JP2003245083A - Vector for extremely amplifying target gene in mammal cell, and method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple method for mass-producing a protein product, applicable to every mammal cell by which the target gene can be amplified in a mammal cell regardless of the chain length and the structure. <P>SOLUTION: The vector having the origin of replication of a mammal, and a nucleus-matrix binding region is used. The target gene to be amplified is arranged in cis (the same structure as the vector) or trans (the different structure to the vector) to the vector, and the arranged product is introduced into a mammal cell. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for highly amplifying a gene of interest in mammalian cells, and a vector used for carrying out the method. INDUSTRIAL APPLICABILITY The present invention is useful for producing useful proteins such as pharmaceuticals in mammalian cells by genetic engineering techniques.

[0002]

2. Description of the Related Art The only known method for increasing the intracellular copy number of a transgene in mammalian cells is
Chinese Hamster Ovary (CHO; Chinese Hamster O
vary) cells as a host, into which the target gene is introduced simultaneously with the dihydrofolate reductase (DHFR) gene, and methotrexate (MTx; Methotrex) is an inhibitor of the dihydrofolate reductase gene product.
ate) is added to the culture solution for selection. This method is now widely used for mass production of useful substances such as pharmaceuticals, and has become an important technique.

However, this method is applicable to host cells
Limited to CHO cells etc., there is a problem of lacking versatility. In addition, it is necessary to select using methotrexate, which is a selective drug, for a long period of time.Moreover, it is necessary to consider the cytotoxic effect of methotrexate while gradually increasing the concentration. And also requires skill. Furthermore, this method is applicable only to relatively short DNA as a target gene, and has a problem that its application range is narrow.

In general, a protein is often subjected to various modifications such as phosphorylation and acetylation depending on its type, and even with the same protein, the type and modification of the modification depend on the cell in which it is expressed, the time of expression, the conditions for stimulation of cells, etc. It is known that the part receiving the heat is different. Therefore, when a gene encoding a target protein to be expressed is to be ectopically and / or atypically expressed in a host cell or the like derived from a tissue in which the protein is not originally expressed, the original There is no guarantee that it will accurately reflect the nature of. In general, a functional modification of a protein often requires a specific modification. Therefore, it is preferable that the host cell for expressing the target protein can be appropriately selected depending on the protein gene to be introduced. Therefore, as in the above-mentioned conventional technique, the applicable cells are limited to CHO cells and the like, which is a major obstacle to the production of useful proteins such as pharmaceuticals in mammalian cells.

[0005]

The present invention has been made in view of the above circumstances, and an object of the present invention is to use a wide range of mammalian cells as a host, without requiring experience and skill, It is to amplify a gene by a simple operation regardless of the chain length of the nucleic acid.

[0006] 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.

[0007]

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

[0008] According to another aspect of the present invention, there is provided a method for highly amplifying and / or expressing a gene of interest in a mammalian cell, wherein the gene of interest is cis with respect to the vector of the present invention. Once placed, it is transfected into a suitable mammalian host cell for amplification and / or
Alternatively, a method characterized by expressing is provided.

[0009] According to another aspect of the present invention, there is provided a method for highly amplifying and / or expressing a gene of interest in a mammalian cell, which gene is placed in trans with respect to the above vector of the present invention. In addition, a method is provided which comprises transfecting this with a vector into a suitable mammalian host cell for amplification and / or expression.

In the present invention, "arranging the target gene in cis with respect to the vector" means arranging the target gene in the vector. In addition, "arranging the target gene in trans with respect to the vector" means arranging the target gene in a structure different from that of the vector.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
In the following description, various publicly known documents are referred to by the numbers in parentheses. Bibliographic items of these documents are listed at the end of the section of Detailed Description of the Invention.

