JP4862156B2 - Method for controlling amplified form of target gene - Google Patents

Description

  The present invention provides a method for controlling the amplified form of a gene when a desired gene is amplified in a mammalian cell. More specifically, when the present invention amplifies a desired gene using the “advanced gene amplification system” developed by the present inventor, the gene is (a) homogenously stained on a double-new chromosome or chromosome. And (b) a method of amplifying the gene as a large amplification region in a uniform chromosome staining region.

The present inventor used a plasmid having a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) (hereinafter referred to as “IR / MAR plasmid”) as a human-derived cancer cell (COLO). 320 Introduced by lipofection into colon cancer cell lines and HeLa cell lines) and selected using drug resistance genes (Blasticidine or Neomycine) present on the plasmid,
(1) The ability to amplify the number of intracellular copies of a gene encoding a desired protein (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 another 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 (COLO 320 colorectal cancer cell lines and HeLa cell lines), CHO cells). The gene can be amplified to about 10,000 copies by simply using the lipofection method and selecting using a drug resistance gene (Blasticidine or Neomycine) present on the plasmid (hereinafter referred to as “advanced gene amplification system”). ") Was completed.

When the target gene is highly amplified using the above advanced gene amplification system, the target gene is amplified mainly by the following amplification forms:
(A) Amplified as a small amplification region on an extrachromosomal double minute chromosome (hereinafter referred to as “DM” where appropriate) or in a homogenous staining region (Homogeneously staining regeon; hereinafter referred to as “HSR” where appropriate); or (B) Amplified as a large amplification region by HSR.

At this time, it is not clear what mechanism determines the amplification form of the gene. However, in the case of performing gene amplification, (a) amplification as a small amplification region on DM or HSR, or (B) It can be arbitrarily controlled according to the purpose whether the HSR is amplified as a large amplification region, that is, the gene amplification form can be controlled not only academically but industrially. That is, if gene amplification can be performed in the form of (a) above, it is less likely to be affected by transcriptional repression caused by repetitive sequences in the amplified gene, and the advantage that the target protein can be highly expressed can be enjoyed. On the other hand, if gene amplification can be performed in the form of (b) above, it is possible to enjoy the advantage that the number of copies of the target gene per cell can be significantly increased.
JP 2003-245083 A (publication date: September 2, 2003) 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.

  However, when a target gene is amplified in a mammalian cell, a method of amplifying the target gene in the form (a) or amplifying the target gene in the form (b), that is, a method for controlling the amplification form, Until now, it was never known. Similarly, there is no known method for controlling the amplification form of a gene when performing gene amplification using the above advanced gene amplification system.

  Therefore, the present invention controls the amplification form of a gene of interest when amplifying a target gene in mammalian cells in order to more efficiently and highly express an amplified gene, particularly a gene amplified using an advanced gene amplification system. The purpose was to provide a method for doing this. In other words, the present invention, when amplifying a target gene using an advanced gene amplification system, (a) amplifies the target gene as a small amplification region on DM or HSR, or (b) An object of the present invention is to provide a method for amplifying as a large amplification region by HSR.

  The present inventor has conducted extensive studies using the advanced gene amplification system in order to solve the above-mentioned problems. As a result, the transcriptional activity state of the promoter contained in the vector during gene transfer (activated state, In addition, the inventors have found that the amplification form of the target gene can be controlled by controlling the inactive state, and have completed the present invention.

That is, the method according to the present invention solves the above-described problems,
(i) a first polynucleotide comprising an origin of replication and a nuclear matrix binding region that function in eukaryotic cells;
(ii) a second polynucleotide encoding a protein to be expressed, and a polynucleotide comprising a transcriptional activity-regulated promoter whose transcriptional activity is activated or inactivated by a predetermined operation, and the second polynucleotide is An introduction step of simultaneously introducing into a mammalian cell a third polynucleotide that is controllably linked to a transcriptional activity-regulated promoter; and a selection for selecting a mammalian cell into which the first and third polynucleotides have been introduced It is a method for controlling the amplification form of the second polynucleotide, comprising the steps and satisfying the following conditions (a) or (b):
(A) When the second polynucleotide is amplified as a small amplification region on a double mint chromosome or in a uniform chromosomal region, the introduction step and the selection step are performed with the transcriptional activity of the transcriptional activity-regulated promoter activated. .
(B) When the second polynucleotide is amplified as a large amplification region in the uniformly stained region of the chromosome, the introduction step and the selection step are performed in a state where the transcription activity of the transcriptional activity-regulated promoter is inactivated.

