JP2006345703A - Mammalian host cell of overexpression type for general use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for conveniently and quickly obtaining a gene-amplified mammalian cell for general use and to provide a mammalian cell obtained thereby. <P>SOLUTION: The mammalian cell has a plural number of sites into which a vector containing an arbitrary foreign gene can be inserted by specific site-specific recombination. More particularly speaking, the mammalian cell is produced by introducing an expression vector containing a first site-specific recombinant sequence and a selection marker into a mammalian cell, carrying out gene amplification gene by using a gene amplification and induction factor corresponding to the selection marker and then introducing a vector containing a second site-specific recombinant sequence and a foreign gene and site-specifically recombining the first site-specific recombinant sequence with the second site-specific recombinant sequence by action of a site-specific recombinant induction factor. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing a mammalian host cell for recombinant expression of a foreign gene with high efficiency, a host cell produced by such a method, and a method for producing a protein using the same.

  Proteins expressed in eukaryotes include proteins with added sugar chains, that is, glycoproteins. The function of the sugar chain in glycoproteins has not been fully elucidated, but it plays an important role in interactions between glycoprotein molecules and other molecules, cell-cell interactions through glycoproteins, Moreover, it is thought that the modification of the sugar chain part affects the function, immunological properties, etc. of the glycoprotein. Therefore, in order to produce a glycoprotein having a desired function / characteristic, it is necessary to produce a protein having a target sugar chain.

  The sugar chain portion of a mammalian glycoprotein generally has a complex sugar chain structure in which a plurality of types of sugars such as mannose, N-acetylglucosamine, galactose, and fucose are bonded in various bond modes including branches. In addition, since complex sugar chain structures differ depending on the cell type, it is necessary to use mammalian cells as a host for recombinant expression in order to express glycoproteins derived from mammals (eg, humans). . In particular, when producing a protein pharmaceutical, it is often necessary to produce a protein having an appropriate complex-type sugar chain structure, and thus it is necessary to use mammalian cells as a host.

  However, recombinant expression of glycoproteins using mammalian cells as host cells has the disadvantages of low growth rate and expensive media compared to other host cells such as bacteria and yeast. One means of overcoming such drawbacks is to increase the recombinant expression efficiency of mammalian host cells, i.e. the productivity of proteins per hour per cell, i.e. the specific production rate.

  In order to increase the specific production rate, it is effective to perform gene amplification in which the gene to be recombinantly expressed is linked to a strong promoter and the copy number of the recombinant gene is increased. Strong promoters for recombinant expression include, for example, various natural promoters (eg, SV40 early promoter, adenovirus E1A promoter, human cytomegalovirus (CMV) promoter, human elongation factor-1 (EF -1) Promoter, Drosophia minimal heat shock protein 70 (HSP) promoter, human metallothionein (MT) promoter, Rous sarcoma virus (RSV) promoter, human ubiquitin C (UBC) promoter, human actin promoter), and artificial promoters ( For example, SRα promoter (SV40 early promoter and HTLV LTR promoter fusion), CAG promoter (CMV-IE enhancer and chicken actin promoter) Since the fusion promoter), such as a hybrid) are well known, by using these known promoter or variants thereof, it can be easily increased the expression level of recombinant expression. On the other hand, performing gene amplification for a desired recombinant gene requires a great deal of time and effort.

  Gene amplification refers to an increase in the number of copies of a chromosomal DNA region containing a specific gene. As a method of artificially amplifying a gene and isolating the amplified mammalian cell, for example, by selecting a drug-resistant cell step by step in a cultured cell of the mammal, resistance to the drug is obtained. And a method of isolating a clone having an increased copy number of a drug resistance gene (for example, dihydrofolate reductase (DHFR)), which is a gene responsible for. In mammalian cells isolated using this method, not only drug resistance genes but also other genes in the vicinity thereof are amplified simultaneously. Therefore, isolation of a cell in which a desired gene has been amplified is performed by inserting a nucleic acid fragment containing the desired gene and a drug resistance gene into a chromosome or a nucleic acid fragment containing a partial sequence of such a gene as a minigene. This is made possible by isolating cells that have been inserted into the chromosome and whose drug resistance gene has undergone gene amplification. This gene amplification method is well known to those skilled in the art, for example, see Schimke RT. "Gene amplification in cultured cells." Biol. Chem. 1988 May 5; 263 (13): 5989-92, Weidle UH, Buckel P, Wienberg J. et al. ”Amplified expression constructs for human tissue-type plasminogen activator in Chinese hamster ovary cells: 198 rev. “Selection and co-operation of heterogeneous genes in mammalian cells”, Methods Enzymol. 1990; 185: 537-66.

  However, when this method is used, there is no versatility with respect to genes, and it is necessary to select drug-resistant cells step by step for each desired gene. In addition, this selection usually requires about 1 to 3 months, and since the selection is usually performed in 5 stages, the entire selection process requires an average of about 7 months. It takes a lot of time and effort to select a host cell. This is because if a transformed host cell is suddenly exposed to a high concentration of drug, all cells will die before gene amplification occurs. This is because it is necessary to repeat the drug concentration stepwise up to the drug concentration.

  In addition, this gene amplification event does not occur in all cells at the same time, but occurs in some cells at various times, and the resulting cell population is naturally heterogeneous, even at high concentrations. Even if the clone is resistant to any of the above drugs, it is not necessarily a clone that amplifies the desired gene. Therefore, a host cell that carries out recombinant expression with higher efficiency from the heterogeneous population (ie, the gene copy number). More labor and time (usually 1-2 months) is required. Furthermore, when the recombinantly expressed protein is produced industrially, particularly when it is produced for use in pharmaceuticals, strict quality control is required. However, production using the heterogeneous cell population described above causes variations in quality. This cloning work is also required because the resulting quality control is not easy.

  In addition, some cell lines constructed by this conventional method are unstable and some cells change their cytological properties during subculture. Therefore, it is necessary to test the stability of the cloned cells by further culturing in a medium containing no inhibitor for about 2 months for a long time.

  Although a method for rapidly selecting a gene-amplified cell has been developed (Japanese Patent Laid-Open No. 2001-37478), there is still a versatile method for producing a mammalian cell capable of highly expressing a foreign gene, and so on. No such versatile mammalian cell has been developed.

Therefore, a method for obtaining a gene amplification host cell having versatility, simply and rapidly, and a host cell obtained by such a method are desired.
JP 2001-37478 A

  An object of the present invention is to provide a method for obtaining a gene amplification host cell which has versatility and is simply and rapidly, and a mammalian cell obtained by such a method.

  The above object has been achieved by developing a mammalian cell having a plurality of sites into which a vector containing any foreign gene can be inserted by site-specific recombination.

  Specifically, the present invention introduces an expression vector containing a first site-specific recombination sequence and a selection marker into a mammalian cell, and a nucleic acid sequence contained in the vector is amplified by a gene corresponding to the selection marker. Gene amplification using an inducer, and then introducing a vector containing a second site-specific recombination sequence and a foreign gene, the first site-specific recombination sequence and the second site-specific recombination sequence Was achieved by producing mammalian cells by site-specific recombination by the action of site-specific recombination inducers.

  In the present invention, an expression vector containing a first site-specific recombination sequence and a selectable marker is introduced into a mammalian cell, and the gene and nucleic acid sequence contained in the vector are converted into gene amplification inducers corresponding to the selectable marker. Mammalian cells obtained by using and gene amplification are cells having a plurality of site-specific recombination sequences. Therefore, the mammalian cell prepared in this way can be used as a material for easily preparing a mammalian cell for inserting any foreign gene into a chromosome and highly expressing the foreign gene. Specifically, a vector containing an arbitrary foreign gene and a second site-specific recombination sequence is introduced into the mammalian cells thus prepared, and the first site-specific recombination is introduced. Mammalian cells containing multiple copies of any foreign gene in the chromosome are produced by site-specific recombination of the sequence with the second site-specific recombination sequence by the action of a site-specific recombination inducer. .

  In accordance with the production method provided by the present invention, mammalian cells that highly express a foreign gene are provided more simply and more quickly than conventional methods. Furthermore, the mammalian cell of the present invention has the versatility that it can contain multiple copies of any foreign gene expression vector.

  Further, in the present invention, the positions on the chromosome where gene amplification occurs efficiently are in the vicinity of the centromere of the E chromosome, the centromere of the J chromosome, the centromere of the K chromosome, the centromere of the L chromosome, the telomere of the N chromosome, Near the telomere pole of the chromosome, near the P chromosome telomere, near the P chromosome telomere, or near the P chromosome telomere, preferably near the centromere of the K chromosome, near the centromere of the L chromosome, near the telomere of the N chromosome, telomere of the N chromosome Near the pole, near the P chromosome telomere, near the P chromosome telomere, or near the P chromosome telomere, more preferably near the N chromosome telomere, near the N chromosome telomere, near the P chromosome telomere, or near the P chromosome telomere pole And even more preferably, the telomeres of the N chromosome Near or in the vicinity P chromosomal telomeres poles, and most preferably, has been identified as being near P chromosomal telomeres pole.

  Accordingly, in the present invention, there is provided a method for producing a mammalian cell that can be used to express a foreign gene, wherein a nucleic acid fragment comprising a first site-specific recombination sequence and a selectable marker gene is isolated from the mammalian cell. After insertion into a chromosome and culturing the mammalian cell in the presence of a gene amplification inducer to cause gene amplification of a nucleic acid fragment containing a selectable marker gene, the insertion position in the chromosome of the nucleic acid fragment is By selecting mammalian cells near the telomere pole or near the P chromosome telomere pole, the gene containing the first site-specific recombination sequence and the selectable marker gene was amplified at a position where gene amplification was likely to occur in the chromosome. A method of producing a mammalian host cell having a nucleic acid sequence is provided. Mammalian host cells so produced can be (i) cultured in the presence of a gene amplification inducer to produce further gene amplification, or (ii) by the action of a site-specific recombination inducer. Causing site-specific recombination between the first site-specific recombination sequence and a second site-specific recombination sequence homologous to the first site-specific recombination sequence, The nucleic acid fragment containing the site-specific recombination sequence and the foreign gene is inserted into the chromosome and then cultured in the presence of a gene amplification inducer to produce further gene amplification.