The present invention is based on the knowledge found in the gene amplification phenomenon in tumor cells and the like. Gene amplification
Tumor cells are the main mechanism by which unlimited growth or drug resistance is acquired (1). Cytogenetically speaking, amplified genes are the most in vivo on extrachromosomal double minut chromosomes (DM). Frequently detected (but with long-term in vitro passage, usually a homogeneously stained region of the chromosome (HSR)
region) leading to the predominant growth of cells). Previous studies have shown that removal of the amplified gene on DM from tumor cells restores the tumor phenotype and cell differentiation to a normal state (2-4).
This type of clearance process is mediated by the selective uptake of DM into the 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 acentric circular DNA of various sizes (8), and although it is acentric, it attaches to mitotic chromosomes and is stably separated into daughter cells ( 7-9). Recent relevant important findings include bovine papillomavirus (10), EB virus (11), Kaposi's sarcoma-associated herpesvirus (12) and S.
Several viral nuclear plasmids, including V40 (13), have been shown to utilize similar mechanisms during cell division. Moreover, recent interesting studies have shown that plasmids carrying the EB virus replicon can be integrated into DM in tumor cells (14). In addition, expression of the viral large T antigen is required for the establishment of the attachment process in a plasmid having the SV40 origin of replication, but the large plasmid encoded by the large
The T gene is a cell-derived nuclear matrix attachment region (MAR; mat
It has been shown that separation can be achieved if substituted with a rix attachment region (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 genomic replication (15).

In the present invention, a gene of interest is introduced by using a vector having a mammalian origin of replication and a nuclear matrix binding region (MAR) based on the basic knowledge observed in the gene amplification phenomenon described above. De novo formation of DM containing said vector was achieved. The vector of the present invention is capable of amplifying a target gene having a long chain length in mammalian cells, and isolating the vector having the amplified gene into stable daughter cells, and as a result, accompanying the amplification of the gene. The effect of increasing the amount of protein produced can be obtained.

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

The mammalian origin of replication and nuclear matrix binding region are any as long as the gene of interest introduced into the vector in mammalian cells is amplified by the combination of the two and is then separated and distributed to daughter cells. However, preferably, the EB virus latent origin of replication (EB
V latent origin), origin of replication such as c-myc locus, dihydrofolate reductase locus, β-globin locus, etc., and nuclear matrix binding region such as Igκ locus, SV40 early region, dihydrofolate reductase locus It has a sequence that

[0016] In addition, the vector is a sequence necessary for cloning in E. coli, depending on the purpose.
Alternatively, a gene for selecting transformed cells such as a drug resistance gene (blasticidin resistance, neomycin resistance, etc.) or a green fluorescent protein gene may be included as a marker protein. Cells transformed with the vector into which these marker proteins have been introduced can be selected by drug selection based on drug resistance genes or by selecting or separating cells expressing the marker protein using a cell sorter or the like.

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

The host cells into which the vector is introduced may be mammalian cells, but cell lines such as COLO 320 cells and Hela cells are preferably used.

In the present invention, the target gene is amplified in a mammalian host cell by using the above vector. In the first embodiment, the target gene 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, the target gene is incorporated into a nucleic acid such as lambda phage or cosmid which has a sticky end and can self-circularize together with a suitable promoter, and this is used for the above-mentioned autonomous replication. It is mixed with a possible vector (plasmid etc.) and cotransfected and introduced into a mammalian host cell. That is, in this embodiment, the target gene is arranged in trans (a structure different from that of the vector). In this embodiment, two kinds of nucleic acids may be mixed and transfected together, but the weight ratio of the two is most preferably 1: 1. In the second aspect, a target gene having a longer chain length can be amplified as compared with the first aspect.

The vector can be introduced into cells by a method well known to those skilled in the art, such as electroporation and lipofection.

The protein transcribed / 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 being collected, extracted and used as a crude extract, or may be purified by various known methods such as chromatography and used.

[0023]

EXAMPLES Examples of the present invention will be described in detail below. The following examples do not limit the present invention.