  In the method according to the present invention, the origin of replication that functions in the eukaryotic cell is derived from the origin of replication of the c-myc locus, the dihydrofolate reductase locus, or the β-globin locus. preferable.

  Furthermore, in the method according to the present invention, the nuclear matrix-binding region is preferably derived from the nuclear matrix-binding region of the Igκ locus, SV40 early region, or dihydrofolate reductase locus.

  According to the present invention, when a target gene is amplified using an advanced gene amplification system, (a) the target gene is amplified as a small amplification region on DM or HSR, or (b) the target gene is large amplified by HSR. It can be amplified as a region. Therefore, according to the present invention, it is possible to amplify the target gene in a form according to the purpose, and as a result, the desired protein (for example, useful protein) can be produced in a large amount.

  The embodiment of the present invention will be described as follows. Note that the present invention is not limited to this.

  The method according to the present invention relates to a method for controlling the amplification form of a target gene when a polynucleotide encoding a protein to be expressed using an advanced gene amplification system (ie, “target gene”) is amplified. It is.

  Here, in the present invention, “controlling the amplification form of the target gene” means that (a) the target gene is amplified as a small amplification region on DM or HSR depending on the purpose, or (b) the target gene is large in HSR. It means to be amplified as an amplification region. First, a method for determining whether the target gene is amplified on DM or HSR is not particularly limited. For example, a known FISH method (fluorescence in situ hybridization) is used for chromosomes at mitosis. The target gene introduced into the mammalian cell is detected, and judgment can be made based on whether fluorescence is shown on DM or fluorescence is shown by HSR. Since HSR is a region on the chromosome while DM exists outside the chromosome, a person skilled in the art can determine whether the target gene was amplified on DM or HSR by performing fluorescence microscopy. Can be easily determined. A specific method for carrying out the FISH method is not particularly limited, and a conventionally known method may be appropriately selected and adopted.

  When the target gene is amplified on DM, it is usually amplified as a small amplification region. On the other hand, when the target gene is amplified by HSR, it may be amplified as a large amplification region, may be amplified as a small amplification region, and may form a structure in which the small amplification regions are arranged in a ladder shape. Whether the target gene was amplified as a large amplification region or a small amplification region was described in, for example, “The ACT Cytogenetics Laboratory Manual (second edition” edited by Margaret J. Barch, Raven Press (1991) Create an interphase chromosome sample according to the method, and in the interphase chromosome sample, if the length of the amplified region is longer than that of the chromosome arm, the size is larger, and if it is shorter, the size is smaller. Judgment can be made.

  The above-mentioned “amplify the target gene as a small amplification region on DM or HSR” means a clone contained in a polyclonal population of transformed cells into which the target gene has been introduced under the same conditions and gene amplification has occurred. Means that the target gene is a clone amplified as a small amplification region on DM or HSR. The above-mentioned “amplifying the target gene as a large amplification region with HSR” means that the majority of clones contained in the polyclonal population of transformed cells into which the target gene has been introduced under the same conditions and gene amplification has occurred, This means that the target gene is a clone amplified by HSR as a large amplification region.

  In the polyclonal population of transformed cells in which gene amplification has occurred, the percentage of transformed cells in which the target gene is amplified as a small amplification region on DM or HSR is the ratio of the target gene in the polyclonal population of transformed cells. The number of clones amplified as a small amplification region on DM, the number of clones amplified as a small amplification region on HSR, and the number of clones whose target gene is amplified as a large amplification region on HSR are detected by the method described above. The number of clones undergoing gene amplification (that is, the number of clones in which the target gene is amplified as a small amplification region on DM, the number of clones in which the target gene is amplified as a small amplification region in HSR, and the target gene is large in amplification with HSR The target gene for the total number of clones amplified as a region) is on DM or H It is determined by calculating the ratio of the number of clones that are amplified as a small amplification region in R.

