JP4923244B2 - Method for increasing gene amplification efficiency - Google Patents

Description

  The present invention provides a method for increasing the efficiency of gene amplification when a desired gene is amplified in a mammalian cell, and a kit for performing the method. More preferably, the present invention relates to a method for further increasing gene amplification efficiency when a desired gene is amplified using the “advanced gene amplification system” developed by the present inventor, and a kit for performing the method.

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 protein to be expressed (hereinafter referred to as “target gene” as appropriate) to about 10,000 copies, and
(2) The target gene must be highly amplified, whether it is introduced as the same gene construct (cis) or 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.
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.

  An object of the present invention is to further improve the gene amplification efficiency in the above advanced gene amplification system. Another object of the present invention is to provide a method for improving the amplification efficiency of the introduced target gene and a kit for performing the method even when the above-described advanced gene amplification system is not used.

The present inventor conducted intensive studies using the above-described advanced gene amplification system to solve the above-described problems, and found that gene amplification was achieved by satisfying at least one of the following conditions (1) to (3). It was discovered that efficiency could be further improved and led to the completion of the present invention:
(1) culturing transformed mammalian cells under low oxygen concentration conditions;
(2) introducing an inverted repeat sequence (eg, a batch sequence) into a mammalian cell;
(3) Gene transfer and selection of transformed cells in a state where the transcriptional activity of the transcriptional activity-regulated promoter is inactivated.

That is, the method according to the present invention is a method for increasing the efficiency of gene amplification, and in order to solve the above 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 promoter, and the third polynucleotide to which the second polynucleotide is controllably linked to the promoter; In the case of amplifying the second polynucleotide by performing an introduction step of simultaneously introducing into a cell and a selection step of selecting a mammalian cell into which the first and third polynucleotides have been introduced, the following is shown (1) Or it is characterized by satisfying one or more of the conditions of (2):
(1) In the selection step, the mammalian cells into which the first polynucleotide and the third polynucleotide are introduced are cultured under a low oxygen concentration condition that is less than the oxygen concentration at which the mammalian cells are normally cultured;
(2) In the introduction step, an inverted repeat sequence is introduced into a mammalian cell simultaneously with the first polynucleotide and the third polynucleotide.

The method according to the present invention may be a method that further satisfies the following condition (3):
(3) The promoter is a transcriptional activity-regulated promoter whose transcriptional activity is activated or inactivated by a predetermined operation, and the introduction step and selection are performed in a state where the transcriptional activity of the transcriptional activity-regulated promoter is inactivated. Perform the process.

  In the method according to the present invention, the inverted repeat sequence may be a palindromic sequence.

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

  In the method according to the present invention, the nuclear matrix-binding region may be derived from the nuclear matrix-binding region of the Igκ locus, SV40 early region, or dihydrofolate reductase locus.

  In the method according to the present invention, the low oxygen concentration condition may be an oxygen concentration of less than 20% (v / v).

The present invention also provides a kit for performing the above method,
Also included is a kit comprising a polynucleotide comprising an origin of replication and a nuclear matrix binding region that functions in eukaryotic cells, and an inverted repeat sequence.

  The present invention also includes a step of culturing a mammalian cell into which a polynucleotide encoding a protein to be expressed is introduced under a low oxygen concentration condition that is less than the oxygen concentration under which the mammalian cell is normally cultured. And a method for increasing the efficiency of gene amplification.

  The present invention also includes a method for increasing the efficiency of gene amplification, which comprises the step of simultaneously introducing a polynucleotide encoding a protein to be expressed and an inverted repeat sequence into a mammalian cell. Furthermore, the present invention includes a kit for performing the above-described method, wherein the kit includes an inverted repeat sequence.

  According to the present invention, it is possible to increase gene amplification efficiency when a target gene is amplified using an advanced gene amplification system or without using the advanced gene amplification system. Therefore, according to the present invention, the desired protein (for example, useful protein) can be produced in large quantities.

  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 increasing gene amplification efficiency when a target gene is amplified using an advanced gene amplification system or without using the advanced gene amplification system.

  Here, “increasing the efficiency of gene amplification” in the present invention means that the frequency of gene amplification structure formation in transformed cells is improved by carrying out the method of the present invention compared to the case where the present invention is not carried out. It means that The “gene amplification structure formation frequency” can be determined by calculating the ratio of clones in which the amplification structure is included in the polyclonal population. The amplification structure of the amplified gene is amplified as a small amplification region on an extrachromosomal double-minute chromosome (hereinafter referred to as “DM” where appropriate) or on a homogeneous staining region of the chromosome (hereinafter referred to as “HSR” where appropriate). In some cases, the HSR may be amplified as a large amplification region or a medium amplification region. A method for determining whether an amplified structure is formed for a clone of a transformed cell is not particularly limited. For example, a known FISH method (fluorescence in situ hybridization) is performed on chromosomes at mitosis, and mammalian cells are obtained. This can be determined by detecting the gene introduced into the. The above judgment can be easily made by those skilled in the art. A specific method for carrying out the FISH method is not particularly limited, and a conventionally known method may be appropriately selected and adopted.