Materials and Methods Plasmids The plasmid construct used in Figure 1 is described. Plasmids pEPBG (11.0kbp) and pSFVdhfr (11.0kbp) are Joh
n Dr. Kolman and Dr. Geoffrey M. Wahl (The Salk
Institute, San Diego, CA). The former plasmid is used for the EB virus latent origin of replication (EBV late
nt origin) (OriP) and EBNA-1, as well as green fluorescent protein and G-associated polypeptid
e) carrying the fusion gene (GFP-GAP), the latter plasmid carrying a 4.6 kbp fragment containing Oriβ from the region 3'-downstream to dihydrofolate reductase (16).
The pSFV-V plasmid lacking the origin of replication (6.4 kbp) was constructed from pSFVdhfr by digesting all dihydrofolate reductase derived sequences with NotI digestion. pNeo.Myc-2.4
The plasmid (9.0kbp; ref. 17) is Michael Leffa
Granted by Dr. K (Wright State University, Dayton, OH). This plasmid contains the 2.4 kbp HindIII / XhoI fragment derived from the promoter region of c-myc, NotI /
It was used to construct pNeo-V (lacking the origin of replication) which was HindIII digested and deleted almost all of the sequence derived from c-myc.
The pNeo.MycΔSV plasmid (lacking 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 MAR binding region of the Igκ gene (Intronic enhancer MAR) was amplified by PCR using pG19 / 45 plasmid as a template, and pAR1 plasmid was constructed from the amplified product (19). pNeo.MycΔ
The SV AR1 plasmid was created by inserting the nuclear matrix binding region sequence (AR1) of pAR1 into pNeo.MycΔSV. By this operation, the nuclear matrix binding region-like SV
40 derived sequences are replaced.

Other Experimental Methods Human colorectal COLO 320DM and COLO 320HSR tumor cell lines were obtained and maintained as described in the literature (5).

The HeLa cell line is the American Type Culture Collection.
(CCL-2). All plasmids were purified using the Qiagen Plasmid Purification Kit (Qiagen Inc., Valencia, CA) and the GenePorter 2 Lipofection Kit (Gene
Cells were transfected by Therapy Systems, San Diego, CA).

[0027] Blasticidine (10
μg / ml; Funakoshi, Tokyo, Japan; pEPBG, pSFVdhf
r and its variants) or 500 μg / ml Neomycin (Life Technologies, Inc., Roc
kville, MD; pNeo.Myc-2.4 and its variants) were used to select for transformants. A biotin-labeled micronucleus probe detecting COLO 320 amplicons was prepared according to our published protocol (5). Preparation of metaphase samples, preparation of DIG-labeled probe and FISH were performed according to previously published protocol (20) (similar to in vitro nuclear matrix binding assay (19)).

<Results and Discussion> Plasmids carrying the EB virus replicon are integrated into DM Before examining the mammalian replicon, we have examined the most characterized episomal vector (ie EB virus replicon). (Compliant with (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 mitotic chromosomes (11). We transfected this plasmid into human COLO 320DM tumor cells with multiple DM and analyzed the stably transformed colony mixture by FISH method. As a result, the sequence of the plasmid was mainly detected in DM of various sizes in some metaphase cells (Fig.
2, A and B; Table 1). No signs of chromosome integration were found. These observations are in line with the results described in a recent report, indicating that the plasmid carrying the EB virus replicon selectively recombines with DM (14). In the experiment using mixed colonies, the percentage of cells in which the plasmid was integrated into DM was small (13%), but the GFP-G encoded by the plasmid was small.
When clones showing high level expression of AP fusion protein were examined, 10 out of 10 clones showed integration into DM. This phenomenon suggests that the sequences integrated in DM are expressed at high levels. Moreover, this result is consistent with the observation that most of the amplified genes in tumor cells are localized in DM.
(1).