  The ratio of transformants in which the target gene in the polyclonal population of transformed cells in which gene amplification has occurred is amplified as a large amplification region by HSR is the ratio of the target gene in DM The number of clones amplified as a small amplification region, the number of clones amplified as a small amplification region by HSR, and the number of clones whose target gene is amplified as a large amplification region by HSR are detected by the above-described method. It is obtained by calculating the ratio of the number of clones in which the target gene is amplified as a large amplified gene by HSR with respect to the number of clones in which gene amplification has occurred.

The method according to the present invention comprises: (i) a first polynucleotide comprising an origin of replication and a nuclear matrix binding region that functions in eukaryotic cells;
(ii) a second polynucleotide encoding a protein to be expressed, and a polynucleotide comprising a transcriptional activity-regulated promoter whose transcriptional activity is activated or inactivated by a predetermined operation, and the second polynucleotide is An introduction step of simultaneously introducing into a mammalian cell a third polynucleotide that is controllably linked to a transcriptional activity-regulated promoter; and a selection for selecting a mammalian cell into which the first and third polynucleotides have been introduced It is a method for controlling the amplification form of the second polynucleotide, comprising the steps and satisfying the following conditions (a) or (b):
(A) When the second polynucleotide is amplified as a small amplification region on a double mint chromosome or in a uniform chromosomal region, the introduction step and the selection step are performed with the transcriptional activity of the transcriptional activity-regulated promoter activated. .
(B) When the second polynucleotide is amplified as a large amplification region in the uniformly stained region of the chromosome, the introduction step and the selection step are performed in a state where the transcription activity of the transcriptional activity-regulated promoter is inactivated.

  The method according to the present invention may include a culture step of culturing the mammalian cell selected by the selection step. Hereinafter, the method according to the present invention will be described step by step.

<Introduction process>
The introduction step in the method according to the present invention includes:
(i) a first polynucleotide comprising an origin of replication and a nuclear matrix binding region that function in eukaryotic cells;
(ii) a second polynucleotide encoding a protein to be expressed, and a polynucleotide comprising a transcriptional activity-regulated promoter whose transcriptional activity is activated or inactivated by a predetermined operation, and the second polynucleotide is This is a step of simultaneously introducing a third polynucleotide operably linked to a transcriptional activity-regulated promoter into a mammalian cell.

  The first polynucleotide includes an origin of replication and a nuclear matrix binding region that function in eukaryotic cells. The origin of replication contained in the first polynucleotide is not particularly limited as long as it functions in eukaryotic cells, such as c-myc locus, dihydrofolate reductase locus, β-globin locus, etc. Is the origin of replication. For the origin of replication of the c-myc locus, see “McWhinney, C. et al., Nucleic Acids Res. Vol. 18, p1233-1242 (1990)”. For the origin of replication of the dihydrofolate reductase locus, see “Dijkwel, P.A. et al., Mol. Cell. Biol. Vol.8, p5398-5409 (1988)”. For the origin of replication of the β-globin locus, see “Aladjem, M. et al., Science vol. 281, p1005-1009 (1998)”.

  The nuclear matrix-binding region contained in the first polynucleotide is not particularly limited as long as it functions in eukaryotic cells, such as Igκ locus, SV40 early region, dihydrofolate reductase locus, etc. Examples include sequences derived from the nuclear matrix binding region. 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.

  In addition to the above-mentioned first polynucleotide, a sequence necessary for cloning in Escherichia coli and / or a drug resistance gene (blasticidin resistance) as a selection marker (marker protein) may be used depending on the purpose. Sex gene, neomycin resistance gene, hygromycin resistance gene, etc.) or green fluorescent protein gene. By using these selection markers as indices, mammalian cells into which the first polynucleotide has been introduced can be selected.

  On the other hand, the second polynucleotide is a polynucleotide that encodes a protein to be expressed (that is, “target gene”), and is not particularly limited, and a polynucleotide encoding a desired protein is appropriately selected and adopted. do it. The second polynucleotide may be obtained using a known technique such as PCR based on the base sequence information. In the description of the present invention, “protein to be expressed” is appropriately referred to as “target protein”.