  Also, by performing the above FISH method, it is determined whether it is amplified as a small amplification region on DM, amplified as a small amplification region with HSR, amplified as a medium amplification region with HSR, or amplified as a large amplification region. Can do. More specifically, it can be first determined whether the gene of interest has been amplified on DM or HSR by detecting whether it exhibits fluorescence on DM or fluorescence on HSR. HSR is a region on the chromosome, whereas DM exists outside the chromosome, so that the two can be easily distinguished by observation with a fluorescence microscope. Whether the target gene was amplified as a large amplification region, a medium amplification region, or a small amplification region, for example, “The ACT Cytogenetics Laboratory Manual (second edition” edited by Margaret J. Barch, An interphase chromosome sample is prepared according to the method described in Raven Press (1991), and if the length of the amplified region is longer than the width of the chromosome arm in the interphase chromosome sample, the size is larger. It can be judged as medium size if it is about the same, small size if it is shorter.

  Further, “increasing the efficiency of gene amplification” means increasing the frequency of gene amplification structure formation compared to the case where the method of the present invention is not performed. More specifically, when the frequency of gene amplification performed on DM is increased as compared with the case where the method of the present invention is not performed, or when the gene is amplified by HSR, This means that the frequency of taking a longer amplification form (that is, the amplification region becomes longer) is increased as compared with the case where the method of the present invention is not performed.

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 promoter, and the third polynucleotide to which the second polynucleotide is controllably linked to the promoter; In the case of amplifying the second polynucleotide by performing an introduction step of simultaneously introducing into a cell and a selection step of selecting a mammalian cell into which the first and third polynucleotides have been introduced, the following is shown (1) Or it is characterized by satisfying one or more of the conditions of (2):
(1) In the selection step, the mammalian cells into which the first polynucleotide and the third polynucleotide are introduced are cultured under a low oxygen concentration condition that is less than the oxygen concentration at which the mammalian cells are normally cultured;
(2) In the introduction step, the palindromic sequence of the second polynucleotide is introduced into the mammalian cell simultaneously with the first polynucleotide and the third polynucleotide.

Further, the method according to the present invention may be a method further comprising the following step (3):
(3) The promoter is a transcriptional activity-regulated promoter whose transcriptional activity is activated or inactivated by a predetermined operation, and the introduction step and selection are performed in a state where the transcriptional activity of the transcriptional activity-regulated promoter is inactivated. Perform the process. That is, the method according to the present invention includes the conditions (1), (2), (1) + (2), (1) + (3), (2) + (3) The case where the condition or the condition of (1) + (2) + (3) is satisfied is included.

  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 promoter, and the third polynucleotide to which the second polynucleotide is controllably linked to the promoter; It is a process of introducing simultaneously into cells.

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

  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.

  The method according to the present invention may include a step of introducing a polynucleotide (fourth polynucleotide) encoding a transcription factor of the promoter into a mammalian cell to express the transcriptional activator. 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, and may be a transcriptional activity-regulated promoter or a constitutive promoter.

  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.

  In the method according to the present invention, in the introduction step, an inverted repeat sequence including a palindromic sequence (also referred to as “Palindrome”) is introduced into a mammalian cell simultaneously with the first polynucleotide and the third polynucleotide. This is one feature. Here, in the description of the present invention, “inverted repeat sequence” and “batch sequence” mean “polynucleotide consisting of inverted repeat sequence” and “polynucleotide consisting of batch sequence”, respectively. The inverted repeat sequence and the batch sequence are described, for example, in the Molecular Biology Dictionary, 1st edition, 4th edition (issued by Tokyo Kagaku Dojin, published date: July 1, 1999). In addition, the inverted repeat sequence including the palindromic sequence can be prepared using a normal genetic engineering technique, and thus can be easily prepared by those skilled in the art.

When introducing inverted repeats, including palindromic sequences, into mammalian cells,
(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 promoter, and a third polynucleotide in which the second polynucleotide is controllably linked to the promoter;
(iii) An inverted repeat sequence (batch sequence) may be introduced simultaneously into mammalian cells.
In such a case, the first polynucleotide, the third polynucleotide, and the inverted repeat sequence (batch sequence) may all be ligated and introduced into mammalian cells as the same gene construct, or separately without ligation. It may be introduced as a gene construct. Alternatively, the first polynucleotide and the inverted repeat sequence (batch sequence) may be the same gene construct and the third polynucleotide may be introduced into the mammalian cell as another gene construct. Alternatively, the third polynucleotide and the inverted repeat sequence (batch sequence) may be introduced into a mammalian cell as the same gene construct and the first polynucleotide as another gene construct. Further, the first polynucleotide and the third polynucleotide may be the same gene construct, and the inverted repeat sequence (batch sequence) may be introduced into the mammalian cell as another gene construct.