Accordingly, the present invention provides the following.
1. A mammalian cell containing at least three nucleic acid fragments containing a site-specific recombination sequence and a selectable marker gene inserted into a chromosome.
2. The mammalian cell according to Item 1, further comprising a site-specific recombination inducer.
3. The mammalian cell according to Item 1, wherein the selectable marker gene is selected from the group consisting of: dihydrofolate reductase gene, glutamine synthetase gene, aspartate transaminase, metallothionein, adenosine deaminase (ADA), adenosine deaminase ( AMPD1, 2), xanthine-guanine-phosphoribosyltransferase, UMP synthase, P-glycoprotein, asparagine synthetase, ornithine decarboxylase.
4). The mammalian cell according to item 3, wherein the selectable marker gene is dihydrofolate reductase.
5. Item 3. The mammalian cell according to Item 2, wherein the site-specific recombination inducer is Cre protein, and the site-specific recombination sequence is a wild type loxP sequence or a mutant type loxP sequence.
6). The mammalian cell according to item 1, wherein the insertion position in the chromosome of the nucleic acid fragment is inserted near the centromere, near the telomere, or near the telomere pole of the chromosome of the mammalian cell.
7). The mammalian cell according to item 1, which is a CHO cell.
8). The insertion position in the chromosome of the nucleic acid fragment is in the vicinity of the centromere of the E chromosome, the centromere of the J chromosome, the centromere of the K chromosome, the centromere of the L chromosome, the telomere of the N chromosome, the telomere pole of the N chromosome of the CHO cell. Item 8. The mammalian cell according to item 7, which is near, P chromosome telomere side, P chromosome telomere vicinity, or P chromosome telomere pole vicinity.
9. Item 9. The mammalian cell according to item 8, wherein the insertion position in the chromosome of the nucleic acid fragment is in the vicinity of the telomer pole of the N chromosome or the pole of the P chromosome telomere in the CHO cell.
10. The mammalian cell according to item 1, wherein a nucleic acid fragment containing a sequence homologous to the site-specific recombination sequence inserted into a chromosome can be inserted into the chromosome by site-specific recombination.
11. Item 2. The mammalian cell according to item 1, wherein a nucleic acid fragment containing a sequence homologous to the site-specific recombination sequence inserted into a chromosome is inserted into the chromosome by site-specific recombination.
12 Item 12. The mammalian cell according to Item 11, wherein the nucleic acid fragment inserted into the chromosome by site-specific recombination contains a foreign gene operably linked to a promoter.
13. Item 13. A method for producing a foreign gene product, comprising culturing the mammalian cell according to Item 12 under conditions under which the mammalian cell expresses the foreign gene.
14 A kit comprising the mammalian cell according to item 1 or 2.
15. A method for producing a mammalian cell that can be used to express a foreign gene comprising the following steps:
(A) inserting a nucleic acid fragment containing a first site-specific recombination sequence and a selectable marker gene into the chromosome of the mammalian cell; and (b) culturing the mammalian cell in the presence of a gene amplification inducer. And causing gene amplification of a nucleic acid fragment containing a selectable marker gene;
Including the method.
16. Furthermore, the manufacturing method of item 15 including the following processes:
(C) A step of selecting a mammalian cell in which the insertion position in the chromosome of the nucleic acid fragment is near the N-chromosome telomere pole or near the P-chromosome telomere pole.
17. In addition, any of the following steps:
(D) introducing a site-specific recombination inducer into the mammalian cell; or (d ′) introducing a vector capable of expressing the site-specific recombination inducer into the mammalian cell. ,
18. The production method according to item 15, comprising In addition, the following steps:
(E) introducing a nucleic acid fragment containing a foreign gene operably linked to a promoter and a second site-specific recombination sequence homologous to the first site-specific recombination sequence into the mammalian cell; Item 18. The production method according to Item 17, comprising a step of site-specific recombination of the first site-specific recombination sequence and the second site-specific recombination sequence with the site-specific recombination inducer. .
19. 16. The production method according to item 15, wherein the selection marker gene is selected from the group consisting of: dihydrofolate reductase gene, glutamine synthetase gene, aspartate transaminase, metallothionein, adenosine deaminase (ADA), adenosine deaminase (AMPD1) 2), xanthine-guanine-phosphoribosyltransferase, UMP synthase, P-glycoprotein, asparagine synthetase, ornithine decarboxylase.
20. Item 20. The production method according to Item 19, wherein the selectable marker gene is dihydrofolate reductase.
21. Item wherein the site-specific recombination inducer is Cre protein, and at least one of the first site-specific recombination sequence and the second site-specific recombination sequence is a wild type or mutant loxP sequence. 18. The production method according to 17.
22. Item 16. The production method according to Item 15, wherein the mammalian cell is a CHO cell or a CHO cell-derived cell.
23. Item 16. The production method according to Item 15, wherein the step (b) of causing gene amplification is performed under conditions in which cells in which a gene contained in a vector inserted in the vicinity of telomeres is amplified selectively appear.
24. A mammalian cell produced by the method according to any one of items 15 to 18.
25. 19. A method for producing a foreign gene product, the method comprising culturing a mammalian cell produced by the method according to Item 18 under conditions under which the mammalian cell expresses the foreign gene.

  The present invention provides a method for producing a mammalian cell that highly expresses a foreign gene in a simpler and quicker manner than conventional methods, and a mammalian cell produced by such a method. Furthermore, general-purpose mammalian cells that can be easily applied to any foreign gene without being limited to a specific gene are also provided.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.

  Listed below are definitions of terms particularly used in the present specification.

  As used herein, the term “site-specific recombination sequence” means that site-specific recombination occurs in mammalian cells depending on the action of the site-specific recombination inducer. A sequence that does not cause site-specific recombination when does not act. Site-specific recombination refers to recombination that occurs depending on the action of a site-specific recombination inducer in a portion having a homologous base sequence of a pair of double-stranded DNAs.

Whether or not “site-specific recombination” has occurred can be easily confirmed using, for example, a method including the following steps:
(1) introducing a nucleic acid fragment containing a site-specific recombination sequence and a foreign gene into a cell; and (2) whether the foreign gene has been inserted in the vicinity of the site-specific recombination sequence in the chromosome. And a step of detecting using a general genomic Southern hybridization method or a genomic PCR method.

  As used herein, “gene amplification” refers to a phenomenon in which the number of specific genes in a set of chromosomes is amplified in the life cycle of an organism. As used herein, “gene amplification inducing factor” refers to a factor that causes gene amplification of a specific gene by culturing mammalian cells in the presence thereof. A gene amplification inducer results in gene amplification of a particular gene or genes, but not other genes. In the present specification, a specific gene corresponding to a gene amplification inducer is also referred to as a “selection marker” gene. When the gene amplification inducing factor is a drug, examples of the selectable marker gene include a gene resistant to the drug.

The combination of a gene amplification inducing factor and a selectable marker gene is, for example, a combination of a factor that inhibits and / or suppresses the growth of mammalian cells and a gene that expresses a protein that removes, suppresses and / or inhibits the action of the factor It is. Preferred combinations of gene amplification inducers and selectable marker genes include, but are not limited to: a combination of dihydrofolate reductase gene (DHFR) and methotrexate (MTX), glutamine synthetase (GS) and A combination of methionine sulfoximine (Msx), a combination of aspartate transaminase (CAD) and N-phosphonacetyl-L-aspartate (PALA), metallothionein (MT) and cadmium ( combination of cd2 ^+), adenine adenosine deaminase (ADA) and adenosine, alanosine, the combination of the 2'-deoxycoformycin, an adenosine deaminase (AMPD1,2), A combination of zaserine and coformycin, a combination of xanthine-guanine-phosphoribosyltransferase and mycophenolic acid, a combination of UMP synthase with 6-azauridine, pyrazofuran, P-glycoprotein (P-gp, MDR) and Combination with multiple drugs, combination of asparagine synthetase (AS) and β-aspartyl hydroxamic acid or albizzinin, ornithine decarboxylase (ODC) and α-difluoromethyl-ornithine (DFMO).

In gene amplification, it is known that by adjusting the concentration of a gene amplification inducing factor, a condition in which cells in which a gene contained in a vector inserted into a telomere is amplified appears selectively can be obtained (Japanese Patent Application Laid-Open No. 2005-318867). 2001-37478)
In this specification, the “telomere side” of the chromosome means a half region on the telomere side when the centromere and the telomere are equally divided into two for each of the short arm and the long arm of the chromosome. Say. In the present specification, the “centromere side” of a chromosome refers to a half region on the centromere side when the centromere and the telomere are equally divided into two for each of the short arm and the long arm of the chromosome. . The term “equally divided” as used herein means that the chromosomes are equally divided based on the images of the chromosomes when the chromosomes are observed using the FISH method.

  In the present specification, the “near telomere” of the chromosome means a region of one third on the telomere side when the centromere and the telomere are equally divided into three for each of the short arm and the long arm of the chromosome. Say. In the present specification, the “near centromere” of the chromosome means a centromere side one-third region when the centromere and telomere are equally divided into three for each of the short arm and long arm of the chromosome. Say.

  In the present specification, the “near telomere pole” of the chromosome means a region of a quarter on the telomere side when the centromere and the telomere are equally divided into four for each of the short arm and the long arm of the chromosome. Say. In this specification, the “centromere pole vicinity” of a chromosome means a centromere side quarter region when the centromere and telomere are equally divided into four for each of the short arm and long arm of the chromosome. Say.

  A typical method for mapping a foreign gene insertion position in a chromosome is the FISH method (Fluorescence in situ hybridization; fluorescence in situ hybridization). In the FISH method, the introduced target gene is used as a probe, and DAPI (4 ', 6'-diamidino-2-phenylindole) is used for counterstaining. Chromosomes are stained and dark bands are produced by using DAPI, so that the insertion position of the chromosome can be specified by superimposing the image by FISH and the image by DAPI using the introduced target gene as a probe. In order to specify which chromosome has entered, it is necessary to stain using a chromosome-specific fluorescent probe. Since there is no such probe in the case of CHO cells, image analysis is performed using the band pattern obtained by DAPI to classify chromosomes.

  In the present invention, site-specific recombination sequences are inserted into the host chromosome. The insertion position of the site-specific recombination sequence is not particularly limited, and may be on the centromere side of the chromosome or on the telomere side. The insertion position of the site-specific recombination sequence is preferably near the centromere of the chromosome or near the telomere of the chromosome, more preferably near the telomere of the chromosome, and even more preferably near the telomere pole of the chromosome.

  As used herein, “site-specific recombination inducer” refers to a factor that causes site-specific recombination of a site-specific recombination sequence in a mammalian cell. Typically, this site-specific recombination inducer is a protein.

  The “site-specific recombination inducer” and the “site-specific recombination sequence” have specificity for each other. Therefore, in order to cause site-specific recombination with a specific site-specific recombination sequence, it is necessary to select a site-specific recombination inducer suitable for the site-specific recombination sequence. Exemplary “site-specific recombination inducers” and “site-specific recombination sequences” combinations include, but are not limited to: loxP from bacteriophage P1 (locus of crossover of P1) ) Sequence and Cre (cyclization recombination) protein, Flp protein and FRT site, φC31 and attB, attP (Thorpe, Helena M .; Wilson, Stuart E .; Smith, Margaret C.M. , Control of directionality in the site-specific recombination system of the Streptomyces phase φC31., Molecular Microbiology (2000), 38 (2), 232-241.), a combination of a resolvase and a res site (Sadowski P., Site-specific recombinases: changing partners and doting. 1986; 165 (2) 341-7) (see generally Sauer B. Site-specific recombination: developments and applications., Curr Opin Biotechnol. 1994 Oct; 5 (5): 521-7. )

  Whether or not the combination of the site-specific recombination inducer and the site-specific recombination sequence of the present invention is appropriate is determined by “site-specific recombination” using genomic Southern hybridization or genomic PCR. It can be confirmed by testing whether it has occurred.

  As long as it retains the same site-specific recombination function as that of the site-specific recombination inducer, it is also possible to use a variant of the site-specific recombination inducer or a homolog or ortholog instead of the site-specific recombination inducer. it can. Alternatively, sequences homologous to site-specific recombination sequences can be used as target sequences that cause site-specific recombination sequences and site-specific recombination, or in place of site-specific recombination sequences.

  As used herein, the terms “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acid may be natural or non-natural and may be a modified amino acid. The term can also be assembled into a complex of multiple polypeptide chains. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) and other modifications known in the art. Is included.

  As used herein, the terms “polynucleotide”, “oligonucleotide”, and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “derivative oligonucleotide” or “derivative polynucleotide”. A “derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or that has an unusual linkage between nucleotides and is used interchangeably. Specific examples of such an oligonucleotide include, for example, 2′-O-methyl-ribonucleotide, a derivative oligonucleotide in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in an oligonucleotide. Is an N3′-P5 ′ phosphoramidate bond derivative oligonucleotide, a ribose and phosphodiester bond in the oligonucleotide converted to a peptide nucleic acid bond, uracil in the oligonucleotide is C− A derivative oligonucleotide substituted with 5-propynyluracil, a derivative oligonucleotide where uracil in the oligonucleotide is substituted with C-5 thiazole uracil, and cytosine in the oligonucleotide is C-5 propynylcytosine Substituted derivative oligonucleotides, derivative oligonucleotides in which the cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, derivative oligonucleotides in which the ribose in the DNA is replaced with 2'-O-propylribose Examples thereof include derivative oligonucleotides in which ribose in the oligonucleotide is substituted with 2′-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-). 98 (1994)). The term “nucleic acid” is also used herein interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. Particular nucleic acid sequences also include “splice variants”. Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. As the name suggests, a “splice variant” is the product of alternative splicing of a gene. After transcription, the initial nucleic acid transcript can be spliced such that different (another) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants varies, but includes exon alternative splicing. Other polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition.