  The third polynucleotide used in the method according to the present invention is a polynucleotide in which the second polynucleotide is controllably linked to a transcriptional activity-regulated promoter whose promoter activity is activated by a predetermined induction operation. Is included. Here, the above-mentioned “transcriptional activity-regulated promoter” refers to a promoter that functions in mammalian cells into which it is introduced and whose transcriptional activity is activated or inactivated by a predetermined operation using a transcription factor or the like. That means. That is, the “transcriptional activity-regulated promoter” may be a promoter whose transcriptional activity is activated by a transcriptional activator or a promoter whose transcriptional activity is inactivated by a transcriptional inactivation factor. The “transcriptional activity regulating 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 present invention.

  By introducing the first polynucleotide and the third polynucleotide into mammalian cells at the same time, the present inventors can constitute the “advanced gene amplification system” disclosed in Patent Document 1 and the like. The second polynucleotide can be highly amplified in animal cells. The mammalian cells are not particularly limited, and examples thereof include CHO-K1 cells (source: for example, ATCC CCL-61, RIKEN RCB0285, RIKEN RCB0403, etc.), various tumor cells, and the like. However, as the mammalian cells, tumor cells having infinite proliferation ability are particularly preferable. Examples of the tumor cells include HeLa cells (obtained from, for example, ATCC CCL-2, ATCC CCL-2.2, RIKEN RCB0007, RIKEN RCB0191, etc.), human colon cancer COLO 320DM cells (obtained from, for example, ATCC CCL- 220), human colon cancer COLO 320HSR cells (source: eg, ATCC CCL-220.1), NS0 cells (source: eg, RIKEN RCB0213), and the like. For human colon cancer COLO 320DM cells, see `` Shimizu, N., Kanda, T., and Wahl, GM Selective capture of acentricfragments by micronuclei provides a rapid method for purifying extrachromosomally amplified DNA. Nat. Genet., 12: 65 -71, 1996.

  The introduction of the first and third polynucleotides into mammalian cells is not particularly limited as long as both polynucleotides are introduced into mammalian cells at the same time. May be introduced as the same gene construct, or may be introduced as separate gene constructs. The form of the gene construct may be a plasmid or a cosmid. The method for introducing the first and third polynucleotides into the mammalian cells is not particularly limited, and a known method such as lipofection, electroporation method, particle gun method may be appropriately selected and employed. .

  When the first and third polynucleotides 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 gene construct containing the first polynucleotide is preferably different from the selection marker contained in the gene construct containing the third polynucleotide.

  In addition, the method according to the present invention may include a step of expressing a transcriptional activator by introducing a polynucleotide (fourth polynucleotide) encoding the transcription factor of the promoter into a mammalian cell. According to this process, the transcription activity of the promoter that controls the expression of the target protein can be adjusted, and the expression of the target protein can be appropriately adjusted. Here, the fourth polynucleotide is a polynucleotide encoding a transcription factor of a promoter contained in the third polynucleotide. For example, when a TRE promoter is used as the promoter, a polynucleotide encoding a Tet-ON protein (referred to as “Tet-ON gene (Clontech)”) or a polynucleotide encoding a Tet-OFF protein (“Tet-ON”). -OFF gene (Clontech) ") may be used as the fourth polynucleotide. In the step of introducing a fourth polynucleotide into the mammalian cell to express a transcriptional activator, the fourth polynucleotide and a promoter for expressing the fourth polynucleotide were linked in a controllable manner. A gene construct may be introduced into a mammalian cell. The promoter for expressing the fourth polynucleotide is not particularly limited as long as it functions in mammalian cells. Even if it is a transcriptional activity-regulated promoter, it is a non-transcriptional activity-regulated promoter. Also good. In addition, the time when the gene construct containing the fourth polynucleotide is introduced into the mammalian cell may be the fourth polynucleotide introduced after the introduction of the first and third polynucleotides, or vice versa. Good. Furthermore, the fourth polynucleotide may be introduced into the mammalian cell simultaneously with the introduction of the first and third polynucleotides. According to the study of the present inventor, since the knowledge that the target protein is highly expressed is obtained when the fourth polynucleotide is introduced into the mammalian cell simultaneously with the introduction of the first and third polynucleotides, Most preferably, they are introduced simultaneously. When the fourth polynucleotide is introduced into a mammalian cell, the gene construct for introducing the fourth polynucleotide preferably 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 gene construct containing the first and / or 3 polynucleotides is preferably different from the selection marker contained in the gene construct containing the fourth polynucleotide. The form of the gene construct and the method for introducing it into mammalian cells may be the same as those for the first and third polynucleotides described above.

