matrix binding region sequence (AR1) of pAR1 into pNeo.MycΔSV. This operation replaces the SV40-derived sequence similar to the nuclear matrix binding region.

[Other experimental methods]
Human colorectal COLO 320DM and COLO 320HSR tumor cell lines were acquired and maintained as described in the literature (5).

  The HeLa cell line was obtained from the American Type Culture Collection (CCL-2). All plasmids were purified with Qiagen plasmid purification kit (Qiagen Inc., Valencia, Calif.) And transfected into cells with GenePorter 2 lipofection kit (Gene Therapy Systems, San Diego, Calif.).

  Blasticidine (10 μg / ml; Funakoshi, Tokyo, Japan; for pEPBG, pSFVdhfr, and variants thereof) or 500 μg / ml Neomycin (Life Technologies, Inc., Rockville, MD; pNeo.Myc-2.4 and its variants) were used to select transformants. Biotin-labeled micronuclear probes that detect COLO 320 amplicons were prepared according to the protocol we published (5). Preparation of metaphase samples, preparation of DIG-labeled probes and FISH were performed according to the published protocol (20) (similar to in vitro nuclear matrix binding assay (19)).

<Results and discussion>
Before investigating mammalian replicons that incorporate EB virus replicons into DM, we will examine the most characterized episomal vectors (ie compliant with EB virus replicons (pEPBG)) Was tested. This plasmid replicates autonomously from the viral cis-acting OriP sequence and requires expression of the viral EBNA-1 gene for attachment to the mitotic chromosome (11). We transfected this plasmid into human COLO 320DM tumor cells with a large number of DM and analyzed the stably transformed colony mixture by FISH method. As a result, the sequence of the plasmid was mainly detected in various sizes of DM in some metaphase cells (FIGS. 2, A and B; Table 1). There were no signs of chromosome integration. These observations are similar to the results described in a recent report, indicating that plasmids carrying the EB virus replicon selectively recombine with DM (14). In experiments using mixed colonies, the percentage of cells that incorporated the plasmid into DM was small (13%), but when clones exhibiting high level expression of the GFP-GAP fusion protein encoded by the plasmid were examined. 10 clones out of 10 exhibited integration into DM. This phenomenon suggests that sequences incorporated into DM are expressed at high levels. This result is also consistent with the observation that most of the tumor cell amplification genes are localized in DM (1).

  Similarly, plasmids with mammalian replicons were also incorporated into DM, and we tested the pSFVdhfr and pNeo.Myc-2.4 plasmids with origin sequences for the dihydrofolate reductase (16) and c-myc loci (17), respectively ( Figure 1). We have found that these plasmids are also incorporated into various sizes of DM in COLO 320DM cells and that they are selectively incorporated into micronuclei (FIG. 2C).

  Also, simultaneous detection of plasmid and amplicon sequences showed that the DM with the plasmid sequence was derived from DM present in untransfected COLO 320DM cells (FIG. 2D). The frequency of DM integration in mixed transformed colonies was high in pSFVdhfr (60%) and low in pNeo.Myc-2.4 (4.3%) (how the integrated sequence affects the frequency) Is not clear).

Integration into DM causes the plasmid to recombine with DM (Table 1) when the mammalian origin sequences that require autonomous replication of the plasmid are deleted from the plasmid (pSFV-V and pNeo-V; Figure 1). It is an important finding that it did not occur. We have also found that the transformation efficiency of pSFV-V (~ 30 colonies / 10 ⁶ cells) is very low compared to pSFVdhfr (~ 800 colonies / 10 ⁶ cells), and these facts are autonomous This suggests that the ability to replicate specifically is important for plasmid integration into DM and that this ability increases transformation efficiency. While the question of why autonomously replicating plasmids frequently recombine with DM is an ongoing issue, one hypothesis is based on the fact that plasmids and DM share a common mode of distribution. . DM tends to localize in the periphery of the nucleus during the G1 phase of the subsequent cell cycle, moves to the inside of the nucleus during replication, and then is separated by attaching to the chromosome (14, 20, 21) . Thus, DM and plasmid colocalization that can occur in interphase nuclei may increase the frequency of recombination between these two DNA species.