  As used herein, “gene” refers to a factor that defines a genetic trait. Usually arranged in a certain order on the chromosome. A gene that defines the primary structure of a protein is called a structural gene, and a regulatory gene that affects its expression. As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide” and “nucleic acid” and / or “protein” “polypeptide”, “oligopeptide” and “peptide”.

  As used herein, “homology” of genes refers to the degree of identity of two or more gene sequences with respect to each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous.

  As used herein, a site-specific recombination sequence is “homologous” when two or more site-specific recombination sequences are recombined with each other in a mammalian cell depending on a site-specific recombination inducer. It means having homology to such an extent that the change occurs. Thus, one of ordinary skill in the art would be able to introduce one to several deletions, additions and / or substitutions into a site-specific recombination sequence and still be “homologous” to the original site-specific recombination sequence. Can be easily obtained.

  In this specification, comparison of base sequence identity and calculation of homology are calculated using BLAST, which is a sequence analysis tool, using default parameters.

  In the present specification, “expression” of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo to take another form. Preferably, a gene, polynucleotide or the like is transcribed and translated into a polypeptide form. However, transcription and production of mRNA can also be an aspect of expression. More preferably, such a polypeptide form may be post-translationally processed.

  In the present specification, the “amino acid” may be natural or non-natural. “Derivative amino acid” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such derivative amino acids and amino acid analogs are well known in the art. The term “natural amino acid” refers to the L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine , And lysine. Unless otherwise indicated, all amino acids referred to in this specification are in L form. The term “unnatural amino acid” refers to an amino acid that is not normally found in proteins in nature. Examples of non-natural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homo-arginine D-form or L-form and D-phenylalanine. “Amino acid analog” refers to a molecule that is not an amino acid but is similar to the physical properties and / or function of an amino acid. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by the generally accepted single letter code.

  In the present specification, a “corresponding” amino acid has or is predicted to have the same action as a predetermined amino acid in a protein or polypeptide as a reference for comparison in a protein molecule or polypeptide molecule. In particular, in the case of an enzyme molecule, it means an amino acid that is present at the same position in the active site and contributes similarly to the catalytic activity.

  In the present specification, the “nucleotide” may be natural or non-natural. “Derivative nucleotide” or “nucleotide analog” refers to a nucleotide that is different from a naturally occurring nucleotide but has the same function as the original nucleotide. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA). .

  In the present specification, the “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit.

  As used herein, “biological activity” refers to activity that a certain nucleic acid sequence or a factor (eg, polypeptide or protein) can have in vivo, and includes activities that exhibit various functions. Is done. For example, if a nucleic acid is a site-specific recombination sequence, its biological activity is specific to a sequence that is homologous to that site-specific recombination sequence by the action of a site-specific recombination inducer. It is an activity that causes site-specific recombination. When a factor is an enzyme, its biological activity includes that enzyme activity. In another example, when an agent is a ligand, the ligand includes binding to the corresponding receptor.

  When the site-specific recombination inducer is a protein, the amino acids in the protein can be expressed in a protein structure such as, for example, a cationic region or a binding site of a substrate molecule, without a clear decrease or loss of interaction binding capacity. It can be substituted with other amino acids. It is the protein's ability to interact and the nature that defines the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in proteins that still retain their original properties after substitution. Thus, various modifications can be made in the peptide disclosed herein or in the corresponding DNA encoding this peptide without any apparent loss of biological utility.

  In designing such modifications, the hydrophobicity index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ. Mol. Biol. 157 ( 1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the protein produced and then defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1 .8); Glycine (−0.4); Threonine (−0.7); Serine (−0.8); Tryptophan (−0.9); Tyrosine (−1.3); Proline (−1.6) ); Histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) And arginine (-4.5)).

  It is well known in the art that one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still result in a protein having a similar biological function (eg, an equivalent protein in enzymatic activity). It is. In such amino acid substitution, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline ( Alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−0.5 ± 1); -1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). It is understood that an amino acid can be substituted with another that has a similar hydrophilicity index and can still provide a biological equivalent. In such amino acid substitution, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

  In the present invention, “conservative substitution” refers to a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid to be replaced with the original amino acid is similar as described above. Examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within the following groups: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine; However, it is not limited to these.

  In the present specification, the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. In this specification, these original polypeptides or polynucleotides are also referred to as “wild-type” polypeptides or “wild-type” polynucleotides. In the present specification, the terms “variant” and “mutant” are used interchangeably. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse alpha hemoglobin genes are orthologs, but the human alpha and beta hemoglobin genes are paralogs (genes generated by gene duplication). . Since orthologs are useful for the estimation of molecular phylogenetic trees, the orthologs of the present invention may also be useful in the present invention.

  “Conservative (modified) variants” applies to both amino acid and nucleic acid sequences. A conservatively modified variant with respect to a particular nucleic acid sequence refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, and if the nucleic acid does not encode an amino acid sequence, its biological function An array that holds When encoding an amino acid sequence, a large number of functionally identical nucleic acids encode any given protein due to the degeneracy of the genetic code. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent modifications (mutations)” which are one species of conservatively modified mutations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of that nucleic acid. In the art, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan), produces a functionally identical molecule. It is understood that it can be modified. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

  As used herein, in addition to amino acid substitutions, amino acid additions, deletions, or modifications can also be made to produce functionally equivalent polypeptides. Amino acid substitution means that the original peptide is substituted with one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids. The addition of amino acids means that one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids are added to the original peptide chain. Deletion of amino acids means deletion of one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids from the original peptide. Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation), and the like. The amino acid to be substituted or added may be a natural amino acid, a non-natural amino acid, or an amino acid analog. Natural amino acids are preferred.

  As used herein, the term “peptide analog” refers to a compound that is different from a peptide but is equivalent to the peptide in at least one chemical or biological function. Thus, peptide analogs include those in which one or more amino acid analogs are added or substituted to the original peptide. Peptide analogs have functions similar to those of the original peptide (for example, similar pKa values, similar functional groups, similar binding modes with other molecules, Such additions or substitutions are made so as to be substantially similar to (eg, similarity in sex). Such peptide analogs can be made using techniques well known in the art. Thus, a peptide analog can be a polymer that includes an amino acid analog.

  As used herein, a nucleic acid form of a polypeptide refers to a nucleic acid molecule that can express the protein form of the polypeptide. As long as the expressed polypeptide has substantially the same activity as that of the naturally occurring polypeptide (including activity that causes site-specific recombination), one of the nucleic acid sequences described above is used. The part may be deleted or substituted with another base, or another nucleic acid sequence may be partially inserted. Alternatively, another nucleic acid may be bound to the 5 'end and / or the 3' end. Alternatively, it may be a nucleic acid molecule that hybridizes under stringent conditions with a gene encoding a polypeptide and encodes a polypeptide having substantially the same function as the polypeptide. Such genes are known in the art and can be used in the present invention.

  Such a nucleic acid can be obtained by a well-known PCR method, and can also be chemically synthesized. These methods may be combined with, for example, a site-specific displacement induction method or a hybridization method.

  As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. There may be any number of substitutions, additions or deletions, as long as it is one or more, and such numbers may be used for the desired function in the variant having that substitution, addition or deletion (eg, site-specific recombination). As long as the functions that cause For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

  Macromolecular structures (eg, polypeptide structures) can be described in terms of various levels of configuration. For a general discussion of this configuration, see, for example, Alberts et al., Molecular Biology of the Cell (3rd edition, 1994), and Canter and Schimmel, Biophysical Chemistry Part I: The Conformation of Biologic 80 (Molecular 80). “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally arranged three-dimensional structures within a polypeptide. These structures are generally known as domains. A domain is a portion of a polypeptide that forms a compact unit of the polypeptide and is typically 50-350 amino acids long. Exemplary domains are made up of parts such as β-sheets (such as β-strands) and α-helix stretches. “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide monomer. “Quaternary structure” refers to a three-dimensional structure formed by non-covalent association of independent tertiary units. Terms relating to anisotropy are used in the same way as terms known in the energy field.

  As used herein, when used for expression of a gene, “site specificity” generally refers to the specificity of expression of the gene at a mammalian site. “Time specificity” refers to the specificity of expression of the gene according to the developmental stage of the mammal. Such specificity can be introduced into a desired organism by selecting an appropriate promoter.

  In the present specification, the expression of the promoter of the present invention is "constitutive" means that it is expressed in almost all amounts in all tissues of an organism regardless of whether the organism is growing or proliferating. Say. Specifically, when Northern blot analysis is performed, for example, at any time point (for example, 2 points or more (for example, 5th day and 15th day)), the same or corresponding sites have almost the same level. When the expression level is observed, the expression is constitutive by definition of the present invention. Constitutive promoters are thought to play a role in maintaining homeostasis of organisms in normal growth environments. The expression “responsiveness” of the promoter of the present invention refers to the property that the expression level changes when at least one factor is given to an organism. In particular, the property of increasing the expression level is called “inducibility” with respect to the factor, and the property of decreasing the expression level is called “decreasing” with respect to the factor. The expression of “decreasing” is a concept that overlaps with “constitutive” expression because it is assumed that expression is observed in the normal state. These properties can be determined by extracting RNA from any part of the organism and analyzing the expression level by Northern blot analysis or quantifying the expressed protein by Western blot. Mammalian cells or mammals (including specific tissues etc.) transformed with a vector incorporating a promoter inducible to the factor together with a nucleic acid encoding the site-specific recombination inducing factor of the present invention, the promoter By using a stimulating factor having an inducing activity, site-specific recombination of site-specific recombination sequences can be performed under certain conditions.

  In the present specification, the expression "specifically expresses" a gene means that the gene is expressed at a different level (preferably higher) from another site or time in a specific site or time of an animal. . With specific expression, it may be expressed only at a certain site (specific site) or may be expressed at other sites. The expression specifically preferably means that it is expressed only at a certain site.

  General molecular biology techniques that can be utilized in the present invention include Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, etc. can be easily implemented by those skilled in the art.

  In the present specification, when referring to a gene, a “vector” refers to one that can transfer a target polynucleotide sequence into a target cell. Such a vector can be autonomously replicated in a host cell such as a mammalian cell or can be integrated into a chromosome, and contains a promoter at a position suitable for transcription of the polynucleotide of the present invention. What is present is exemplified.

  “Expression vector” refers to a nucleic acid sequence in which various regulatory elements are operably linked in a host cell in addition to a structural gene and a promoter that regulates its expression. Regulatory elements can preferably include a terminator, a selectable marker such as a drug resistance gene (eg, a neomycin resistance gene) and an enhancer. It is well known to those skilled in the art that the type of mammalian expression vector and the type of regulatory element used can vary depending on the host cell.

  “Recombinant vector” refers to a vector capable of transferring a target polynucleotide sequence into a target cell. Examples of such vectors include those that can replicate autonomously in mammalian host cells or can be integrated into a chromosome and contain a promoter at a position suitable for transcription of the polynucleotide of the present invention. .

  Examples of “recombinant vectors” that can be used in the present invention include pcDNAI / Amp, pcDNAI, pCDM8 (all available from Funakoshi), pAGE107 (Japanese Patent Laid-Open No. 3-22979, Cytotechnology, 3, 133 (1990)), pREP4 (Invitrogen) ), PAGE103 (J. Biochem., 101, 1307 (1987)), pAMo, pAMoA (J. Biol. Chem., 268, 2272-222787 (1993)), pSV2-dhfr (Mol. Cell. Biol. 1,). 854, (1981)), pSV 1GT5-dhfr (Mol. Cell. Biol. 1, 854, (1981)) and the like.