  The length of the inverted repeat sequence (batch sequence) used in the method of the present invention is not particularly limited, but is preferably 50 bp to 1000 bp, more preferably 100 bp to 400 bp. If it is the said preferable range, it will become easy to make high cell growth efficiency and high gene amplification efficiency compatible.

  The base sequence of the inverted repeat sequence (batch sequence) used in the method of the present invention is not particularly limited except that it is an inverted repeat sequence (batch sequence), and encodes a protein. Or may not be coded. In the examples described later, inverted repeat sequences (batch sequences) are prepared using the d2EGFP structural gene. Note that the inverted repeat sequence (batch sequence) may be appropriately prepared by known methods such as chemical synthesis of any nucleotide chain.

  The present inventor has found that the efficiency of gene amplification increases when inverted repeats (batch sequences) are introduced into mammalian cells. The details of the mechanism by which the amplification efficiency of the gene is improved by introducing the inverted repeat sequence (pamphlet sequence) into the host cell as described above is unknown at present, but the DNA at the position of the inverted repeat sequence (batch sequence) is unknown. The inventor speculates that this may cause a double-strand break, which triggers gene amplification.

The method according to the present invention may be a method that satisfies the following conditions.
The introduction step and the selection step are performed in a state where the promoter contained in the third polynucleotide is a transcriptional activity-regulated promoter and the transcriptional activity of the transcriptional activity-regulated promoter is inactivated.

  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 the promoter is a promoter whose transcriptional activity is activated by the presence of the transcriptional activator, it is introduced without the presence of the transcriptional activator in the system that performs the introduction step and the selection step. In the case where the promoter is a promoter whose transcription activity is inactivated by the presence of a transcription inactivating factor, transcription is not performed in the system that performs the introduction step and the selection step. The introduction step and the selection step may be performed in the presence of an activator.

  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 the absence of tetracycline (Doxycycline (Clontech)), the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are introduced into mammalian cells, and tetracycline (Doxycycline (Clontech)). The selection process may be carried out in the absence of a company))). 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 the presence of tetracycline (Doxycycline (Clontech)), the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are introduced into mammalian cells, and tetracycline (Doxycycline (Clontech) The selection step may be carried out in the presence of the production)). 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.

  Although the details of the mechanism by which the efficiency of gene amplification is improved by performing the introduction and selection steps while the transcriptional activity of the transcriptional activity-regulated promoter is inactivated are unknown at this time, transcription from the promoter occurs. In contrast, the double-strand breaks of DNA frequently and violently occur everywhere due to the frontal collision with the replication fork from the IR, so that the gene amplification structure once formed is destroyed. The inventors speculate that the gene amplification efficiency may be improved because the gene amplification structure is unlikely to break down in the absence of transcription from the promoter. DNA double-strand breaks trigger gene amplification, but as described above, if double-strand breaks occur frequently and violently everywhere, it can no longer be repaired, leading to the destruction of the gene amplification structure. .

<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.

  As described above, the method according to the present invention is an embodiment in which the selection step includes the step of culturing mammalian cells into which the first polynucleotide and the third polynucleotide have been introduced. One of the characteristics is that the culture is performed under a certain low oxygen concentration condition. The “under low oxygen concentration condition” means that the oxygen concentration in the atmosphere when culturing mammalian cells is lower than the oxygen concentration at which the mammalian cells are normally cultured. The “oxygen concentration at which mammalian cells are usually cultured” is not uniquely determined because it varies depending on the cells, but is 20% (v / v) for normal mammalian cells. Therefore, under low oxygen concentration conditions, the oxygen concentration in the atmosphere when culturing mammalian cells may be less than 20% (v / v). The oxygen concentration when culturing mammalian cells under hypoxic conditions is preferably 5% (v / v) or more and less than 20% (v / v), preferably 5% (v / v) or more and 10% (v / v). More preferably less than v). If it is in the said preferable range, a mammalian cell will be easy to proliferate and it will be easy to acquire the increase effect of gene amplification efficiency. “% (V / v)” means “volume percent” and is also expressed as “% v / v” or “% vol / vol”.

  Although details of the mechanism by which gene amplification efficiency is improved by culturing mammalian cells under conditions of low oxygen concentration, which is less than the normal oxygen concentration, are unknown at this time, DNA double-strand breaks under low oxygen concentration The inventor speculates that this may trigger gene amplification.