The nuclear matrix binding region is required for extrachromosomal replication driven by the mammalian origin. There is a report that the nuclear matrix binding region exists near the replication initiation site on the dihydrofolate reductase locus (16), 4.6 kbp of pSFVdhfr. There is no report on the insert. Nevertheless, nuclear matrix binding region search program (22) analysis predicted the potential presence of a potential nuclear matrix binding region within the insert, and the actual predicted fragment was detected in vitro in the nuclear matrix (Fig. 3). Moreover, the nuclear matrix binding region search program could not detect a candidate nuclear matrix binding region in the 2.4 kbp insert derived from the c-myc locus of pNeo.Myc-2.4. However, the plasmid contains a sequence derived from the SV40 early region that exhibits nuclear matrix binding activity (18) and was demonstrated to have nuclear matrix binding activity by our in vitro matrix binding assay (FIG. 3). To investigate the role of nuclear matrix binding region in transformation efficiency and integration into DM, we deleted the SV40-derived nuclear matrix binding region-like sequence of pNeo.Myc-2.4 (this created pNeo.MycΔSV Figure 1), on the other hand, we replaced the site of another construct (pNeo.MycΔSV AR1) with a nuclear matrix binding region from the Igκ gene (19). As expected, by in vitro binding assay, the NcoI / HindIII fragment generated from the former plasmid (pNeo.MycΔSV) does not show nuclear matrix binding activity, whereas that generated from the latter (pNeo.MycΔSV AR1) It was shown to bind (Figure 3). When COLO 320DM cells were transfected with these plasmids, the transformation efficiency was pNeo.MycΔSV (˜90 colonies / 10 ⁶ cells) compared to pNeo.MycΔSV AR1 (˜800 colonies / 10 ⁶ cells). It was very low. Furthermore, the former plasmid (pNeo.Myc ΔSV) was not incorporated into DM, while the latter (pNeo.Myc ΔSV AR1) was incorporated (Table 1; FIG. 2E). These observations indicate that DM incorporation is dependent on the cis-acting nuclear matrix binding region. Thus, the nuclear matrix binding region appears to play an important role in autonomous replication of the plasmid.

[A self-replicating plasmid causes a phenomenon similar to gene amplification]
An unexpected phenomenon was observed when different cell lines (ie COLO 320HSR cells) were used for transfection. This cell line usually does not contain DM, but DM with many plasmid sequences was observed when transfected with pSFVdhfr or pNeo.MycΔSV AR1 (FIGS. 2G and H, respectively) (ie, many microchromatin Pairs were observed at metaphase). Two-color FISH analysis shows that these DMs do not contain the original amplicon sequence found in COLO 320 cells (FIG. 2G), which means that DM occurred de novo. Cells with DM were frequently present only when transfected with a plasmid having both an origin and a nuclear matrix binding region (Table 1). These phenomena were also observed when HeLa (another tumor cell line lacking DM) was used (FIG. 2I). The interpretation that plasmids frequently recombine with endogenous extrachromosomal DNA is a plausible interpretation that can explain this phenomenon. Indeed, many mammalian somatic cells contain extrachromosomal closed-loop DNA that relies on genomic flexibility (23). Our observations directly support a model that suggests that DM occurs by recombination between episomes that cannot be detected under a microscope (24).

  We also found a group of cells exhibiting a very strong plasmid signal along the chromosomal arm similar to the homogenous chromosome staining region (found in tumor cells) (Figure 2F, arrow). Such cell populations were observed only when a plasmid having both an origin and a nuclear matrix binding region was used. Interestingly, the frequency of this cell population was much higher when transfected into COLO 320DM cells compared to COLO 320HSR cells (Table 1). This finding is consistent with a model that suggests that the uniformly stained region is formed by tandem integration of DM into the chromosome arm (24).

