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  Method and kit for expressing protein under regulation of the expression from repeated sequence formed by gene amplification, and transformantUSPTO Application #: 20070298458Title: Method and kit for expressing protein under regulation of the expression from repeated sequence formed by gene amplification, and transformant Abstract: A method is disclosed for releasing the transcriptional regulation caused by a repeated sequence in a gene, a kit therefor and so on to thereby establish a system capable of producing a protein in a large amount. At least one embodiment of the method can be achieved by any one or more of the following methods: (a) in the amplification of a gene encoding a target protein, co-amplifying a polynucleotide of 10 kbp or more such as a λ-phage DNA or an insulator sequence; (b) selecting by culturing cells having undergone gene amplification in media containing a drug with a gradual increase in concentration; (c) elevating the promoter activity of inducing the expression of a gene encoding a target protein; (d) excising an amplified gene region from a chromosome with the use of Cre-LoxP System; (e) treating cells having undergone gene amplification with 5-aza-2′-deoxycytidine to thereby lower the methylation degree of DNA; and (f) selecting the mammalian cells having undergone gene amplification on double minute chromosomes. (end of abstract) Agent: Harness, Dickey & Pierce, P.L.C - Reston, VA, US Inventor: Noriaki Shimizu USPTO Applicaton #: 20070298458 - Class: 435069100 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Recombinant Dna Technique Included In Method Of Making A Protein Or Polypeptide The Patent Description & Claims data below is from USPTO Patent Application 20070298458. Brief Patent Description - Full Patent Description - Patent Application Claims
TECHNICAL FIELD [0001] The present invention relates to a method of expressing a protein, whose expression is repressed, from a repeated sequence formed in mammalian cells in which gene amplification is induced. BACKGROUND ART [0002] The inventor of the present invention has found that it becomes possible to copy a gene (target gene), which encodes a target protein, in an intercellular copy number increased to approximately 100,000 copies simply by plasmid conduction into human-derived cancer cells (COLO 320 colon cancer cell line, and HeLa cell line) by lipofection, the plasmid (hereinafter, "IR/MAR plasmid") having mammalian copying initiation region (IR; initiation region) and a matrix attachment region (MAR; matrix attachment region), and selecting by utilizing a gene tolerant to a chemical (Blasticidine or Neomycine), and that the mass amplification of the target gene can be attained regardless of whether the target gene has a gene structure identical to that of the IR/MAR plasmid (i.e., the target gene has a cis gene structure) or the target gene has a gene structure different from that of the IR/MAR plasmid (i.e., the target gene has a trans gene structure) (see Patent Document 1, Patent Document 2, Non-Patent Document 1, and Non-Patent Document 2). [0003] A cell line in which an IR/MAR plasmid and a target gene were transfected, was analyzed quantitatively as to a transcription amount of mRNA from the target gene. This analysis found that the transcription amount of mRNA was not increased while the copy number of the target gene was increased. It was deduced that the transcription was repressed due to a repeated sequence produced by the mass amplification of a region including the target gene. [0004] One known method of releasing the transcription repression caused by the repeated gene sequence is, for example, treating the cells with histone acetylating enzyme inhibitor such as trichostatin A (see Non-Patent Document 3). [0005] [Patent Document 1] [0006] Japanese Unexamined Patent Application Publication, Tokukai, No. 2003-245083 (published on Sep. 2, 2003) [0007] [Patent Document 2] [0008] Japanese Unexamined Patent Application Publication, Tokukai, No. 2004-337066 (published on Dec. 2, 2004) [0009] [Non-Patent Document 1] [0010] Noriaki Shimizu, et al. (2001) Plasmids with a Mammalian Replication Origin and a Matrix Attachment Region Initiate the Event Similar to Gene Amplification. Cancer Research vol. 61, no. 19, p6987-6990. [0011] [Non-Patent Document 2] [0012] Noriaki Shimizu, et al (2003) Amplification of plasmids containing a mammalian replication initiation region is mediated by controllable conflict between replication and transcription. Cancer Research, vol. 63, no. 17, p5281-5290. [0013] [Non-Patent Document 3] [0014] McBurney, M. W. et al, Exp Cell Res (2002), vol 274, p1-8 [0015] Therefore, there is such a problem in that even if a polynucleotide containing the amplified target gene is amplified in order to mass-produce a protein via the amplification of the target gene, the transcription of the target gene is repressed once the repeated sequence is created thereby failing to express the protein of the final target. [0016] Sole application of the method of treating the cells with histone acetylating enzyme inhibitor such as trichostatin A as disclosed in Non-Patent Document 3 could not sufficiently release the transcription repression caused by the repeated sequence of the gene amplified to several thousand to approximately 10 thousand copies. [0017] An object of the present invention is to provide a method and kit etc. for releasing the transcription repression caused by the repeated sequence of the gene, and to establish a system for mass production of useful protein by gene amplification. DISCLOSURE OF INVENTION [0018] In order to attain the object, the inventor of the present invention diligently worked to find a method capable of amplifying a target gene by using the IR/MAR plasmid, and expressing a protein without transcription repression, even in case where repeated sequence occurs. The inventor accomplished the present invention based on the result of the diligent work. [0019] In order to attain the object, a method according to the present invention is a method of expressing a protein from a repeated sequence formed in mammalian cells in which gene amplification is induced, the protein having been under expression repression, the method including: transfecting a first polynucleotide and a second polynucleotide simultaneously into the mammalian cells, where the first polynucleotide includes an origin of replication and a nucleic matrix attachment region that function in eukaryotic cells, and the second polynucleotide encodes a protein to be expressed, the method further comprising: transfecting at least one of a third polynucleotide and a fourth polynucleotide into the mammalian cells in transfecting the first polynucleotide and the second polynucleotide into the mammalian cells, where the third polynucleotide has a length of 10 kbp or more, and the fourth polynucleotide includes an insulator sequence. [0020] Moreover, in order to attain the object, a method according to the present invention is a method of expressing a protein from a repeated sequence formed in mammalian cells in which gene amplification is induced, the protein having been under expression repression, the method including: transfecting a first polynucleotide and a fifth polynucleotide simultaneously in the mammalian cells, where the first polynucleotide includes an origin of replication and a nucleic matrix attachment region that function in eukaryotic cells, and the fifth polynucleotide includes a second polynucleotide and a chemical tolerant gene, the second polynucleotide encoding a protein to be expressed, the method further comprising: culturing the mammalian cells sequentially in a medium of increasing concentrations of a chemical. [0021] Moreover, in order to attain the object, a method according to the present invention is a method of expressing a protein from a repeated sequence formed in mammalian cells in which gene amplification is induced, the protein having been under expression repression, the method including: transfecting a first polynucleotide and a sixth polynucleotide simultaneously in the mammalian cells where the first polynucleotide includes an origin of replication and a nucleic matrix attachment region that function in eukaryotic cells, and the sixth polynucleotide includes a promoter region and a second polynucleotide controllably linked with each other, the second polynucleotide encoding a protein to be expressed, the method further comprising: transfecting into the mammalian cells a seventh polynucleotide, which encodes a transcription activation factor of the promoter, and expressing the transcription activation factor. Continue reading... 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