By the way, the method according to the present invention is as follows.
(A) When the second polynucleotide is amplified as a small amplification region on a double mint chromosome or in a uniform chromosomal region, the introduction step and the selection step are performed with the transcriptional activity of the transcriptional activity-regulated promoter activated. Or
(B) When the second polynucleotide is amplified as a large amplification region in the uniformly stained region of the chromosome, the introduction step and the selection step are performed in a state where the transcription activity of the transcriptional activity-regulated promoter is inactivated. It is said.

  A method of activating or inactivating the transcription activity of the promoter may be performed by a predetermined method corresponding to the promoter. More specifically, when performing (a) above, if the promoter is a promoter whose transcriptional activity is activated by the presence of a transcriptional activator, the introduction process and the selection process are performed in the system. In the case where the promoter is a promoter whose transcription activity is inactivated by the presence of the transcription inactivating factor, the introduction step and the selection step may be performed in the presence of the transcription activating factor. In addition, the introduction step and the selection step may be performed without the presence of the transcription inactivating factor in the system for performing the selection step.

  On the other hand, when performing (b) above, if the promoter is a promoter whose transcriptional activity is activated by the presence of a transcriptional activator, transcriptional activation is carried out in the system that performs the introduction step and the selection step. The introduction step and the selection step may be performed without the presence of the factor. Conversely, when the promoter is a promoter whose transcription activity is inactivated by the presence of the transcription inactivating factor, the introduction step and the selection step. The introduction step and the selection step may be performed in the presence of a transcription inactivating factor in the system.

  For example, a TRE promoter (manufactured by Clontech) is used as a transcriptional activity-regulated promoter contained in the third polynucleotide, and a Tet-ON gene (manufactured by Clontech) encoding a transcriptional activator of the promoter is used as the fourth polynucleotide. In order to perform the above (a), the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are introduced into mammalian cells in the presence of tetracycline (Clontech). In addition, the selection step may be performed in the presence of tetracycline (Doxycycline (Clontech)). In order to perform the above (b), the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are introduced into a mammalian cell in the absence of tetracycline (Doxycycline (Clontech)), and tetracycline ( The selection process may be performed in the absence of Doxycycline (Clontech). Tet-ON protein (Clontech) is a transcriptional activator that binds to the TRE promoter, and the transcriptional activity of the TRE promoter (Clontech) is activated in the presence of tetracycline (Doxycycline (Clontech)). The

  On the other hand, a TRE promoter (Clontech) is used as a transcriptional activity-regulated promoter contained in the third polynucleotide, and a Tet-OFF gene (Clontech) encoding a transcriptional activator of the promoter is used as the fourth polynucleotide. In order to carry out the above (a), the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are added to mammalian cells in the absence of tetracycline (Clontech). Then, the selection step may be performed in the absence of tetracycline (Doxycycline (Clontech)). In order to perform the above (b), the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are introduced into a mammalian cell in the presence of tetracycline (Doxycycline (Clontech)), and further tetracycline (Doxycycline) is introduced. The selection process may be performed in the presence of (Clontech). Tet-OFF protein (Clontech) is a transcriptional activator that binds to TRE promoter (Clontech), and transcription of TRE promoter (Clontech) in the presence of tetracycline (Doxycycline). The activity is inactivated.

  At this time, it is unclear why the gene amplification form can be controlled by the method according to the present invention. However, the inventor has previously stated that `` the structure where the replication fork and the transcription device collide, In the presence of MAR, double-strand breaks are frequently induced, and the structure at that position is destabilized. Therefore, such double-strand breaks are frequently induced by transcriptional activation. It is speculated that this is because the structure was changed.

<Selection process>
The “selection step” of the method according to the present invention is a step of selecting mammalian cells into which the first and third polynucleotides have been introduced. More particularly, this step comprises a polyclonal population of cells comprising mammalian cells into which the first and third polynucleotides have not been introduced, and mammalian cells into which the first and third polynucleotides have been introduced. The latter cell is selected from the above. In addition, when 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 first and third polynucleotides are not introduced , And a mixture of cells containing mammalian cells into which the first and third polynucleotides have been introduced, whereas the culture process described below is performed as mammalian cells into which the first and third polynucleotides have been introduced. The steps are clearly different in that the cells that have already been selected are cultured. By such a selection step, a mammalian cell in which the second polynucleotide (target gene) introduced into the mammalian cell is highly amplified can be selected.