  As described above, in the basic research process of the present invention, we have clarified a novel in vitro model in the mechanism of gene amplification that plays a critical role in human tumorigenesis. This model appears to be useful for functional analysis of mammalian origins of replication, since most of the work to date has been done using sequences integrated on chromosomes that are affected by surrounding chromatin. It is.

  In the present invention we have shown by the in vitro model that DM integration or DM production requires a cis-acting origin of replication and a nuclear matrix binding region on the plasmid. This result strongly suggests that recombination requires a plasmid capable of autonomous replication. Such extrachromosomal replication studies are likely to facilitate the development of stable plasmids with mammalian replicons required for gene therapy.

<References>
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18.1233-1242, 1990.18. Pommier, Y., Cockerill, PN, Kohn, KW, and Garrard, WT Identification within the simian virus 40 genome of a chromosomal loop attachment site that contains topoisomerase II cleavage sites. J. Virol., 64: 419-423, 1990.
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  The present invention is useful for producing useful proteins such as pharmaceuticals in mammalian cells by genetic engineering techniques.

FIG. 1 shows the plasmids used in this study. The figure shows the organization of pEPBG, pSFVdhfr and pNeo.Myc-2.4 with mammalian origins of replication from the EB virus replicon (OriP and EBNA-1), the dihydrofolate reductase locus, and the c-myc locus Yes. Other plasmids in this study were constructed from pSFVdhfr or pNeo.Myc-2.4. Intronic sequences derived from the SV40 early region or Igκ gene (AR1) exhibited nuclear matrix binding activity (indicated by black squares). Dihydrofolate reductase Oriβ contains a sequence exhibiting nuclear matrix binding activity therein (see FIG. 3). GFP-GAP (GFP and GAP fusion gene); BS (blasticidin S resistance); Hyg (hygromycin resistance); Neo (neomycin resistance); MMTI (5 'flanking DNA and promoter region of mouse metallothionein). Open squares represent vector regions necessary for bacterial cell growth. FIG. 2 is a fluorescence micrograph showing the results of FISH analysis of tumor cells transformed with a plasmid. COLO 320DM (AF), COLO 320HSR (G and H), and HeLa (I) cells become pEPBG (A and B), pSFVdhfr (C, D, G, and I), or pNeo.Myc ΔSV AR1 (E, F, and H). Stable transformants were selected after 30 days of culture, and metaphase chromosome samples were prepared. A DIG-labeled probe prepared from the plasmid to be transfected was used for hybridization with a chromosome specimen and detected with green fluorescence. D and G show the results of detection of red fluorescence by simultaneously hybridizing a biotin-labeled probe prepared from purified micronuclei of untransfected COLO 320DM cells and a DIG-labeled plasmid probe. White arrows indicate DM (A-E and G-I) or a large signal (F inset) in the interphase nucleus (F) corresponding to a homogeneous staining region at metaphase. Arrowheads (F and H) indicate micronuclei that have accumulated plasmid-derived sequences. DNA in red (AC and I) with propidium iodide or blue (DH) with 4 ', 6-diamino-2-phenylindole (4', 6-diamidino-2-phenylindole) Counterstained. Green and red overlaps are yellow, green and blue overlaps are cyan, and all three overlaps are white. In D, the plasmid sequence (green) coincided with the amplicon sequence (red) localized in DM (detected in blue, white arrow). In G, the amplicon sequence (red) was detected mainly in the marker uniformly stained region in the cells (yellow arrows I, II and III), but DM with the plasmid sequence (shown in green, white arrows) It was not detected in (blue). Bar represents 10 μm. FIG. 3 is a PAGE electrophoresis photograph showing the results of an in vitro matrix binding assay. The plasmids were digested with BamHI / HindIII (pAR1), NcoI / HindIII (pNeo.Myc-2.4, pNeo.Myc ΔSV and pNeo.Myc ΔSV AR1, respectively; see FIG. 1 for restriction enzyme recognition sites), HinfI (pSFVdhfr), It was labeled with [α-32P] dATP by an end-filling reaction. The labeled DNA fragments were incubated with the nuclear matrix in the presence of various concentrations of unlabeled competitor (20: 1 mixture of E. coli DNA and pUC18 DNA). After washing the matrix by repeated centrifugation, DNA fragments bound to the matrix were separated by 5% -PAGE and visualized by autoradiography. Lane 1, input DNA; reaction in the presence of Lanes 2-4, competing DNA (4, 8, 16 μg, respectively). The arrowhead shows a band specifically bound to the matrix. pAR1 is a plasmid used as a control and contains a nuclear matrix-binding region derived from the mouse Igκ gene (arrowhead). The pNeo.Myc-2.4 fragment exhibiting nuclear matrix binding activity has an SV40 early region that has proven to be a nuclear matrix binding region (18). Deletion of this sequence completely destroyed nuclear matrix binding activity (pNeo.MycΔSV). However, insertion of the Igκ nuclear matrix binding region sequence into this plasmid restored nuclear matrix binding activity (pNeo.MycΔSV AR1). The pSFVdhfr fragment (a) bound to the matrix was identified as a 886 bp fragment from the center of the dihydrofolate reductase insert that matches the potential nuclear matrix binding region predicted by the nuclear matrix binding region search program. The identity of fragment (b) was not identified, but it is likely to be interpreted as a fragment derived from the insert as well.