  The selection step in the method of the present invention can also be carried out by detecting the first and third polynucleotides or nucleotide fragments thereof contained in mammalian cells by PCR or Southern blotting. 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. The oxygen concentration in the culture atmosphere in the culturing step is not particularly limited, and may be a normal oxygen concentration condition or a low oxygen condition. However, according to the inventor's study, culturing under the latter condition tends to obtain a higher expression level of the target protein. Therefore, culturing mammalian cells under hypoxic conditions also in the culturing step. It can be said that it is preferable.

  In addition, this invention also includes the kit for performing the method concerning this invention demonstrated above. That is, the kit according to the present invention is characterized by comprising a polynucleotide including a replication origin and a nuclear matrix-binding region that function in eukaryotic cells, and an inverted repeat sequence. The kit according to the present invention is not limited to the above configuration as long as the method according to the present invention can be performed, and may include other configurations. In addition, about description of the articles | goods which comprise the kit concerning this invention, description of the method concerning this invention can be used suitably.

  By the way, the method according to the present invention described above can be applied to a system other than the advanced gene amplification system developed by the present inventor, that is, a normal system that does not use IR and MAR. Specifically, the method includes a step of culturing a mammalian cell into which a polynucleotide encoding a protein to be expressed is introduced under a low oxygen concentration condition that is less than the oxygen concentration at which the mammalian cell is normally cultured. The present invention also includes a method for increasing the efficiency of gene amplification. Furthermore, the present invention also provides a method for increasing the efficiency of gene amplification, comprising the step of simultaneously introducing a polynucleotide encoding a protein to be expressed and an inverted repeat sequence (batch sequence) into a mammalian cell. Include. Furthermore, the present invention also includes a kit for carrying out a method for increasing the efficiency of gene amplification, comprising an inverted repeat sequence (batch sequence). For the description of the embodiment using a system other than the advanced gene amplification system, the above description can be used as appropriate, except for the explanation of IR and MAR.

  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.

[Example 1]
(Method)
Two plasmids (pΔBNAR1 and pΔBNTRE) having a blasticidin resistance gene (referred to as “BSR” as appropriate), IR and MAR are introduced into CHO-K1 cells (hereinafter referred to as “CHO cells”) by lipofection, respectively. did. Thereafter, transformants were selected with 5 μg / ml blasticidin. During selection with blasticidin, the oxygen concentration in the culture atmosphere is set to 20% (v / v) of the normal condition, 10% (v / v) of the hypoxic condition, or 5% (v / v) of the hypoxic condition. ) And CHO cells were cultured. After culturing for 4 weeks, the cells were fixed and FISH was performed using a plasmid probe.

  pΔBNAR1 removes the portion expressing the hygromycin resistance gene by simultaneous digestion of pSFVdhfr with Bam HI and Nru I, and then once cloned, followed by replication origin (“DHFR IR”) of the dihydrofolate reductase locus downstream of BSR. It was constructed by cutting at the Not I site at the part in contact with (where appropriate) and inserting AR1 having MAR activity at that position. The AR1 was obtained by the method described in “N. Shimizu, et al. (2001) Cancer Research, vol. 61, p6987-6990.”.

  In addition, pΔBNTRE removes the portion expressing the hygromycin resistance gene by simultaneous digestion of pSFVdhfr with Bam HI and Nru I, and then at that position, TRE promoter-multicloning site (denoted as “MCS”)-SV40 polyA It was constructed by inserting a sequence. The TRE promoter-MCS-SV40 polyA sequence was obtained by digesting pTRE2Hyg (purchased from Clontech) with Xho I and Ase II.

  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 from the 3′-downstream region of hydrofolate reductase (“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.).

CHO cells were obtained from Tohoku University Institute of Aging Medicine, Medical Cell Resource Center. Cell culture was performed in a medium in which 10% fetal bovine serum was added to D-MEM / F-12 medium (Gibco) and the above cell line in the presence of 37 ° C. and 5% CO ₂ .

  The above plasmid was 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).

  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.

(result)
FIG. 1 shows a gene amplification structure (a gene is amplified as a small amplification region by HSR (indicated by “small HSR”) in a polyclonal population of transformed cells into which pΔBNAR1 has been introduced. Frequency of amplification (indicated by “medium-sized HSR”) and gene being amplified as a large amplification region by HSR (indicated by “large-sized HSR”) (ie, “gene amplification structure formation frequency”) showed that. FIG. 2 shows the frequency of formation of gene amplification structures (small HSR, medium HSR, and large HSR) in a polyclonal population of transformed cells introduced with pΔBNTRE. In addition, it shows that gene amplification efficiency is so high that the sum total of gene amplification structure formation frequency (it is also only called "amplification structure formation frequency") is large. Further, as the amplification structure increases in the order of small HSR → middle HSR → large HSR, it is possible to enjoy the advantage that the copy number of the target gene per cell can be remarkably increased. Regarding the size of the amplified structure, an interphase chromosome sample was prepared according to the method described in “The ACT Cytogenetics Laboratory Manual (second edition” edited by Margaret J. Barch, Raven Press (1991). In the specimen, confirmation was made based on the width of the chromosome arm.