[0029]

[Table 1]

A plasmid having a mammalian replicon is also D
Similarly, we incorporated dihydrofolate reductase (1
6) and pS with origin sequences of c-myc locus (17)
FVdhfr and pNeo.Myc-2.4 plasmids were tested (Fig.
1). We have also shown that these plasmids also integrate into various sized DMs in COLO 320DM cells, and
We found that they were selectively incorporated into micronuclei (Fig. 2C).

Simultaneous detection of plasmid and amplicon sequences also showed that DM with plasmid sequences was derived from DM present in untransfected COLO 320 DM cells (FIG. 2D). The frequency of DM incorporation in mixed transformed colonies was determined by pSFVdhfr (60
%) And low in pNeo.Myc-2.4 (4.3%) (it is not clear how the integrated sequence affects frequency).

Incorporation into DM requires autonomous replication of the plasmid The mammalian origin sequences are plasmids (pSFV-V and pNeo-V;
When deleted from Figure 1), the plasmid is DM
It is an important finding that recombination did not occur with (Table 1). Also we pSFVdhfr (~ 800 colonies / 10 ⁶ cells)
We also found that the transformation efficiency of pSFV-V (~ 30 colonies / 10 ⁶ cells) was very low compared to that, and these facts indicate that the ability to replicate autonomously is important for plasmid integration into DM. And that this ability increases transformation efficiency. It is a question currently inferring why autonomously replicating plasmids frequently recombine with DM, but one hypothesis is based on the fact that plasmids and DM share a common partition mode. . DM tends to be localized in the periphery of the nucleus during the subsequent G1 phase of the cell cycle, migrates into the nucleus during replication, and is then separated by attaching to the chromosome (14, 20, 21). . Therefore, D that can occur in the interphase nucleus
Colocalization of M and plasmids may increase the frequency of recombination between these two DNA species.

Nuclear Matrix Binding Region Is Required for Extrachromosomal Replication Driven by a Mammalian Origin The nuclear matrix binding region has been reported to be located near the replication initiation site on the dihydrofolate reductase locus (16), pSFVdhfr. There is no report on the 4.6 kbp insert of. Nevertheless, a nuclear matrix binding region retrieval program (22) analysis predicted the possible presence of potential nuclear matrix binding regions within the insert, and the actually predicted fragments were found to be nuclear matrix bound in vitro. Was found to bind to (Fig. 3). In addition, the nuclear matrix binding region search program could not detect the nuclear matrix binding region candidate in the 2.4 kbp insert derived from the c-myc locus of pNeo.Myc-2.4. However, the plasmid contained sequences from the SV40 early region which exhibited 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 the nuclear matrix binding region in transformation efficiency and integration into DM, we deleted the nuclear matrix binding region-like sequence from SV40 of pNeo.Myc-2.4 (hence pNeo.M).
ycΔSV is generated; FIG. 1), while we replaced the site of another construct (pNeo.MycΔSV AR1) with the nuclear matrix binding region from the Igκ gene (19). As expected, the NcoI / HindIII fragment generated from the former plasmid (pNeo.MycΔSV) by in vitro binding assay shows no nuclear matrix binding activity, whereas the latter (pNeo.M
Those produced from ycΔSV AR1) were shown to bind to the nuclear matrix (Fig. 3). When COLO 320DM cells were transfected with these plasmids, transformation efficiency in comparison with pNeo.MycΔSV AR1 (~800 colonies / 10 ⁶ cells), in pNeo.MycΔSV (~90 colonies / ¹⁰⁶ cells) It was very low. In addition, the former plasmid (pNeo.Myc ΔS
V) was not incorporated into DM, while the latter (pNeo.
Myc ΔSV AR1) was incorporated (Table 1; Figure 2E). These observations indicate that DM integration depends on the cis-acting nuclear matrix binding region. Therefore, the nuclear matrix binding region seems to play an important role in autonomous replication of plasmid.