  A “terminator” is a sequence that is located downstream of a region encoding a protein of a gene and is involved in termination of transcription when DNA is transcribed into mRNA and addition of a poly A sequence. It is known that the terminator is involved in the stability of mRNA and affects the expression level of the gene.

  As used herein, the term “promoter” refers to a region on DNA that determines the transcription start site of a gene and directly regulates its frequency, and is a base sequence that initiates transcription when RNA polymerase binds. is there. Since the promoter region is usually a region within about 2 kbp upstream of the first exon of the putative protein coding region, if the protein coding region in the genomic nucleotide sequence is predicted using DNA analysis software, The promoter region can be estimated. The putative promoter region varies for each structural gene, but is usually upstream of the structural gene, but is not limited thereto, and may be downstream of the structural gene. Preferably, the putative promoter region is present within about 2 kbp upstream from the first exon translation start.

  An “enhancer” can be used to increase the expression efficiency of a target gene. When used in mammalian cells, representative enhancers include, but are not limited to, the SV40 enhancer. A plurality of enhancers may be used, but one may be used or may not be used.

  As used herein, “operably linked” refers to a transcriptional translational regulatory sequence (eg, promoter, enhancer, etc.) or a translational regulatory sequence that has the expression (operation) of a desired sequence. That means. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but need not necessarily be adjacent.

  Methods for introducing foreign genes into mammals are well known in the art and commonly used. For example, Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J, et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like. Representative gene transfer methods include, but are not limited to, calcium phosphate precipitation, liposome method, electroporation, and DNA transfer using naked DNA.

  When the organism of the present invention is an animal, the transgenic organism may be a microinjection method (microinjection method), a viral vector method, an ES cell method (embryonic stem cell method), a sperm vector method, a method for introducing a chromosome fragment (trans (Zomic method), episomal method, etc., can be used to produce transgenic animals. Techniques for producing such transgenic animals are well known in the art.

  Mammalian cells that can be used in the present invention include mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, CHO cells that are Chinese hamster cells, BHK cells, African green monkey kidney cells, human leukemia cells, HBT5637. (Japanese Patent Laid-Open No. 63-299), human colon cancer cell lines, and the like. Mouse myeloma cells such as ps20 and NSO, rat myeloma cells such as YB2 / 0, human fetal kidney cells such as HEK293 (ATCC: CRL-1573), human leukemia cells such as BALL-1, and Africa Examples of green monkey kidney cells include COS-1 and COS-7, and examples of human colon cancer cell lines include HCT-15.

  As used herein, “animal” is used in the broadest sense in the art and includes vertebrates and invertebrates. Examples of animals include, but are not limited to, mammals, birds, reptiles, amphibians, fishes, insects, and worms.

  In the present specification, the “tissue” of an organism refers to a group of cells having a certain similar action in the group. Thus, the tissue can be part of an organ (organ). In an organ (organ), it often has cells having the same function, but those having slightly different functions may be mixed, so in the present specification, as long as the tissue shares certain characteristics, Various cells may be mixed.

  In the present specification, an “organ” refers to a structure having one independent form and a structure that performs a specific function by combining one or more tissues. Animals include, but are not limited to, stomach, liver, intestine, pancreas, lung, trachea, nose, heart, artery, vein, lymph node (lymphatic system), thymus, ovary, eye, ear, tongue, skin, etc. Not.

  As used herein, “transgenic” refers to an organism in which a specific gene is incorporated into an organism or an organism in which such a gene has been incorporated (for example, an animal such as a mouse).

  The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the above detailed description of the invention nor the following examples, but is limited only by the scope of the claims.

(Preparation of cell clones with gene amplified selection markers)
Using the cells that had been subjected to gene amplification under various conditions, the position of the chromosome into which the gene-amplified foreign gene was inserted and the amplified copy number were examined.
(1. Introduction of vector containing selection marker)
PSV2-dhfr / hGM-CSF (6.8 kb) (FIG. 1) (plasmid pSV2-dhfr (ATCC No. 37146) carrying the SV40 early promoter and mouse dhfr gene was cleaved with EcoRI and PvuII by lipofection, and Plasmid pCD-hGM-CSF (ATCC No. 57594) having SV40 early promoter and hGM-CSF gene (cut by SalI and ClaI and ligated through a linker) was introduced into CHO cell strain DG44 Then, the transformant was obtained by culturing in a medium in which 10% fetal calf serum was added to Iskov medium (containing nucleic acid). The selectable marker when using this vector is dihydrofolate reductase.
(2. Gene amplification of inserted genes)
Since the gene amplification inducing factor corresponding to dihydrofolate reductase is methotrexate (MTX), the cells are placed in Iskov medium (nucleic acid) containing MTX at three different concentrations as follows: 1 × 10 ⁵ ( cells / ml) and when the cells became confluent, it was considered that resistance was acquired, and the MTX concentration was further increased to the next concentration. Gene amplification was performed by culturing from a slowly increasing system to a rapidly increasing system with an upper limit of MTX concentration of 1000 nM:
(1) 0 nM, 50 nM, 200 nM, 500 nM, and 1000 nM;
(2) 0 nM, 100 nM, 500 nM, and 1000 nM; and (3) 0 nM, 100 nM, and 1000 nM.

  In the process of increasing the MTX concentration, no cloning operation was performed at each step of increasing the concentration in order to select cells having various properties caused by these MTX concentration increasing conditions as a population.

After cells that were resistant to 1000 nM MTX were obtained, the cells were cloned by the following method:
Each 96-well microplate was inoculated with one cell (total amount 200 μL). After confirming that the colony was from one cell and discarding the old medium by aspiration, 100 μL of a 0.25% trypsin solution was added and incubated at 37 ° C. for 10 minutes. When the cells were peeled off and rounded, 100 μL of fresh medium was added and well-beveled, and passaged to a 24-well microplate (total volume 2 mL). After culturing to confluence, the old medium was aspirated and discarded, and 1 mL of a 0.25% trypsin solution was added and incubated at 37 ° C. for 10 minutes. Furthermore, when the cells were peeled off and rounded, 1 mL of a new medium was added and well pipeted, and subculture was performed to obtain a clone (total volume 10 mL).

  As a result, in the gene amplification of (1) above, clone L1-20, clone L1-1C, clone L1-B, clone L1-1, clone L1-A, clone L1-F, and clone L1-H are obtained. In the gene amplification of (2) above, clones L4-B, L4-C, L4-I, L4-F, L4-G, L4-K, L4-M, L4-H, and L4-N are obtained. In the gene amplification in (3) above, clones L5-38, L5-55, and L5-3 were obtained. These clones were characterized as described in Example 2.

(Characteristics of clone with gene amplified selection marker)
For the cell clone isolated in Example 1, the insertion position on the chromosome of the gene amplified gene and the copy number amplified as a result of gene amplification were determined using the following method.
(1. Chromosome observation by FISH method)
Using the FISH method, it was confirmed in which position of the chromosome the vector was inserted and how much gene amplification was performed for the mammalian cells that had undergone gene amplification.

The FISH method is performed on the following principle:
(1) Chromosome specimens are prepared by fixing M-phase cells with a fixative and dropping them onto a slide glass.
(2) The plasmid is biotin-labeled by the nick translation method to prepare a probe having a size of 200 to 600 bp.
(3) Hybridize the DNA on the denatured glass slide and the probe.
(4) React with FITC-labeled streptavidin.

  Specifically, the procedure was as follows.

(1.1. Preparation of chromosome specimen)
The colcemid solution (10 μg / mL) used for the chromosome specimen is a solution stored in 4 ° C. after dissolving 1 mg of demecorcine (Sigma: D6279) in 100 mL of ultrapure water and filter sterilizing with a 0.22 μm pore size filter. It was used. The fixative was methanol: acetic acid = 3: 1 prepared as needed. The 75 mM KCl solution was used by dissolving 0.56 g of KCl in 100 mL of ultrapure water.

A chromosome sample was prepared by the following procedure.
1) When the cells were about 80-90% confluent in the medium T-flask, the old medium was aspirated and discarded, and 30 mL of new medium was added.
2) 60 μl of colcemid solution was added and incubated for 4-5 hours.
3) Shake-off method (Kuriki H, Sonta S, Murata K., Flow karyotype analysis and the sorting of the 19th affl. 22.) M cells were suspended.
4) After centrifugation at 95 × g (1,000 rpm) for 10 minutes, the supernatant was discarded. (When the amount of cells was small, it was centrifuged at 1,500 rpm for 20 minutes.)
5) Suspended in 1.5 mL of 75 mM KCl and allowed cells to swell for 20 minutes at room temperature.
(Transferred to a pointed 10 mL centrifuge tube.)
6) One drop of fixative was added and left in ice for 5 minutes.
7) While shaking the centrifuge tube, 3 drops of the fixative was added and allowed to stand in ice for 5 minutes.
8) While shaking the centrifuge tube, 10-15 drops of the fixative was added and allowed to stand in ice for 5 minutes.
9) While shaking the centrifuge tube, 10-15 drops of the fixative solution was added, and further added while shaking the wall until it became two layers (total amount 8-10 mL), and gently mixed with a Pasteur pipette.
10) After centrifuging at 95 × g (1,000 rpm) for 5 minutes, the supernatant was discarded.
11) The suspension was suspended in 5 mL of fresh fixative and centrifuged at 95 × g (1,000 rpm) for 5 minutes, and then the supernatant was discarded. This operation was repeated three times.
12) Diluted with an appropriate amount of fixative, and dropped from about 80 cm onto a slide glass (MATUNAMI: S-2124) placed on a wet paper towel (Kimwipe). By this operation, the nuclear membrane of the cell was destroyed, and the chromosome was exposed and fixed.
13) It was observed with a phase contrast microscope (OLYMPUS CK2), and it was confirmed that no residue remained around the nucleus or the chromosome was not transparent. At this time, if any residue was observed, the cells were dripped and then slowly dried by blowing. Moreover, when the chromosome became transparent, the acetic acid concentration of the fixing solution was increased to a maximum of 2: 1 (methanol: acetic acid).
14) After overnight incubation at 37 ° C, it was stored in a black box at 4 ° C. When opening, the lid was not opened until the temperature inside the box was the same as the room temperature.

(1.2. Labeling of probe)
10 × NT buffer (composition is as follows) 10 μl
0.5 mM dAGC mixture 10 μl
Biotin-16-dUTP (Roche: 1093070) 4 μl
0.1 M mercaptoethanol 10 μl
DNA polymerase (10 U / μL) (Roche: 642711) 2 μl
DNase I (Takara: 2210A) 4 μl
(Diluted to 1/2000 to 10000 with cold water. Prepared on ice water with NaCl added)
RNase solution (10 mg / mL) 2 μl
(Not required if template is RNase treated)
Template DNA (total amount 1 to 10 μg) was added to the above solution, and the reaction was carried out according to the following procedure with a total volume of 100 μl. The reaction time and DNase I concentration were appropriately set for each plasmid preparation.
1) The above solutions were mixed in order. (From the dilution of the enzyme, the operation up to this point was performed in a cold room). After incubation at 15 ° C. (cold room) for about 2 hours (90 minutes to 120 minutes), it was stored at −20 ° C.
2) The reaction solution was incubated at 100 ° C. for 10 minutes (or 68 ° C. for 15 minutes) to inactivate the enzyme, and then allowed to stand for 10 minutes or more in ice.
3) It was confirmed by 2% agarose gel electrophoresis (TBE buffer solution) that it was 200-600 bp.
4) When the fragment length was larger than the target fragment length, 2 μl of DNase I and 1 μl of DNA polymerase were further added to the reaction solution and incubated at 15 ° C. for 30 minutes.
5) The reaction solution was treated at 68 ° C. for 15 minutes to inactivate the enzyme.
6) Stored at -20 ° C. Electrophoresis was performed again to confirm the fragment length.
In addition, the composition of the used solution was as follows.
・ 10 × NT buffer solution was prepared by dissolving 0.5 mg of BSA in 1 mL of 10 × NT B solution (50 mL of 1M Tris-HCl (pH 8.0), 1.02 g of MgCl ₂ .6H ₂ O, 100 mL of ultrapure water) After sterilizing by filtration with a 0.22 μm filter, one stored at −20 ° C. was used.
・ 0.5 mM dAGC mixture is
dATP (Takara: 4026) 4.7 μl
dGTP (Takara: 4027) 4.9 μl
dCTP (Takara: 4028) 4.9 μl
Was mixed with 1 mL of sterilized water and a solution stored at −20 ° C. was used.
-As the RNase solution (10 mg / mL), the following solution was incubated at 100 ° C for 15 minutes and then stored at -20 ° C.
RNase A 10mg
1 M Tris-HCl (pH 8.0) 10 μl
3 μl of 5M NaCl (pH 7.5)
1mL of ultrapure water
The 0.1 M mercaptoethanol solution used was a stock solution (14.3 M) diluted 140 times and stored at 4 ° C.