Claims (14)

A method for amplifying a gene of interest in the form of DM and / or HSR, comprising:
A vector comprising the gene of interest, a mammalian replication origin, a nuclear matrix binding region, and a gene for selecting a transformed cell, and the vector in which the gene of interest is placed in cis with respect to the vector Transfection into cells;
Culturing the transfected mammalian cells;
Selecting or separating based on a gene for selecting the transformed cells, and separating mammalian cells in which the target gene is amplified in the form of DM and / or HSR;
Including methods.   The method according to claim 1, wherein the gene for selecting the transformed cell is one or more genes selected from the group consisting of a blasticidin resistance gene, a neomycin resistance gene, and a green fluorescent protein gene. .   The method according to claim 1 or 2, wherein the origin of mammalian replication is derived from any one of the origin of replication of the c-myc locus, the dihydrofolate reductase locus, and the β-globin locus.   The method 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.   The method according to any one of claims 1 to 4, wherein the mammalian cells are selected from the group consisting of COLO 320DM cells, COLO 320HSR cells, and Hela cells. A vector set for amplifying a gene of interest in the form of DM and / or HSR in mammalian cells,
A vector comprising a mammalian origin of replication, a nuclear matrix binding region, and a gene for selecting transformed cells;
A target gene placed in trans with respect to the vector;
A vector set comprising:   The vector according to claim 6, wherein the gene for selecting transformed cells is one or more genes selected from the group consisting of a blasticidin resistance gene, a neomycin resistance gene, and a green fluorescent protein gene. set.   The vector set according to claim 6 or 7, wherein the origin of mammalian replication is derived from any one of the origin of replication of a c-myc locus, a dihydrofolate reductase locus, and a β-globin locus.   The vector set according to any one of claims 6 to 8, 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 transformant of a mammalian cell into which a vector comprising a gene for selecting a target gene, a mammalian replication origin, a nuclear matrix-binding region, and a transformed cell has been introduced,
A transformant in which the gene of interest is amplified in the form of DM and / or HSR.   The transformant according to claim 10, wherein the mammalian origin of replication is derived from any one of the origin of replication of the c-myc locus, the dihydrofolate reductase locus, and the β-globin locus.   The transformant according to claim 10 or 11, 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.   The transformant according to any one of claims 10 to 12, wherein the mammalian cells are selected from the group consisting of COLO 320DM cells, COLO 320HSR cells, and Hela cells.   The gene for selecting the transformed cells is one or more genes selected from the group consisting of a blasticidin resistance gene, a neomycin resistance gene, and a green fluorescent protein gene. A transformant according to claim 1.