The autonomously replicating plasmid induces a phenomenon similar to gene amplification. A cell line different in unexpected phenomenon (that is, COLO 320HSR).
Cells) were used for transfection. Although this cell line is normally DM-free, DM with many plasmid sequences were observed when transfected with pSFVdhfr or pNeo.MycΔSV AR1 (respectively,
2G and H) (ie, many microchromatin pairs observed in metaphase). These DM are COLO 320
Two-color FISH analysis showed that the cells did not contain the original amplicon sequence found (Fig. 2G), which means that DM occurred de novo. D
M-bearing cells were frequently present only when transfected with a plasmid containing both the origin and the nuclear matrix binding region (Table 1). In addition, these phenomena are
It was also observed when (another tumor cell line lacking DM) was used (Fig. 2I). The interpretation that the plasmid frequently recombines with the endogenous extrachromosomal closed circular DNA is a plausible explanation that may explain this phenomenon. Indeed, many mammalian somatic cells contain extrachromosomal closed circular DNA, which relies on genomic flexibility (23). Our observations directly support a model that suggests that DM occurs by microscopically undetectable episomal recombination (24).

We also found a group of cells displaying a very strong plasmid signal along chromosomal arms that resembled a uniform chromosome staining region (found in tumor cells) (Fig. 2F, arrow). Such groups of cells were only observed when a plasmid with both the origin and the nuclear matrix binding region was used. Interestingly, the frequency of this cell population was much higher when transfected into COLO 320DM cells versus COLO 320HSR cells (Table
1). This finding is consistent with a model that presents that the uniformly stained region is formed by tandem integration of DM into the chromosome arm (24).

As described above, in the course of the basic research of the present invention, we have revealed a novel in vitro model in the mechanism of gene amplification that plays a crucial role in human tumorigenesis. This model appears to be useful for functional analysis of the origin of replication in mammals, since to date much of the work in this subject has been done using sequences integrated on the chromosome that are affected by the surrounding chromatin. Is.

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

[0038]

INDUSTRIAL APPLICABILITY The method according to the present invention can be applied to any mammalian cells, and it is possible to amplify a target gene in cells by several thousand to ten thousand copies or more by an extremely simple operation. Become. Furthermore, since the method according to the present invention can amplify a gene of any length in principle, it is possible to amplify a wide range of target gene regions on the genome.

[0039]

[Reference] 1. Benner, SE, Wahl, GM, and Von
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[Brief description of drawings]

FIG. 1 is a diagram showing the plasmids used in this study. The figure shows the EB virus replicon (OriP and
EBNA-1), the dihydrofolate reductase locus, and
pEPBG and pS with origin of mammalian replication derived from c-myc locus
The configurations of FVdhfr and pNeo.Myc-2.4 are shown. Other plasmids in this study were pSFVdhfr or pNeo.Myc.
-Built from 2.4. Intronic sequences from the SV40 early region or the Igκ gene (AR1) exhibited nuclear matrix binding activity (indicated by filled squares). The dihydrofolate reductase Oriβ contains a sequence exhibiting nuclear matrix binding activity (see FIG. 3). GFP-GAP (GFP and G
AP 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 required for growth of bacterial cells.

FIG. 2 is a fluorescence micrograph showing the results of FISH analysis of tumor cells transformed with the plasmid. COLO 3
20DM (AF), COLO 320HSR (G and H), and HeLa (I)
Cells were transfected with pEPBG (A and B), pSFVdhfr (C, D, G, and I), or pNeo.MycΔSV AR1 (E, F, and H). Stable transformants were selected after 30 days in culture, and metaphase chromosome samples were prepared. The DIG-labeled probe prepared from the transfecting plasmid was used for hybridization with the chromosome preparation and detected by green fluorescence. D and G show the results obtained by simultaneously hybridizing a biotin-labeled probe prepared from purified micronuclei of untransfected COLO 320DM cells and a DIG-labeled plasmid probe and detecting with red fluorescence.
White arrows indicate a giant signal (F inset) of DM (AE and GI) or interphase nuclei (F) corresponding to the uniform staining region of metaphase. Arrowheads (F and H) indicate micronuclei that have accumulated plasmid-derived sequences. Propium iodide produces red (AC and
Blue) (DH) to I) or with 4 ', 6-diamino-2-phenylindole (4', 6-diamidino-2-phenylindole)
DNA was counterstained. The green and red overlaps are yellow, the green and blue overlaps are cyan, and all three overlaps are white. In D, the plasmid sequence (green) was co-localized with the amplicon sequence (red) localized in DM (detected in blue, white arrow). In G, the amplicon sequence (red) was mainly detected in the marker uniform staining region in cells (yellow arrows I, II and III), but DM with plasmid sequence (green, indicated by white arrow). It was not detected in (blue). Bar is 10μ
represents m.