(1.3. Confirmation of probe label)
In order to evaluate how much biotin-labeled nucleotides are contained per unit volume of the probe solution, the label of the probe was checked.

The following were used as reagents.
Buffer 1 (0.1 M maleic acid, 0.15 M NaCl; (pH 7.5))
In order to adjust the pH, 15 g of NaOH was added to the following solution in advance. After pH adjustment, autoclaved:
Maleic acid 23.21 g
NaCl 17.53 g
Distilled water 2 L
Buffer 2; 10 g of a blocking reagent (Roche: 1 096 176) was added to 100 mL of Buffer 1 and the following solutions were dissolved while heating in a microwave oven. After autoclaving, it was stored at 4 ° C.
1M MgCl ₂ ; the following solution was autoclaved and stored at room temperature:
MgCl ₂ .6H ₂ O 101.65 g / 500 mL of ultrapure water
Buffer 3 (adjustment when necessary) (100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 50 mM MgCl ₂ )
After being prepared with the following composition, it was sterilized by filtration through a membrane filter having a pore size of 0.22 μm:
Tris-HCl 2.42 g
NaCl 1.17g
1M MgCl ₂ 10mL
200 mL distilled water.

Using the above reagents, probe labeling was confirmed by the following procedure:
1) The probe solution was diluted with sterilized water to 1/20, 1/200, 1/2000, 1/20000, 1/200000, 1/2000000.
2) Nylon membrane (Roche: 1 209 299) 1 μL was spotted on the prepared dilution series.
3) Bake at 120 ° C. for 30 minutes.
4) Both sides were UV-crosslinked for 3 minutes.
5) The membrane was washed with buffer 1 for 10 minutes.
6) The membrane was washed with 10 mL of Buffer 2 and 90 mL of Buffer 1
7) Immerse the membrane in 2 μL of streptavidin-alkaline phosphatase (Oncogene Research Product: OR04L), 1 mL of Buffer 2 and 9 mL of Buffer 1 and seal using Hybridback Cosmo Bio: S-1001) The membrane was incubated for 30 minutes.
8) The membrane was washed with buffer 1 for 15 minutes. This washing was repeated twice. )
9) CSPD (3- (4-methoxyspiro {l, 2-dioxetane-3,2 '-(5'-chloro) tricyclo [3.3.1.13,7] decane) on saran wrap (Asahi Kasei) } -4-yl) phenyl phosphate disodium (Disodium 3- (4-methoxyspiro {l, 2-dioxetane-3,2 '-(5'-chloro) triticlo [3.3.1.13,7] decane) } -4-yl) Phenyl phosphate) (Roche: CD005) was immersed in 100 μL of Buffer 3 and incubated at room temperature for 10 min.
10) The membrane sealed using the hybrid back was preincubated at 37 ° C. for 5-15 minutes.
11) An X-ray film (Fuji Photo Film: RX-U) was exposed for about 10 minutes.

(1.4. Hybridization)
The following solutions were used for hybridization:
20 × SSC (pH 7.0): NaCl 350.64 g, sodium citrate 176.46 g, ultrapure water 2 L.
-Denatured solution; Formamide (super high quality; Roche 100144) 35 mL, 20 × SSC 5 mL, sterile water 50 mL
Salmon sperm DNA solution (1 μg / μL): A salmon sperm crude extract dissolved in TE buffer, phenol / chloroform extracted, ethanol precipitated, and stored at −20 ° C. was used.
20% dextran sulfate solution (dissolved in 2 × SSC); autoclaved 50% dextran sulfate solution 400 μL, 20 × SSC 100 μL, sterile water 1 mL was used at 4 ° C.

Experiments were performed using the above reagents by the following procedure:
1) While observing with a microscope, the region on the slide glass where the chromosome was present was marked with a diamond pen, and then incubated at 37 ° C. for 3 hours.
2) The denaturing solution placed in the copulin was kept at 68-72 ° C.
(67 + n ° C. (n ≦ 5, n is the number of slides))
3) 8 μL of the probe reaction solution (0.16 to 1.6 μg was used per slide) and 8 μL of salmon sperm DNA solution were mixed and ethanol precipitated.
4) 6.5 μL of 100% formamide (per slide) was added to the purified probe and suspended by pipetting 100 times.
5) Further, 6.5 μL of 20% dextran sulfate solution (per slide) was added, suspended by pipetting 100 times, and then vortexed for 20 minutes.
(Do not spin down after this.)
6) The probe solution was denatured at 80 ° C. for 10 minutes and then left on ice for 10 minutes.
7) The slides were incubated with denaturing solution (68-72 ° C.) for 2 minutes.
8) Incubated with 70% ethanol (−20 ° C.) for 5 minutes using a coplinger.
9) Incubated with 90% ethanol (room temperature) for 5 minutes using a coplinger.
10) After dehydrating with 100% ethanol (room temperature) for 5 minutes using a coplinger, it was air-dried in a test tube stand.
11) The denatured probe was mounted on a cover glass (MATSANAMI: 22 × 22), and a slide glass was placed so as not to cause bubbles.
12) Enclosed with paper bond (Kokuyo), put slide glass in sealed wet box, and incubated at 37 ° C. overnight. Samples for which signal was difficult to detect were incubated for 24 hours.

(1.5. Preparation of immobilized sample for signal detection)
The following solutions were used for the preparation of immobilized samples for signal detection:
・ Cleaning liquid 1 (prepared as needed)
Formamide 80 mL
20 x SSC 16 mL
Sterile water 160 mL
-Cleaning solution 2;
20 x SSC 16 mL
Sterile water 160 mL
-Cleaning solution 3;
20 x SSC 40 mL
Tween20 200 μL
Sterile water 200 mL
Blocking solution: A solution having the following composition was prepared and then filtered through a 0.22 μm pore size filter.
Block Ace (snow mark: UK-B80) 0.3 g
20 x SSC 2 mL
Tween20 10 μL
Sterile water 10 mL
-Detection solution: After preparing a solution having the following composition, a solution filtered with a 0.22 μm pore size filter was used.
Block Ace (snow mark: UK-B80) 0.1 g
20 x SSC 2 mL
Tween20 10 μL
Sterile water 10 mL
Antifade solution;
DABCO (SIGMA: D2522) 0.233 g
Glycerol 9 mL
1M Tris-HCl (pH 7.5) 0.2 mL
Sterile water 10 mL
-DAPI solution;
DAPI stock solution (400 μg / mL) 40 μL
20 x SSC 5 mL
50 mL of sterile water.

Using the above reagents, an immobilized sample for signal detection was prepared by the following procedure:
1) The slide was taken out from the moist chamber, the paper bond was peeled off with a toothpick, and the cover glass was slid and removed. (The moist chamber was again warmed in the incubator.)
2) The slide was washed 3 times for 5 minutes with the washing solution 1 kept at 43 ° C.
3) The slide was washed 3 times for 5 minutes with the washing solution 2 kept at 60 ° C.
4) 150 μL of blocking solution was mounted on a cover glass (24 × 50), and a slide glass was placed thereon.
5) Incubated in a moist chamber at 37 ° C. for 30 minutes.
6) To 600 μL of the detection solution, 1.5 μL of FITC avidin was dissolved and vortexed. 150 μL was mounted on a cover glass, and a slide glass was placed thereon.
7) Incubated in a moist chamber at 37 ° C. for 30 minutes.
8) The slide glass was washed 3 to 4 times for 5 minutes with the washing solution 3 kept at 43 ° C.
9) 50 mL of DAPI solution was made and the slide was incubated for 5 minutes.
10) The slide was washed for 5 minutes with the washing solution 3 diluted twice.
11) 40 μL of antifade solution was mounted on a cover glass (24 × 50), and a slide glass was placed thereon.
12) Fixed with nail polish.

(1.6. Observation with a microscope)
The signal on the chromosome was observed with a fluorescence filter (Carl Zeiss, Axioplan 2) in the cell physiology laboratory using 365 nm excitation and a blue filter. The objective lens used was 63 times. Image capture was performed using a cooled CCD camera (PVCAM camera manufactured by Photometrics) and image analysis software IPLab Spectrum ™ provided in the fluorescence microscope. After use, the lens was wiped with a cleaner (methyl acetate 65 mL, ethanol 30 mL, diethyl ether 5 mL).

(2. Analysis of FISH image)
FISH image data (using 365 nm excitation blue filter) was used on a Macintosh PC (Power Mac. G3), and the NIH Image and Microsoft Excel Analyzing System III (CHIASuM III, CHIASu. III). K., Ohue S., Takeda S., Kimura H, Sakiki S .: A case of chronic sub-human hydrated cerebral hydrated cerrebred cerebral cerebral. (1986), and Kato S., Fukui K .: Condensation pattern (CP) analysis of plant chromosomes by an improvised chromosome image analyzing system, III. By performing the following operation, the position of the amplified gene on the chromosome was identified (the number of the step corresponds to the number in FIG. 2).
1) FISH images in TIFF format were acquired by IPLab Spectrum ^™ and imported into the CHIAS III program.
2) The chromosomal region in the acquired image was determined, and the chromosomal region was extracted according to the program.
3) The extracted chromosome image was normalized to 256 gray scales.
4) The centromere position of the chromosome was determined, a line was drawn on the center of the chromosome, and the concentration value on the line was digitized.
5) Draw a 256-tone band pattern based on the numerical value, and finally determine the chromosome band pattern by binarizing the density (3. Quantification of copy number by dot blot)
Intracellular DNA was prepared according to Nomura's method (Shintaro Nomura Cell Engineering, Separate Volume 9, Deisotope Protocol, Shujunsha 35-43 (1994)). RapidPrep ^™ Micro Genomic DNA Isolation Kit (Pharmacia Biotech: 27-5225-01) ). For the DNA to be used for the probe, only the dhfr gene in the plasmid dhfr-mloxP introduced into the cells was recovered using a Prep-A-Gene DNA purification kit (Bio-rad: 732-6009). Copy number quantification was performed by Dot Blotting using DIG-DNA Labeling and Detection Kit (Roche: 1093657).
(1) DNA is extracted from cells.
(2) A probe is prepared by labeling the plasmid with digoxigenin (DIG) by the random prime method.
(3) DNA is applied to a nylon membrane and the probe is hybridized.
(4) An alkaline phosphatase labeled anti-DIG antibody is bound.
(5) Add a substrate to develop color.
It was done by the process. More specifically, it was performed as follows.