  Although the specific method of the selection step is not particularly limited, for example, when a drug resistance gene is included in the gene construct used when introducing the first and third polynucleotides into mammalian cells, A mammalian cell into which the first and third polynucleotides have been introduced may be selected using the drug resistance. Further, by detecting the first and third polynucleotides or nucleotide fragments thereof contained in the mammalian cells by PCR or Southern blotting, the mammalian cells introduced with the first and third polynucleotides are selected. May be. Specific methods of the drug resistance, PCR method, and Southern blot method are not particularly limited, and known methods can be appropriately used.

<Culture process>
The “culturing step” of the method according to the present invention is a step of culturing mammalian cells that have already been selected by the above selection step. Through this culturing step, a mammalian cell into which the second polynucleotide (target gene) has been introduced and highly amplified can be grown, and the target protein can be produced by a predetermined operation (transcription induction operation). .

  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.

  Embodiments will be described below in more detail with reference to the accompanying drawings. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  The materials and methods used in the following examples are shown below.

(Plasmid)
pSFVdhfr (11.0 kbp) was a gift from Dr. John Kolman and Dr. Geoffrey M. Wahl (The Salk Institute, San Diego, CA). PSFVdhfr has a 4.6 kbp fragment containing Oriβ derived from the 3′-downstream region of the hydrofolate reductase gene (“Dijkwel, PA, and Hamlin, JL Matrix attachment regions are positioned near replication initiation sites, genes , and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells. Mol. Cell Biol., 8: 5398-5409, 1988.).

  pSFVdhfr / d2EGFP removes the hygromycin resistance gene expression unit of pSFVdhfr, and at that position is a TRE promoter (Clontech), d2EGFP structural gene (referred to as “d2EGFP gene”), SV40 poly A sequence (pTRE-d2EGFP (Clontech) (In this order) was constructed by incorporating the transcription so that the transcription was directed in the IR direction (see FIG. 1).

(cell)
The human colorectal COLO 320DM tumor cell line (human colorectal cancer COLO 320DM cell line, hereafter referred to as “COLO 320DM”) is “Shimizu, N., Kanda, T., and Wahl, GM Selective capture of acentricfragments by micronuclei provides a rapid method for purifying extrachromosomally amplified DNA. Nat. Genet., 12: 65-71, 1996. "

The cells and the transformed cells were also cultured according to the above documents. Briefly, the cell line was cultured in RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd.) with 10% fetal bovine serum added at 37 ° C. in the presence of 5% CO ₂ .

(Lipofection)
All the above plasmids were purified with Qiagen plasmid purification kit (Qiagen Inc., Valencia, CA) and transfected into cells with GenePorter 2 lipofection kit (Gene Therapy Systems, San Diego, CA).

(Method and result)
TRE-promoter transcriptional activity with IR and MAR derived from the human DHFR locus and a plasmid (pSFVdhfr / d2EGFP) having the d2EGFP gene under the control of TRE-promoter (tetracycline-inducible promoter). PTet-ON plasmid (manufactured by Clontech) containing the Tet-ON gene encoding the oxidization factor (Tet-ON protein) was mixed in an equal amount and simultaneously transfected into human colon cancer COLO 320DM cells by the lipofection method (Introduction process).

  Each of the cells after the introduction step was cultured in a medium containing 5 μg / ml Blasticidine for 2 weeks to select a polyclonal polyclonal population of transformed cells (selection step).

  The polyclonal population of transformed cells selected by the selection process was inoculated into a fresh medium, and further cultured for 2 weeks (culture process).

In addition, Doxycycline (manufactured by Clontech, hereinafter referred to as “Dox”) was added to the medium at 1 μg / ml according to the following pattern (I) or (II). The transcriptional activity of TRE-promoter is activated by adding Dox to the medium.
(I) Dox is added to the system in all steps of the introduction step, the selection step, and the culture step.
(II) Dox is not added to the system in the introduction step and the selection step. Dox is added to the medium only during the last day of the culture process.