  The horizontal axis of FIGS. 1 and 2 represents the oxygen concentration% (v / v) in the culture atmosphere, and the vertical axis represents the amplification structure formation frequency (%). According to both figures, transformed cells introduced with plasmids (pΔBNAR1 and pΔBNTRE) equipped with IR and MAR were subjected to low oxygen concentration conditions (10% (v / v) or 5% (v / v)). It was shown that the frequency of amplification structure formation was clearly higher in the case of cultivating under the condition (2) than in the case of culturing under normal oxygen concentration conditions (20% (v / v)). In addition, the cells cultured under low oxygen concentration conditions tended to increase in size of the amplified structure (increase in medium-sized HSR or large-sized HSR).

  Note that “L”, “M”, and “S” in FIGS. 1 and 2 indicate “large HSR”, “medium HSR”, and “small HSR”, respectively (the same applies to the following examples).

[Example 2]
(Method)
A plasmid (pSFVdhfr / d2EGFP) having an expression marker d2EGFP structural gene (hereinafter “d2EGFP gene”) downstream of the TRE promoter and having IR and MAR, or having a TRE promoter and d2EGFP gene and having no IR and MAR The plasmid (pSFV-Vd2EGFP) was introduced into CHO cells together with Tet-On plasmid (Clontech) by lipofection. Thereafter, transformants were selected with 5 μg / ml blasticidin. During selection with blasticidin, the oxygen concentration in the culture atmosphere is set to 20% (v / v) of the normal condition, 10% (v / v) of the hypoxic condition, or 5% (v / v) of the hypoxic condition. ) And CHO cells were cultured. After culturing for 4 weeks, the cells were fixed and FISH was performed using a plasmid probe. After 4 weeks of culture, Doxycycline (Clontech, hereinafter referred to as “Dox”) is added to the medium at 1 μg / ml to induce protein expression, and the cell sorter d2EGFP (Green Fluorescent Protein). The expression level was quantified. The measurement of the expression level of d2EGFP by a cell sorter was performed by sorting only 20,000 cells by using only live cells as a gate. The cell sorter was manufactured by Becton Dickinson and operating conditions were in accordance with the attached manual.

  pSFVdhfr / d2EGFP removes the hygromycin-resistant gene expression unit of pSFVdhfr, and in that order the TRE promoter (Clontech), d2EGFP gene, SV40 poly A sequence (derived from pTRE-d2EGFP (Clontech)) Thus, it was constructed by incorporating the transcription so that it was directed in the IR direction. The above pSFVdhfr (11.0 kbp) was provided by Dr. John Kolman and Dr. Geoffrey M. Wahl (The Salk Institute, San Diego, CA). PSFVdhfr has a 4.6 kbp fragment containing Oriβ from the 3′-downstream region of hydrofolate reductase (“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.).

  In addition, pSFV-Vd2EGFP was excised from pSFVdhfr by DHFR IR part by Not I digestion, and once cloned, then the expression cassette of hygromycin resistance gene was removed by Bam HI and Nru I digestion, and the TRE promoter (manufactured by Clontech) was placed at that position. ), D2EGFP gene, SV40 poly A sequence (derived from pTRE-d2EGFP (manufactured by Clontech)) was constructed in this order so that transcription was directed in the direction of Bam HI.

(result)
3 to 7 show the results regarding CHO cells into which pSFVdhfr / d2EGFP had been introduced. FIG. 3 shows the frequency of gene amplification structure formation. FIG. 4 shows the polyclonal population of each transformed cell. FIG. 5 is a diagram showing the results of analyzing the d2EGFP expression level of a polyclonal population of transformed cells cultured under 20% (v / v) oxygen conditions using a cell sorter (FIG. 5). d2EGFP expression level = 2,882), FIG. 6 is a diagram showing the results of analyzing the d2EGFP expression level of a polyclonal population of transformed cells cultured under 10% (v / v) oxygen conditions using a cell sorter ( d2EGFP expression level = 10,825), FIG. 7 is a diagram showing the results of analyzing the d2EGFP expression level of a polyclonal population of transformed cells cultured under 5% (v / v) oxygen conditions using a cell sorter ( d2EGFP expression level = 3,422).