(3.1. Extraction of chromosomal DNA using GenomicPrep ^™ Cells and Tissue DNA Isolation Kit)
Chromosomal DNA was extracted using GenomicPrep ^™ Cells and Tissue DNA Isolation Kit according to the manufacturer's instructions as follows.
1) Collect 1.0-2.0 × 10 ⁶ cells as a medium suspension or PBS suspension in a 1.5 mL polypropylene tube and centrifuge at 8,000 × g (10,000 rpm) for 5 minutes. The supernatant was removed leaving 20-40 μL of the liquid volume.
2) Vortex mixed.
3) Cell Lysis solution 300 μL was added and gently pipetted to lyse the cells. (The sample can be kept in this state at room temperature for 18 months)
4) Add 1.5 μL of RNase A solution to the lysed cells.
5) The tube was mixed by inverting about 25 times and incubated at 37 ° C. for 15 minutes to 1 hour.
6) After cooling the sample to room temperature, 100 μL of Protein Precipitation solution was added.
7) Vortexed for at least 20 seconds until evenly mixed.
8) The protein was precipitated by centrifuging at 15,000 × g (14,000 rpm) for 3 minutes.
9) The supernatant containing the DNA was transferred to a tube containing 300 μL of 100% isopropanol.
10) The tube was mixed by inverting about 50 times until the DNA became a white thread.
11) Centrifugation was performed at 15,000 × g (14,000 rpm) for 1 minute, and the supernatant was discarded.
12) Standing on a paper towel and absorbing the supernatant.
13) 300 μL of 70% ethanol was added and mixed by inversion several times.
14) Centrifugation was performed at 15,000 × g (14,000 rpm) for 1 minute, and the supernatant was discarded.
15) Standing on a paper towel and drying for 15 minutes.
16) To dissolve the DNA, 100 μL of DNA Hydration solution was added.
17) Incubate overnight at room temperature or 1 hour at 65 ° C.
18) Stored refrigerated. In the case of long-term storage, it was stored at -20 ° C or -80 ° C.
19) During use, ethanol precipitation was performed and the buffer was changed.

(3.2. Quantification of DNA)
Chromosomal DNA used as a sample was quantified by filtering and sterilizing 10 × TNE buffer (10 mM EDTA, 2M NaCl, 100 mM Tris-HCl) and H33258 assay solution (100 mL of TNE buffer with a 0.22 μm pore size filter). / ML Hoechst 33258 10 μL was added.) And the following method.
1) A dilution series (0, 20.4, 40.7, 81.3, 162.5, 325 ng / TE 10 μL) of λ-DNA (325 μg / mL) (Takara: 3010) was prepared as a standard sample.
2) 2 mL of H33258 assay solution was added to and mixed with 10 μL of the solution of (1) above in a 1.5 mL polypropylene tube and the sample.
3) The solution of the above (2) was transferred to a fluorescence measurement cell and measured using a fluorometer (excitation wavelength 365 nm, emission wavelength 455 nm).
4) A standard straight line was drawn using Excel ^™ (Microsoft) to calculate the DNA concentration.
5) The DNA concentration in the sample was adjusted from the obtained value.

(3.3. Preparation of probe)
1) According to the gene of interest, dhfr and hGM-CSF genes were cloned using PCR. For PCR, as a reaction solution: Takara Ex-Taq polymerase (TaKaRa: RR001) 0.5 μL, 10 × Taq buffer 10 μL, dNTP mixture (2.5 mM each) 8 μL, primer 1 (sense strand) 100 pmol, primer 2 ( Antisense strand) 100 pmol, template DNA (> 1 μg) (typically 0.1 μg) were used.

The PCR was performed at 94 ° C for 2 minutes in a thermal cycler, followed by 25 to 30 cycles of 94 ° C for 30 seconds, 55 to 65 ° C for 30 seconds and 74 ° C for 1 minute per cycle, and then 74 ° C. After heating for 7 minutes, it was stored at 4 ° C.
2) After the PCR was completed, unreacted substances were removed with GFX ^™ PCR DNA and Gel Band Purification Kit (Amasham Bioscience: 27-9602-01), and the buffer solution was replaced with distilled water.
3) DNA concentration was quantified by absorption at 260 nm.
4) 0.01-3 μg of DNA was used for the DIG label. Depending on the labeling efficiency, the DNA concentration used was varied.
5) The required amount of DNA was dispensed into a polypropylene tube and adjusted to a final volume of 16 μL.
6) The solution of (5) above was denatured at 100 ° C. for 10 minutes and then allowed to stand in ice (ice / NaCl) for 10 minutes or longer.
6) 4 μL of DIG-High Prime (Roche: 1585 606) and 16 μL of DNA immediately after denaturation were mixed in a microtube in ice.
7) After reacting at 37 ° C. for 20 hours, 1 μL of 0.5 M EDTA (pH 8.0) was added to terminate the reaction.

(3.4. Assay of labeled probe concentration)
The following reagents were used for the assay of labeled probe concentration:
Buffer 1 (pH 7.5): 0.1 M maleic acid, 0.15 M NaCl (15 g of NaOH was added in advance for pH adjustment and autoclaved.)
Buffer 2; 10 g of blocking reagent (Roche: 1 096 176) was added to 100 mL of Buffer 1 and the solution was dissolved while heating. After autoclaving, it was stored at 4 ° C.
Buffer 3 (prepared as necessary): 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 50 mM MgCl ₂ were used after being filter sterilized with a filter having a pore size of 0.22 μm.
Wash buffer: Tween 20 1.5 mL / Buffer 1 500 mL.

The labeled probe concentration was assayed using the above reagent and the following method.
1) The standard sample (pBR322, DIG-labeled control DNA) and the sample were added with TE buffer to 1/20 (1 ng / μL), 1/200 (100 pg / μL), 1/2000 (10 pg), respectively. / L), 1/20000 (1 pg / μL), 1 / 2000,000 (0.1 pg / μL), 1 / 2000,000 (0.01 pg / μL).
2) 1 μL of this diluted series was spotted on a nylon membrane (Roche: 1 209 299).
3) Baking was performed at 120 ° C. for 30 minutes in an oven set at 120 ° C. in advance.
4) UV-crosslinked on a transilluminator for 3 minutes on both sides.
5) The membrane was washed with buffer 1 for 10 minutes.
6) The membrane was washed with 10 mL of Buffer 2 and 90 mL of Buffer 1 for 30 minutes.
7) Hybridize with 2 μL of antibody solution (anti-DIG AP Fab fragment) (centrifuged at 14,000 × g for about 3 minutes before use, and the supernatant was used), 1 mL of Buffer 2 and 9 mL of Buffer 1 The sealed membrane was incubated for 30 minutes using a pack (Cosmo Bio: -1001).
8) The membrane was washed twice with buffer 1 for 15 minutes.
9) The membrane was allowed to stand in buffer 3 for 2 minutes.
10) The membrane was immersed in 100 μL of CSPD (Roche: CD005) and 10 mL of Buffer 3 on Saran Wrap (Asahi Kasei), and incubated at room temperature for about 10 minutes.
11) The membrane sealed using the hybrid pack was preincubated at 37 ° C. for 5 to 15 minutes.
12) The X-ray film was exposed.

(3.5. Hybridization)
When quantifying the 10 ² order copy number, 1.0 × 10 ⁵ cells (0.24 μg) of chromosomal DNA is used, and when the 10 ¹ order copy number is quantified, 1.0 × 10 ⁶ cells. Minute (2.38 μg) chromosomal DNA was used. As the hybridization buffer, an aqueous solution containing 25 mL of 20 × SSC, 10 mL of buffer 2 and 0.1 g of N-lauroyl sarcosine was autoclaved, and 0.2 mL of 10% SDS was added and stored at 4 ° C. .

Hybridization was performed using the above reagents and the following method:
1) Plasmid dhfr-mloxP was digested with Bgl II at 37 ° C. for 1.5 hours.
2) A 400 copy / 100 μL solution was prepared, and a dilution series was prepared as a standard sample. (Calculated by changing the dilution series of copy number depending on the experimental purpose. 200 copies to 10 copies were used)
3) The copy number dilution series was changed depending on the cell line and calculated. Evaluation was made with chromosomal DNA for 10 ⁵ to 10 ⁶ cells.
4) A dilution series of a plasmid used as a standard and sample chromosomal DNA were denatured at 100 ° C. for 10 minutes. 5) Cooled in ice (ice / NaCl) for 10 minutes or more.
6) A membrane (Roche: 1 209 229) and three filter papers (BIO-RAD: 1620101), which had been wetted with 10 × SSC in advance, were mounted on a blotting apparatus.
7) DNA was blotted onto the membrane using BIO-DOT ^™ SF (BIO-RAD) while evacuating. (The screw was tightly closed so that there was no gap between the membrane and the slot.)
8) 100 μL of 10 × SSC was poured into the slot and washed twice.
9) Bake at 120 ° C. for 30 minutes.
10) Both surfaces were UV-crosslinked for 3 minutes.
11) Sealed membranes were incubated for 1 hour at 68 ° C. in hybridization buffer (20 mL per 100 cm ² ).
12) Sealed membranes were incubated overnight at 68 ° C. in hybridization buffer (2.5 mL per 100 cm ² ) containing 5 μL of DNA probe in 1 mL.

(3.6. Immunological detection)
The following reagents were used for immunological detection:
10% (w / v) SDS; sodium dodecyl sulfate 10 g / distilled water 100 mL
-2 x SSC-0.1% (w / v) SDS; SDS was added after autoclaving the SSC solution.
• 0.1 × SSC • 0.1% (w / v) SDS; SDS was added after autoclaving the SSC solution.
Hybridization buffer: (20 × SSC 25 mL, 10% SDS 0.2 mL, buffer 2 10 mL, N-lauroyl sarcosine 0.1 g)
Buffer 1 (pH 7.5): 0.1 M maleic acid, 0.15 M NaCl) For pH adjustment, 15 g of NaOH was previously added. Autoclaved.
Buffer 2; 10 g of blocking reagent (Roche: 1 096 176) was added to 100 mL of Buffer 1 and dissolved while heating. After autoclaving, it was stored at 4 ° C.
Buffer 3 (prepared as necessary): Tris 2.42 g, NaCl 1.17 g, 1 M MgCl ₂ 10 mL were dissolved in distilled water 200 mL and used after sterilizing by filtration with a filter having a pore size of 0.22 μm.
Wash buffer: Tween 20 1.5 mL / Buffer 1 500 mL
Using the above reagents, immunological detection was performed using the following method:
1) 2 × SSC · 0.1% SDS was kept at 40 ° C., 0.1 × SSC · 0.1% SDS was kept at 68 ° C., and the washing buffer was kept at 40 ° C.
2) The membrane was washed twice with 250 L of 2 × SSC / 0.1% SDS (40 ° C.) solution for 5 minutes.
3) The membrane was washed twice in 250 ml of 0.1 × SSC / 0.1% SDS (68 ° C.) solution for 15 minutes.
4) The membrane was washed once with 5OmL of wash buffer (40 ° C) for 5 minutes.
5) The mixture was incubated with 10 mL of Buffer 2 and 90 mL of Buffer 1 for 30 minutes at room temperature with shaking.
6) 0.5 μL of antibody solution (anti-DIG-alkaline phosphatase) (14,000 × g before use, supernatant was used after centrifugation for 3 minutes), sealed with 1 mL of buffer 2 and 9 mL of buffer 1 The membrane was incubated with shaking for 30 minutes.
7) The membrane was washed 3 times for 7 minutes with 250 mL wash buffer (40 ° C.).
8) The membrane was left in buffer 3 for 2 minutes.
9) The membrane was immersed in 30 μL of CSPD and 3 mL of buffer 3 on Saran Wrap, and incubated at room temperature for about 10 minutes.
10) The sealed membrane was preincubated for 5-15 minutes at 37 ° C.
11) It exposed to the X-ray film (FUJIFILM: 50NIF).
12) Capture X-ray film signals with a scanner, Gel Plotting Macros (a macro command program in NIH image program developed by NIH (available from http://rsb.info.nih.gov/nih-image/)) Quantified with.