FIG. 3 is a PAGE electrophoretic photograph showing the results of an in vitro matrix binding assay. The plasmids were designated BamHI / HindIII (pAR1) and NcoI / HindIII (pNeo.Myc-
2.4, pNeo.Myc ΔSV and pNeo.Myc ΔSV AR1; see Fig. 1 for restriction enzyme recognition sites), digest with HinfI (pSFVdhfr), and end-filling reaction [α
- ³² P] labeled with dATP. Unlabeled competitor of labeled DNA fragments at various concentrations (20: 1 mixture of E. coli DNA and pUC18 DNA)
Incubation with nuclear matrix in the presence of After repeating centrifugation to wash the matrix,
The matrix-bound DNA fragments were separated by 5% -PAGE and visualized by autoradiography. Lane 1,
Input DNA; Lanes 2-4, competitive DNA (4, respectively,
8, 16 μg) Reaction in the presence. Arrowheads show bands specifically bound to the matrix. pAR1 is the plasmid used as a control and contains the nuclear matrix binding region derived from the mouse Igκ gene (Arrowhead). The pNeo.Myc-2.4 fragment that exhibits nuclear matrix-binding activity contains the SV40 early region, which has been shown to be the nuclear matrix-binding region (18). Deletion of this sequence completely abolished nuclear matrix binding activity (pNeo.M
ycΔSV). However, insertion of Igκ nuclear matrix binding region sequences into this plasmid restored nuclear matrix binding activity (pNeo.MycΔSV AR1). The matrix-bound pSFVdhfr fragment (a) was identified as a 886 bp fragment from the core of the dihydrofolate reductase insert that matched 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 that the fragment is also derived from the insert.

Claims (8)

[Claims] 1. A vector for highly amplifying and / or expressing a gene of interest in a mammalian cell, comprising:
A vector comprising an origin of replication that functions in a eukaryotic cell and a nuclear matrix binding region, capable of autonomous replication in a transfected cell, and stably distributed to daughter cells. 2. The origin of replication that functions in the eukaryotic cell,
EB virus latent origin of replication (EBV latent origin), c-myc
The vector according to claim 1, which is derived from any one of the origin of replication of the locus, the dihydrofolate reductase locus, and the β-globin locus. 3. The vector according to claim 1, wherein the nuclear matrix binding region is derived from any one of the Igκ locus, the SV40 early region, and the dihydrofolate reductase locus nuclear matrix binding region. 4. The vector according to claim 1, wherein the gene of interest to be amplified and / or expressed is arranged in cis with respect to the vector. 5. A method for highly amplifying and / or expressing a target gene in a mammalian cell, comprising transfecting the recombinant vector according to claim 4 into a suitable mammalian host cell to obtain the target gene. Amplify and / or gene
Alternatively, a method of expressing. 6. A method for highly amplifying and / or expressing a target gene in a mammalian cell, wherein the target gene is arranged in trans with respect to the vector according to any one of claims 1 to 4. And transfecting it with a suitable mammalian host cell for amplification and / or expression. 7. A transformed cell transfected with the vector according to claim 1. 8. A transformed cell transfected in the method according to claim 6.