  According to FIG. 3, transformed cells introduced with a plasmid (pSFVdhfr / d2EGFP) equipped with IR and MAR were subjected to low oxygen concentration conditions (10% (v / v) or 5% (v / v)). In the case of culturing, the frequency of amplification structure formation tends to be higher than that in the case of culturing under normal oxygen concentration conditions (20% (v / v)), and cells cultured under low oxygen concentration conditions. Showed that the amplification structure was enlarged (large HSR increased). 4-7, the transformed cells into which the plasmid (pSFVdhfr / d2EGFP) provided with IR and MAR was introduced were subjected to low oxygen concentration conditions (10% (v / v) or 5% (v / v). It was shown that the expression level of d2EGFP was higher when cultured in ()) than when cultured under normal oxygen concentration conditions (20% (v / v)).

  8 to 12 show the results regarding CHO cells into which pSFV-Vd2EGFP has been introduced. FIG. 8 shows the frequency of gene amplification structure formation. FIG. 9 shows the polyclonal population of each transformed cell. FIG. 10 is a diagram showing the results of analyzing the d2EGFP expression level of a polyclonal population of transformed cells cultured under 20% (v / v) oxygen conditions using a cell sorter. (D2EGFP expression level = 136), FIG. 11 is a figure which shows the result of having analyzed the d2EGFP expression level of the polyclonal population of the transformed cell cultured by oxygen condition of 10% (v / v) by the cell sorter (d2EGFP Expression level = 165), FIG. 12 is a diagram showing the results of analyzing the expression level of d2EGFP in a polyclonal population of transformed cells cultured under oxygen conditions of 5% (v / v) using a cell sorter (d2EGFP expression level) = 740).

  According to FIG. 8, transformed cells into which a plasmid (pSFV-Vd2EGFP) without IR and MAR was introduced were subjected to low oxygen concentration conditions (10% (v / v) or 5% (v / v)). In the case of culturing, the frequency of amplification structure formation tends to be higher than that in the case of culturing under normal oxygen concentration conditions (20% (v / v)), and cells cultured under low oxygen concentration conditions. Showed that the amplification structure was enlarged (medium-sized HSR was increased). Further, according to FIGS. 9 to 12, transformed cells into which a plasmid (pSFVdhfr / d2EGFP) equipped with IR and MAR was introduced were subjected to low oxygen concentration conditions (10% (v / v) or 5% (v / v). It was shown that the expression level of d2EGFP was higher when cultured in ()) than when cultured under normal oxygen concentration conditions (20% (v / v)).

Example 3
A plasmid (pΔBN.Palimd) having a palindromic sequence of the d2EGFP gene was constructed. Briefly, it was as follows. First, by ligating the d2EGFP gene obtained by digesting pSFVdhfr / d2EGFP with Sac II and Bam HI, a gene construct having a repetitive structure of palindrome of the gene was obtained. Next, a palindromic sequence (1 unit) of the d2EGFP gene was prepared by digesting the gene construct with Bam HI. The obtained d2EGFP gene batch sequence (1 unit) was ligated to the SacII and Bam HI digests of pSFVdhfr / d2EGFP, and the palindromic sequence of the blasticidin resistance gene, IR, MAR, TRE promoter, and d2EGFP gene ( A plasmid (pΔBN.Palind) having 1 unit) was constructed. A schematic diagram of the pΔBN.Palind is shown in FIG. In pΔBN.Palind, the target gene can be said to be a blasticidin resistance gene (“BSR” in FIG. 13).

  pΔBN.Palind having the palindromic sequence of the d2EGFP gene and pSFVdhfr / d2EGFP not having the palindromic sequence of the d2EGFP gene were introduced into CHO cells by lipofection together with the Tet-On plasmid (manufactured by Clontech) (introduction step).

  Each cell after the gene transfer was cultured in a medium containing 5 μg / ml blasticidin for 2 weeks to select a polyclonal population of stable 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, according to the pattern shown below, Dox was added to the culture medium so that it might become 1 microgram / ml.
(I) Dox is added to the system in all steps of the introduction step, the selection step, and the culture step.
(II) Dox is added in the system in the introduction step and the selection step. Dox is not added to the medium during the culture process.
(III) Dox is not added to the system in the introduction step and the selection step. Dox is added to the medium during the 2 weeks of the culture process.
(IV) Dox is not added to the system in the introduction process and the selection process. Dox is added to the medium only in the latter half of the culture process.
(V) Dox is not added to the system in the introduction process and the selection process. Dox is added to the medium only during the last day of the culture process.

  The transformed cells that had undergone the above steps were fixed, and FISH was performed using a plasmid probe. Moreover, about the said cell, the expression level of d2EGFP was quantified with the cell sorter.