(4. Results of FISH method and dot blotting method)
As a result of the FISH experiment according to the above-described example, gene amplification in the chromosome was observed in a specific region indicated by the squares 1 to 9 in the upper part of FIG. 3 (near the centromere of E chromosome, J chromosome Centromere vicinity, K chromosome centromere vicinity, L chromosome centromere vicinity, N chromosome telomere pole vicinity, P chromosome telomere side, P chromosome telomere pole vicinity). The number of copies of gene amplification observed in the specific region indicated by the squares 1 to 9 in FIG. 3 is shown for each clone (lower row in FIG. 3).

  Gene amplification that produced the highest copy number (95, 108, 117, 119, 125, 141, 166 copies) was confirmed near the telomere pole of the P chromosome.

  From the above results, it has been clarified that gene amplification is efficient when site-specific recombination sequences are inserted in the vicinity of the N-chromosome telomere pole and the P-chromosome telomere pole. In particular, when a site-specific recombination sequence was inserted in the vicinity of the telomere of the P chromosome, the gene amplified copy number was the highest.

  It can be understood that the optimum gene amplification conditions are conditions under which a clone in which a gene inserted in the vicinity of the telomere in the P chromosome is amplified appears selectively. Specifically, when MTX is used as a gene amplification inducer, (1) not only when the MTX concentration is increased in the order of 0 nM, 50 nM, 200 nM, 500 nM, and 1000 nM, but (2) 0 nM, 100 nM, Even when the MTX concentration was increased in the order of 500 nM and 1000 nM, it was possible to obtain cell clones in which the gene inserted in the vicinity of the telomere pole of the P chromosome was amplified. Even when 100 nM MTX was used after 0 nM, cells (clone L4-N) in which the gene inserted in the vicinity of the telomere pole of the P chromosome was obtained were obtained because only the initial MTX concentration was important. No, it was shown that a clone having a nucleic acid fragment inserted in the vicinity of the telomere pole could be obtained even by relaxing the subsequent conditions for increasing the MTX concentration. Therefore, the condition for easily obtaining a cell clone in which a nucleic acid fragment is inserted in the vicinity of the telomere pole is a condition for moderately increasing the MTX concentration.

  Although not intending to be bound by theory, when the MTX concentration is rapidly increased, the gene amplification rate of the selectable marker gene cannot catch up with the increase in MTX concentration. It is thought that the possibility that the acquired clone appears will increase.

(Preparation of a cell clone having a nucleic acid fragment containing the first site-specific recombination sequence and a selection marker in the chromosome at a high copy number)
In order to prepare a versatile high-expression mammalian host cell, a cell clone having a high copy number of a nucleic acid fragment containing a site-specific recombination sequence and a selectable marker is used for gene amplification as follows. Prepared.

(1. Introduction of vector containing first site-specific recombination sequence and selectable marker)
As the first site-specific recombination sequence, the mutant loxP sequence described in SEQ ID NO: 3 that can suppress excision of the inserted sequence was used (Albert et al., 1995 Araki et al., 1997).

  As a selectable marker, the dihydrofolate reductase (DHFR) gene operably linked to the SV40 promoter and polyA + addition signal was used. A strong promoter, cytomegalovirus (CMV) promoter, was used. A vector dhfr-mloxP (5.8 kb) containing these sequences was constructed (FIG. 4) and introduced into CHO cells by the lipofection method.

(2. Gene amplification of inserted genes)
First, using Iscof's medium containing 50 nM MTX and no nucleic acid, the cells were cultured with cells having an initial cell number of 1 × 10 ⁵ cells / ml. When the cells became confluent, it was considered that the cells had acquired resistance, and 70 cell lines were cloned. Among these clones, about 40 strains, the insertion position on the chromosome could be confirmed by the FISH method.

  In this experiment, in order to select cells having various properties caused by these MTX concentration increasing conditions as a population, no cloning operation is performed at each concentration increasing step, and resistance to 1000 nM MTX is exhibited. After the cells were obtained, the cells were cloned.

(3. Characterization of isolated gene amplified clones by FISH method and dot blotting method)
Characterization of the isolated gene amplified clone was performed according to the method described in Example 2.

(4. Results of FISH method and dot blotting method)
Of the 36 isolated strains, 9 were cell strains that had been amplified in the vicinity of the telomere pole of the P chromosome. Among these nine cell lines, one cell line had a high gene copy number (about 20 to 30 copies). This cell clone is designated “C20”.

  Four cell lines with gene amplification in the vicinity of the centromere of the chromosome were isolated, but all these four strains had a gene copy number as low as several copies. On the other hand, 23 cell lines that had been amplified in the vicinity of telomeres of chromosomes other than the P chromosome were isolated. In these cell lines, the gene amplified chromosomal sites are the vicinity of A chromosome telomere, B chromosome telomere, D chromosome telomere, F chromosome telomere, G chromosome telomere, I chromosome telomere, J chromosome telomere, K It was near chromosome telomere, near N chromosome telomere, and near P chromosome telomere. Among these 23 strains, 9 clones had a nucleic acid fragment inserted in the vicinity of the P chromosome telomere. Among the nine strains, one cell strain had a high gene copy number (20 to 30 copies). A typical result is shown in FIG.

  Therefore, even when the nucleic acid fragment containing the first site-specific recombination sequence and the selection marker was gene amplified, the same results as in the case of gene amplification of only the selection marker were obtained.

(Preparation of a mammalian host cell having the desired foreign gene in the chromosome at a high copy number using the general-purpose mammalian host cell prepared in Example 3)
(1) operably linked to a promoter to a general-purpose mammalian host cell strain C20 having a nucleic acid fragment containing the first site-specific recombination sequence prepared in Example 3 and a selection marker in the chromosome at a high copy number By introducing a nucleic acid fragment containing a foreign gene and a second site-specific recombination sequence homologous to the first site-specific recombination sequence, and (2) introducing a site-specific recombination inducer A mammalian host cell having a desired foreign gene in the chromosome at a high copy number is prepared.

  The following procedure introduces (1) a nucleic acid fragment containing a foreign gene operably linked to a promoter and a second site-specific recombination sequence homologous to the first site-specific recombination sequence. Thereafter, (2) the procedure for introducing a site-specific recombination inducer is described, but it is also possible to carry out (1) and (2) in the reverse order.

  Specifically, the following procedure is performed.

(1. Introduction of a nucleic acid fragment containing a foreign gene into a general-purpose mammalian host cell prepared in Example 3)
As a nucleic acid fragment containing a foreign gene operably linked to a promoter and a second site-specific recombination sequence homologous to the first site-specific recombination sequence, the pIRESneo2 vector (Clontech code number: 6938-1) A pIRES-PromoxP vector (5.3 kb) containing IRES derived from encephalomyocarditis virus (ECMV), human GM-CSF gene, polyadenylation signal of SV40, and mutant loxP sequence (SEQ ID NO: 5), Then, it is introduced into the general-purpose mammalian host cell strain C20 prepared in Example 3 by the lipofection method, seeded in a 35 mm petri dish and cultured.

(2. Introduction of site-specific recombination inducer into host cells)
(2.1. Preparation of recombinant Cre protein)
A variant of Cre, a protein that recombines the loxP sequence in a site-specific manner, is prepared as follows.

  When the Cre protein as a gene amplification inducer is directly introduced into cells (“direct introduction method”), in order to introduce the Cre recombinant protein into the C20 cell and the nucleus with high efficiency, the Cre protein is added to Arg. -Rich sequence was added.

  This Arg-rich sequence has been reported to be transferred to the nucleus with high efficiency. The Tat protein and Rev protein of the HIV-I virus (Futaki et al., 2001, Suzuki et al., 2002, Vives et al. , 1997), and the ability to transport proteins into cells and nuclei has been confirmed with only 9 residues of the 49-57th Arg-rich sequence (Shimizu et al., 2000, Wender et al.). , 2000, Yoon et al., 2002). Therefore, an Arg-rich sequence was used to efficiently transfer the Cre protein into the nucleus.

(2.1.1. Preparation of Cre protein expression vector with Arg-rich sequence added)
In order to prepare a Cre protein expression vector to which an Arg-rich sequence was added, a base sequence encoding the Arg-rich sequence in the Tat sequence (SEQ ID NO: 6: 5′-cgtagaagacggcgtcagcggcgcaga-3 ′) was inserted into the primer and A directional primer “Tat-Cre-forward” (SEQ ID NO: 7: 5′-CGACCGCATGCGATAGAAGCGGCCGTCAGCGGCGCAGATCTATTATTACTGACCGTACACC-3 ′) was prepared. To the reverse primer “Tat-Cre-reverse”, His _6- Tag was added to facilitate the purification of the recombinant Cre protein (SEQ ID NO: 8: 5′-CGCGGGTCACTTAGTGTGGTGGTGTGGTGATGCATCATGCCATCATTCCCAGCAGCGC-3 ′).

  Using the above primers, PCR reaction was performed using pBS185 (Invitrogen 10347-011;) as a template. A target fragment was confirmed by subjecting a part of the reaction solution to agarose gel electrophoresis. The PCR product was cloned into TA cloning (Invitrogen: 45-0046) to obtain Tat49-57-Cre integrated into the pCR2.1 vector.

  From this pCR2.1, a DNA fragment encoding Tat49-57-Cre was obtained by cleaving restriction enzymes SphI and SalI, ligated with vector pET17b (Novagen NV509), and Tat49- which is a Cre protein to which an Arg-rich sequence was added. An expression vector for 57-Cre protein is used.

(2.1.2. Expression of Cre protein added with Arg-rich sequence)
Using Escherichia coli as a host cell, the Cre protein added with the Arg-rich sequence is expressed as follows:
1) E. coli BL21 (DE3) pLysS is transformed using the Tat49-57-Cre protein expression vector.
2) This E. coli is cultured at 37 ° C. in 100 mL LB medium (kanamycin added) until OD ₆₀₀ = about 0.6.
3) Add IPTG to a final concentration of 1 mM, and further culture for 3 hours.
4) The culture is ice-cooled, centrifuged at 7000 × g for 10 minutes at 4 ° C., and collected.
5) The collected E. coli is suspended in 0.25 times as much chilled 20 mM Tris-HCl (pH 8.0) as the culture solution, and collected again under the same conditions.

(2.1.3. Extraction and purification of Cre protein added with Arg-rich sequence)
Above 1.1.2. The Escherichia coli collected in is sonicated as follows.
1) E. coli is suspended in a sonication buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM PMSF) in 1/20 volume of the culture solution, and transferred to a polypropylene tube.
2) Apply ultrasonic waves in several batches for 10-15 seconds while cooling with ice.
3) Centrifuge at 11,000 × g for 30 minutes at 4 ° C. to remove cell debris and insoluble matter.
4) Filter the cell extract (supernatant) with a 0.45 μm filter.

From the filtrate, an Arg-rich sequence-added Cre protein to which His-tag has been added is purified using HiTrap Chelating HP (Amasham Pharmacia: 17-0408-01) as follows. The following buffers are used for purification:
Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, pH 7.4,
Elution buffer: 20 mM sodium phosphate, 0.5 M NaC, 500 mM imidazole, pH 7.4.
1M nickel sulfate solution.

Using these buffers, purify as follows:
1) Connect the column to a syringe and feed 0.5 ml of 0.1M nickel sulfate solution at a flow rate of 1 drop / 2 seconds.
2) Pump 5 ml of ultrapure water at a flow rate of 1 drop / second.
3) Pump 10 ml of binding buffer at a flow rate of 1 drop / second.
4) Send the cell extract at a flow rate of 1 drop / 2 seconds and collect the eluate (non-adsorbed fraction).
5) Pump the binding buffer at a flow rate of 1 drop / second. Measure A280 every 5 mL and continue to flow binding buffer until the value is constant.
6) Send 5 ml of elution buffer at a flow rate of 1 drop / second, collect the eluate, and use it as a purified protein solution.
7) Confirm the Arg-rich sequence-added Cre protein in the eluate by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).
8) Quantify the Arg-rich sequence-added Cre protein by the Bradford method.