  For the transformed cells after completion of the culture step in the pattern (I) or (II), the amplified gene was detected by the FISH method. The FISH method uses the same plasmid DNA introduced into cells as biotin or DIG (digoxigenin) labeled as a probe, hybridizes with a chromosomal specimen prepared from a transformant cell, and then hybridizes. This was done by detecting the soy probe with a fluorescent dye.

  The results are shown in FIG. Also, (a) and (b) in each figure are the results of COLO 320DM treated with the pattern (I) above, (a) is a clone in which the d2EGFP gene is amplified as a small amplification region by HSR. (B) is a fluorescence microscope image of a clone in which the d2EGFP gene is amplified as a small amplification region on DM. Moreover, (c) of FIG. 2 is the result of COLO 320DM processed with the pattern of (II), and is a fluorescence microscope image showing that the d2EGFP gene was amplified as a large amplification region by HSR. The amplified gene is a part indicated by an arrow and / or white in the figure, and whether the amplification region is small or large is described in “The ACT Cytogenetics Laboratory Manual (second edition” edited by Margaret J. Barch, An interphase chromosome sample was prepared according to the method described in Raven Press (1991), and confirmed in the interphase chromosome sample based on the width of the chromosome arm.

  For COLO 320DM treated with the pattern of (I) or (II), the ratio of transformed cells in which the target gene in the polyclonal population of transformed cells is amplified as a small amplification region on DM (“DM in FIG. 3) ), The ratio of transformants in which the target gene is amplified as a small amplification region by HSR (indicated by “small HSR” in FIG. 3), and the traits in which the target gene is amplified by HSR as a large amplification region The ratio of the converter (indicated by “large HSR” in FIG. 3) is shown in FIG.

  According to FIG. 3, in the clone of 26% of the transformed cell population of COLO 320DM treated with the pattern (I), the d2EGFP gene was amplified as a small amplification region by HSR. In addition, in 23% of the clones of the same transformed cell population, the d2EGFP gene was amplified as a small amplification region on DM. In the transformed cell population of COLO 320DM treated with the pattern (I), no clone was found in which the d2EGFP gene was amplified as a large amplification region by HSR. That is, the above result means that the majority of clones contained in the transformed cell population in which gene amplification of the d2EGFP gene occurred is a clone in which the d2EGFP gene is amplified as a small amplification region on DM.

  On the other hand, in the clone of 28% of the transformed cell population of COLO 320DM treated with the pattern (II), the d2EGFP gene was amplified as a large amplification region by HSR. Further, in 13% of the clones of the transformed cell population, the d2EGFP gene was amplified as a small amplification region on DM. In the transformed cell population of COLO 320DM treated with the pattern (II), no clone was found in which the d2EGFP gene was amplified as a small amplification region by HSR. That is, the above results mean that the majority of clones contained in the transformed cell population in which gene amplification of the d2EGFP gene occurred was clones in which the d2EGFP gene was amplified as a large amplification region by HSR.

  Therefore, it can be seen that the target gene (d2EGFP gene) can be amplified as a small amplification region on DM or HSR by treating cells with the pattern (I). It was found that the target gene (d2EGFP gene) can be amplified with HSR as a large amplification region by performing the treatment with the pattern of In other words, it is shown that the target gene (d2EGFP gene) can be amplified as a small amplification region on DM or HSR by performing the introduction step and the selection step with the transcriptional activity of the promoter activated, The above results indicate that the target gene (d2EGFP gene) can be amplified as a large amplification region on the HSR by performing the introduction step and the selection step in a state where the transcriptional activity of the promoter is inactivated.

  As described above, according to the method of the present invention, when a target gene is amplified using an advanced gene amplification system, (a) the target gene is used as a small amplification region on a double mint chromosome or a uniform chromosome region. Or (b) the target gene can be amplified as a large amplification region in the uniformly stained region of the chromosome. Therefore, according to the present invention, it is possible to amplify the target gene in a form according to the purpose, and as a result, the desired protein (for example, useful protein) can be produced in a large amount.