(result)
FIG. 14 shows the result of the amplification structure formation frequency of the CHO cell introduced with pΔBN.Palind, and FIG. 15 shows the result of the amplification structure formation frequency of the CHO cell introduced with pSFVdhfr / d2EGFP. In each figure, the horizontal axes (I) to (V) correspond to the Dox addition patterns (I) to (V), respectively. Comparing FIG. 14 with FIG. 15, it can be seen that the amplification structure formation frequency of the CHO cells introduced with pΔBN.Palind is clearly higher than that in the case of pSFVdhfr / d2EGFP. That is, the above results indicate that gene amplification efficiency was increased by introducing palindromic sequences.

  Further, according to the results of FIGS. 14 and 15, the amplified structure of cells added with Dox in the patterns of (III), (IV) and (V) compared to the patterns of (I) and (II). The frequency of formation tended to increase, and the amplification structure was shown to be larger (medium-sized HSR and large-sized HSR were increased). That is, this result indicates that the gene amplification efficiency was improved by carrying out the introduction step and the selection step in a state where the transcription activity of the transcriptional activity-regulated promoter was inactivated.

  16 to 21 show the expression level of d2EGFP in CHO cells into which pSFVdhfr / d2EGFP has been introduced. FIG. 16 is a bar graph showing the expression level of d2EGFP in the Dox addition patterns of (I) to (V), and FIGS. 17 to 21 are cases where Dox is added in the pattern patterns of (I) to (V). (2) d2EGFP expression level = 686, (II) d2EGFP expression level = 1,003, (III) d2EGFP expression level = 3,277, (IV) ) D2EGFP expression level = 3,459, (V) d2EGFP expression level = 3,020).

  According to FIGS. 16 to 21, the expression level of d2EGFP is also higher in the cells to which Dox is added in the patterns (III), (IV) and (V) than in the patterns (I) and (II). However, it was shown to be higher. In other words, this result indicates that the gene amplification efficiency was improved and the d2EGFP was highly expressed in the final stage by performing the introduction and selection steps while the transcriptional activity of the transcriptional activity-regulated promoter was inactivated. ing.

  As described above, according to the method of the present invention, it is possible to increase gene amplification efficiency when a target gene is amplified using an advanced gene amplification system or without using the advanced gene amplification system. Become. Therefore, according to the present invention, the desired protein (for example, useful protein) can be produced in large quantities.

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

It is a bar graph which shows the gene amplification structure formation frequency regarding the polyclonal population of the transformed cell into which p (DELTA) BNAR1 was introduce | transduced. It is a bar graph which shows the gene amplification structure formation frequency regarding the polyclonal population of the transformed cell which introduce | transduced p (DELTA) BNTRE. When CHO cells introduced with pSFVdhfr / d2EGFP are cultured under low oxygen concentration conditions (10% (v / v) or 5% (v / v)), or under normal oxygen concentration conditions (20% (v / v) It is a bar graph which shows the gene amplification structure formation frequency at the time of culture | cultivating in ()). When CHO cells introduced with pSFVdhfr / d2EGFP are cultured under low oxygen concentration conditions (10% (v / v) or 5% (v / v)), or under normal oxygen concentration conditions (20% (v / v) It is a bar graph which shows the d2EGFP expression level when it culture | cultivates in ()). It is a figure which shows the result of having analyzed the expression level of d2EGFP with the cell sorter in the case of culture | cultivating the CHO cell introduce | transduced pSFVdhfr / d2EGFP on normal oxygen concentration conditions (20% (v / v)). It is a figure which shows the result of having analyzed the expression level of d2EGFP with the cell sorter in the case of culture | cultivating the CHO cell introduce | transduced pSFVdhfr / d2EGFP on normal low oxygen concentration conditions (10% (v / v)). It is a figure which shows the result of having analyzed the expression level of d2EGFP with the cell sorter in the case of culture | cultivating the CHO cell in which pSFVdhfr / d2EGFP was introduce | transduced by normal low oxygen concentration conditions (5% (v / v)). When CHO cells introduced with pSFV-Vd2EGFP are cultured under low oxygen concentration conditions (10% (v / v) or 5% (v / v)), or under normal oxygen concentration conditions (20% (v / v) It is a bar graph which shows the gene amplification structure formation frequency at the time of culture | cultivating in ()). When CHO cells introduced with pSFV-Vd2EGFP are cultured under low oxygen concentration conditions (10% (v / v) or 5% (v / v)), or under normal oxygen concentration conditions (20% (v / v) It is a bar graph which shows the d2EGFP expression level when it culture | cultivates in ()). It is a figure which shows the result of having analyzed the expression level of d2EGFP with the cell sorter in the case of culture | cultivating the CHO cell introduce | transduced pSFV-Vd2EGFP on normal oxygen concentration conditions (20% (v / v)). It is a figure which shows the result of having analyzed the expression level of d2EGFP with the cell sorter in the case of culture | cultivating the CHO cell in which pSFV-Vd2EGFP was introduce | transduced on normal low oxygen concentration conditions (10% (v / v)). It is a figure which shows the result of having analyzed the expression level of d2EGFP with the cell sorter in the case of culture | cultivating the CHO cell by which pSFV-Vd2EGFP was introduce | transduced on normal low oxygen concentration conditions (5% (v / v)). It is a schematic diagram which shows the structure of p (DELTA) BN.Palimd. It is a bar graph which shows the result of the amplification structure formation frequency of the CHO cell which introduce | transduced p (DELTA) BN.Palind. It is a bar graph which shows the result of the amplification structure formation frequency of the CHO cell which introduce | transduced pSFVdhfr / d2EGFP. It is a bar graph which shows d2EGFP expression level at the time of adding Dox in each pattern of (I)-(V) with respect to the CHO cell into which pSFVdhfr / d2EGFP was introduced. It is the result of having analyzed the expression level of d2EGFP by the cell sorter when Dox is added in the pattern of (I) to the CHO cell introduced with pSFVdhfr / d2EGFP. It is the result of having analyzed the expression level of d2EGFP by the cell sorter when Dox is added in the pattern of (II) to the CHO cell introduced with pSFVdhfr / d2EGFP. It is the result of having analyzed the expression level of d2EGFP by the cell sorter when Dox is added in the pattern of (III) to the CHO cell into which pSFVdhfr / d2EGFP is introduced. It is the result of having analyzed the expression level of d2EGFP with the cell sorter when Dox is added in the pattern of (IV) to the CHO cell introduced with pSFVdhfr / d2EGFP. It is the result of having analyzed the expression level of d2EGFP with the cell sorter when Dox is added to the CHO cell introduced with pSFVdhfr / d2EGFP in the pattern of (V).