(2.2. Site-specific recombination by introducing the prepared site-specific recombination inducer into a host cell)
By introducing the prepared Cre protein, which is a site-specific recombination inducer, into the host cell strain C20, site-specific recombination occurs.

A nucleic acid containing a foreign gene operably linked to a promoter and a second site-specific recombination sequence homologous to the first site-specific recombination sequence for the general-purpose mammalian host cell prepared in Example 3 After the fragment is introduced, the modified Cre protein, which is a site-specific recombination inducer, is allowed to act as described below, and the first site-specific recombination sequence in the chromosome and the nucleic acid fragment introduced into the cell A site-specific recombination between the first site-specific recombination sequence and a second site homologous to the foreign gene and the first site-specific recombination sequence operably linked to the promoter. The nucleic acid fragment containing the site-specific recombination sequence is inserted into the mammalian host cell chromosome.
1) Above 1. C20 cells transfected by the lipofection method are cultured until reaching 80-90% confluence.
2) Change to a new medium and Tat49-57-Cre protein prepared in step 1 is added to a final concentration of 3 μM.
3) Incubate at 37 ° C. for 3 hours and wash cells 3 times with PBS (−).

(2.3. Confirmation of site-specific recombinants)
Site-specific recombinants are identified using any one of the following three methods, or any combination of the following three methods:
(1) Confirmation with G418 selective medium Although the G418 resistance gene exists on pIRES-PromoxP inserted into the chromosome by recombination, its expression depends on the CMV promoter introduced into the chromosome by dhfr-mloxP. Therefore, unless pIRES-PromoxP is inserted into the chromosome by site-specific recombination, the obtained transformant does not have G418 resistance. Using this principle, clones that grow on G418 selective media are identified as clones that have undergone site-specific recombination.
(2) Confirmation by PCR method PCR can be performed using the expected chromosomal sequence as a template when site-specific recombination occurs, and the success or failure of recombination can be determined by whether or not the target band is obtained.
(3) Confirmation of expression of hGM-CSF Similar to the G418 resistance gene, the expression of hGM-CSF also depends on the CMV promoter introduced into the chromosome by dhfr-mloxP. Therefore, hGM-CSF is expressed only when pIRES-PromoxP is inserted into the chromosome by site-specific recombination.

(Preparation of a mammalian host cell having the desired foreign gene in the chromosome at a high copy number using the general-purpose mammalian host cell strain C20 prepared in Example 3-2)
A general mammalian host cell having a nucleic acid fragment containing the first site-specific recombination sequence prepared in Example 3 and a selection marker in the chromosome at a high copy number, (1) a site-specific recombination inducer A vector capable of expression, and (2) a nucleic acid fragment containing a foreign gene operably linked to a promoter and a second site-specific recombination sequence homologous to the first site-specific recombination sequence Thus, a mammalian host cell having a desired foreign gene in the chromosome at a high copy number is prepared.

  In addition, the gene introduction (1) and (2) described above may be performed either in transfection first or simultaneously (co-transfection).

  Specifically, the following procedure is performed.

(1. Introduction of a nucleic acid fragment containing a foreign gene into the general-purpose mammalian host cell strain C20 prepared in Example 3)
As a nucleic acid fragment containing a foreign gene operably linked to a promoter and a second site-specific recombination sequence homologous to the first site-specific recombination sequence, the pIRESneo2 vector (Clontech code number: 6938-1) A pIRES-PromoxP vector (5.3 kb) containing IRES derived from encephalomyocarditis virus (ECMV), human GM-CSF gene, polyadenylation signal of SV40, and mutant loxP sequence (SEQ ID NO: 5), Then, it is introduced into the general-purpose mammalian host cell strain C20 prepared in Example 3 by the lipofection method, seeded in a 35 mm petri dish and cultured.

(2. Introduction of vector capable of expressing site-specific recombination inducer into host cell)
Using a Cre-expressing adenovirus AxCANCre (RIKEN Genebank, RBD: 1748, expressing the Cre protein under the CAG promoter) as a vector for expressing the Cre protein as a site-specific recombination inducer, as follows: Introduce into the host cell.
1) Collagen-coated 75 cm ³ bottles were mixed with 1 × 10 ⁷ cells of CHO cells prepared in Example 3 and adenovirus AxCANCre 2 × 10 ⁸ PFU in 15 ml of IMDM (Iskov) medium (nucleic acid) at 37 ° C. Incubate in a CO ₂ incubator.
2) Gently shake the bottle every few seconds like a seesaw and spread the virus solution evenly. This operation is performed 3 times every 20 minutes.
3) Add 14 ml of medium and continue incubation at 37 ° C. for 2 days.

(3. Confirmation of site-specific recombinants)
Site-specific recombinants are identified using any one of the following three methods, or any combination of the following three methods:
(1) Confirmation with G418 selective medium Although the G418 resistance gene exists on pIRES-PromoxP inserted into the chromosome by recombination, its expression depends on the CMV promoter introduced into the chromosome by dhfr-mloxP. Therefore, unless pIRES-PromoxP is inserted into the chromosome by site-specific recombination, the obtained transformant does not have G418 resistance. Using this principle, clones that grow on G418 selective media are identified as clones that have undergone site-specific recombination.
(2) Confirmation by PCR method PCR can be performed using the expected chromosomal sequence as a template when site-specific recombination occurs, and the success or failure of recombination can be determined by whether or not the target band is obtained.
(3) Confirmation of expression of hGM-CSF Similar to the G418 resistance gene, the expression of hGM-CSF also depends on the CMV promoter introduced into the chromosome by dhfr-mloxP. Therefore, hGM-CSF is expressed only when pIRES-PromoxP is inserted into the chromosome by site-specific recombination.

FIG. 1 is a diagram schematically showing a plasmid pSV2-dhfr / hGM-CSF. FIG. 2 is a diagram schematically showing a chromosomal image analysis using Chromosome Image Analyzing System (CHIAS III), which is a method for identifying the position of the amplified gene on the chromosome by the FISH method used in the present invention. FIG. 3 is a schematic diagram showing the chromosome position where gene amplification was confirmed as a result of Example 2 and the copy number of gene amplification. FIG. 4 is a diagram schematically showing the plasmid dhfr-mloxP. FIG. 5 shows the results of Example 3. Cells transformed with dhfr-mloxP were selected by adding 50 nM MTX, 60 cells were cloned, FISH method and dot blotting method were performed on each cell line, and gene amplification was confirmed. The chromosomal position where gene amplification was confirmed and the copy number of gene amplification are schematically shown.

SEQ ID NO: 1 Cre (nucleic acid sequence)
SEQ ID NO: 2 Cre (amino acid sequence)
SEQ ID NO: 3 loxP (wild type nucleic acid sequence)
SEQ ID NO: 4 loxP (mutant nucleic acid sequence 1)
SEQ ID NO: 5 loxP (mutant nucleic acid sequence 2)
SEQ ID NO: 6 Arg-rich sequence derived from Tat sequence SEQ ID NO: 7 Tat-Cre-Forward primer SEQ ID NO: 8 Tat-Cre-Reverse primer

Claims (25)

A mammalian cell containing at least three nucleic acid fragments containing a site-specific recombination sequence and a selectable marker gene inserted into a chromosome. The mammalian cell according to claim 1, further comprising a site-specific recombination inducer. The mammalian cell of claim 1, wherein the selectable marker gene is selected from the group consisting of: dihydrofolate reductase gene, glutamine synthetase gene, aspartate transaminase, metallothionein, adenosine deaminase (ADA), adenosine deaminase (AMPD1, 2), xanthine-guanine-phosphoribosyltransferase, UMP synthase, P-glycoprotein, asparagine synthetase, ornithine decarboxylase. The mammalian cell according to claim 3, wherein the selectable marker gene is dihydrofolate reductase. The mammalian cell according to claim 2, wherein the site-specific recombination inducer is Cre protein, and the site-specific recombination sequence is a wild type loxP sequence or a mutant loxP sequence. The mammalian cell according to claim 1, wherein the insertion position in the chromosome of the nucleic acid fragment is inserted near the centromere, near the telomere, or near the telomere pole of the chromosome of the mammalian cell. The mammalian cell according to claim 1, which is a CHO cell. The insertion position in the chromosome of the nucleic acid fragment is in the vicinity of the centromere of the E chromosome, the centromere of the J chromosome, the centromere of the K chromosome, the centromere of the L chromosome, the telomere of the N chromosome, the telomere pole of the N chromosome of the CHO cell. The mammalian cell according to claim 7, which is in the vicinity, the P chromosome telomere side, the P chromosome telomere vicinity, or the P chromosome telomere pole vicinity. The mammalian cell according to claim 8, wherein the insertion position in the chromosome of the nucleic acid fragment is in the vicinity of the telomer pole of the N chromosome or the pole of the P chromosome telomere in the CHO cell. The mammalian cell according to claim 1, wherein a nucleic acid fragment containing a sequence homologous to the site-specific recombination sequence inserted into a chromosome can be inserted into the chromosome by site-specific recombination. The mammalian cell according to claim 1, wherein a nucleic acid fragment containing a sequence homologous to the site-specific recombination sequence inserted into a chromosome is inserted into the chromosome by site-specific recombination. The mammalian cell according to claim 11, wherein the nucleic acid fragment inserted into the chromosome by site-specific recombination contains a foreign gene operably linked to a promoter. A method for producing a foreign gene product, comprising culturing the mammalian cell according to claim 12 under conditions in which the mammalian cell expresses the foreign gene. A kit comprising the mammalian cell according to claim 1 or 2. A method for producing a mammalian cell that can be used to express a foreign gene comprising the following steps:
(A) inserting a nucleic acid fragment containing a first site-specific recombination sequence and a selectable marker gene into the chromosome of the mammalian cell; and (b) culturing the mammalian cell in the presence of a gene amplification inducer. And causing gene amplification of a nucleic acid fragment containing a selectable marker gene;
Including the method. Furthermore, the manufacturing method of Claim 15 including the following processes:
(C) A step of selecting a mammalian cell in which the insertion position in the chromosome of the nucleic acid fragment is near the N-chromosome telomere pole or near the P-chromosome telomere pole. In addition, any of the following steps:
(D) introducing a site-specific recombination inducer into the mammalian cell; or (d ′) introducing a vector capable of expressing the site-specific recombination inducer into the mammalian cell. ,
The manufacturing method of Claim 15 including In addition, the following steps:
(E) introducing a nucleic acid fragment containing a foreign gene operably linked to a promoter and a second site-specific recombination sequence homologous to the first site-specific recombination sequence into the mammalian cell; 18. The production according to claim 17, comprising the step of site-specific recombination of the first site-specific recombination sequence and the second site-specific recombination sequence by the site-specific recombination inducer. Method. The production method according to claim 15, wherein the selection marker gene is selected from the group consisting of: dihydrofolate reductase gene, glutamine synthetase gene, aspartate transaminase, metallothionein, adenosine deaminase (ADA), adenosine deaminase ( AMPD1, 2), xanthine-guanine-phosphoribosyltransferase, UMP synthase, P-glycoprotein, asparagine synthetase, ornithine decarboxylase. The production method according to claim 19, wherein the selectable marker gene is dihydrofolate reductase. The site-specific recombination inducer is a Cre protein, and at least one of the first site-specific recombination sequence and the second site-specific recombination sequence is a wild type or mutant loxP sequence. Item 18. The manufacturing method according to Item 17. The production method according to claim 15, wherein the mammalian cell is a CHO cell or a cell derived from a CHO cell. The production method according to claim 15, wherein the step (b) of causing gene amplification is performed under conditions in which a cell in which a gene contained in a vector inserted in the vicinity of telomeres is amplified appears selectively. A mammalian cell produced by the method according to any one of claims 15 to 18. A method for producing a foreign gene product, comprising the step of culturing mammalian cells produced by the method according to claim 18 under conditions in which the mammalian cells express the foreign gene.