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

It is a figure which shows typically the structure of the plasmid (pSFVdhfr / d2EGFP) used in the Example. It is the fluorescence microscope image (x600) of the clone (COLO 320DM) of the transformed cell acquired in the Example, (a) is the fluorescence microscope image of the clone in which the d2EGFP gene is amplified as a small amplification region by HSR, (b ) Is a fluorescence microscope image of a clone in which the d2EGFP gene is amplified as a small amplification region on DM, and (c) is a fluorescence microscope image of a clone in which the d2EGFP gene is amplified as a large amplification region by HSR. In Examples, for COLO 320DM treated with the pattern (I) or (II), the ratio of transformed cells in which the target gene relative to all transformed cells is amplified as a small amplification region on DM, It is a bar graph which shows the ratio of the transformant by which the target gene was amplified as a small amplification region by HSR, and the ratio of the transformant by which the target gene was amplified by HSR as a large amplification region with respect to all the transformed cells.

Claims (3)

(i) a first polynucleotide comprising an origin of replication and a nuclear matrix binding region that function in eukaryotic cells;
(ii) a second polynucleotide encoding a protein to be expressed, and a polynucleotide comprising a transcriptional activity-regulated promoter whose transcriptional activity is activated or inactivated by a predetermined operation, and the second polynucleotide is An introduction step of simultaneously introducing into a mammalian cell a third polynucleotide that is controllably linked to a transcriptional activity-regulated promoter; and a selection for selecting a mammalian cell into which the first and third polynucleotides have been introduced And (a) in the case where the second polynucleotide is amplified as a small amplification region on a double mint chromosome or a uniform chromosomal region, the introduction step in a state where the transcription activity of the transcriptional activity-regulated promoter is activated. and we have a selection process row,
(B) when amplifying the second polynucleotide as a large amplification region a uniform dyed region of the chromosome, rows that have the introduction process and selection process in a state of transcriptional activity of transcription activity regulated promoter has been inactivated,
The origin of replication that functions in the eukaryotic cell is derived from the origin of replication of the c-myc locus, the dihydrofolate reductase locus, or the β-globin locus,
A method for controlling an amplified form of a second polynucleotide, wherein the nuclear matrix-binding region is derived from a nuclear matrix-binding region of an Igκ locus, an SV40 early region, or a dihydrofolate reductase locus . (i) a first polynucleotide comprising an origin of replication and a nuclear matrix binding region that function in eukaryotic cells;
(ii) a second polynucleotide encoding a protein to be expressed, and a polynucleotide comprising a transcriptional activity-regulated promoter whose transcriptional activity is activated or inactivated by a predetermined operation, and the second polynucleotide is An introduction step of simultaneously introducing into a mammalian cell a third polynucleotide operably linked to a transcriptional activity-regulated promoter; and
A selection step of selecting mammalian cells into which the first and third polynucleotides have been introduced, and
In the state in which the transcriptional activity of the transcriptional activity-regulated promoter is activated, the introduction step and the selection step are performed,
The origin of replication that functions in the eukaryotic cell is derived from the origin of replication of the c-myc locus, the dihydrofolate reductase locus, or the β-globin locus ,
The second polynucleotide is a double chromosomal chromosome or a homogeneous chromosomal region characterized in that the nuclear matrix binding region is derived from the Igκ locus, the SV40 early region, or the nuclear matrix binding region of the dihydrofolate reductase locus. A method of amplification as a small amplification region . (i) a first polynucleotide comprising an origin of replication and a nuclear matrix binding region that function in eukaryotic cells;
(ii) a second polynucleotide encoding a protein to be expressed, and a polynucleotide comprising a transcriptional activity-regulated promoter whose transcriptional activity is activated or inactivated by a predetermined operation, and the second polynucleotide is An introduction step of simultaneously introducing into a mammalian cell a third polynucleotide operably linked to a transcriptional activity-regulated promoter; and
A selection step of selecting mammalian cells into which the first and third polynucleotides have been introduced, and
The introduction step and the selection step are performed in a state where the transcriptional activity of the transcriptional activity-regulated promoter is inactivated,
The origin of replication that functions in the eukaryotic cell is derived from the origin of replication of the c-myc locus, the dihydrofolate reductase locus, or the β-globin locus,
The nucleic matrix attachment region, Ig kappa locus, characterized in that from the SV40 early region, or the nuclear matrix attachment region of the dihydrofolate reductase locus, a second polynucleotide as a large amplification region a uniform dyed region of the chromosome How to amplify .