Claims (13)

(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 promoter; And the introduction process which introduce | transduces into the mammalian cell simultaneously the 3rd polynucleotide by which the said 2nd polynucleotide is connected controllably with the said promoter, and the mammalian cell into which the said 1st and 3rd polynucleotide was introduce | transduced In the case of amplifying the second polynucleotide by performing a selection step of selecting
A method for increasing gene amplification efficiency characterized by satisfying the following condition (1):
(1) In the above selection step, a mixture of cells containing 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, Is cultured under a low oxygen concentration condition, which is less than the normal oxygen concentration. The method according to claim 1, further satisfying the following condition (3):
(3) The promoter is a transcriptional activity-regulated promoter whose transcriptional activity is activated or inactivated by a predetermined operation, and the introduction step and selection are performed in a state where the transcriptional activity of the transcriptional activity-regulated promoter is inactivated. Perform the process.   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. Method.   The method according to any one of claims 1 to 3, 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.   The method according to any one of claims 1 to 4, wherein the low oxygen concentration condition in the selection step is an oxygen concentration of less than 20% (v / v).   6. The method according to any one of claims 1 to 5, comprising a culturing step of culturing mammalian cells selected by the selection step and introduced with the first and third polynucleotides.   The method according to claim 6, wherein the culturing step is performed under a low oxygen concentration condition that is lower than an oxygen concentration under which normal mammalian cells are cultured.   The method according to claim 7, wherein the low oxygen concentration condition in the culturing step is an oxygen concentration of less than 20% (v / v). Gene of interest encoding a protein to be expressed, and a polynucleotide comprising a promoter and a polynucleotide in which the target gene is operably linked to the promoter, introducing step for introducing into mammalian cells, and
In the case of amplifying the target gene by performing a selection step of selecting mammalian cells into which the polynucleotide has been introduced, the gene amplification efficiency characterized by satisfying the following condition (1) is increased. Method:
(1) In the selection step, mammalian cells in which the polynucleotide is not introduced, and the mixture of cells in which the polynucleotide include mammalian cells transfected, mammalian cells is often less than the oxygen concentration of the culture Incubate under certain hypoxic conditions.   The method according to claim 9, wherein the low oxygen concentration condition in the selection step is an oxygen concentration of less than 20% (v / v). The method according to claim 9 or 10, comprising a culturing step of culturing the mammalian cell selected by the selection step and introduced with the polynucleotide .   The method according to claim 11, wherein the culturing step is performed under a low oxygen concentration condition that is lower than an oxygen concentration under which normal mammalian cells are cultured.   The method according to claim 12, wherein the low oxygen concentration condition in the culturing step is an oxygen concentration of less than 20% (v / v).

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