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.

1 . 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 a third 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.    
     
     
         2 . The method as set forth in  claim 1 , wherein: 
 the second polynucleotide is transfected as a fifth polynucleotide that includes the second polynucleotide and a chemical tolerant gene; and    the method further comprises:    culturing the mammalian cells sequentially in a medium of increasing concentrations of a chemical.    
     
     
         3 . The method as set forth in  claim 1 , comprising: 
 selecting mammalian cells that the gene amplification occurs on a double minute chromosome.    
     
     
         4 . The method as set forth in  claim 1 , comprising: 
 treating the mammalian cells with 5-aza-2′-deoxycytidine.    
     
     
         5 . The method as set forth in  claim 1 , wherein the origin of replication of c-myc locus, dihydrofolate reductase locus, or β-globin locus.  
     
     
         6 . The method as set forth in  claim 1 , wherein the nucleic matrix attachment of region is derived from a nucleic matrix attachment region of Igκ locus, SV40 initial region, or dihydrofolate reductase locus.  
     
     
         7 . A kit for 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 kit comprising: 
 a first polynucleotide including an origin of replication and a nucleic matrix attachment region that function in eukaryotic cells; and    a third polynucleotide, which has a length of 10 kbp or more.    
     
     
         8 . The kit as set forth in  claim 7 , the kit further comprising 5-aza-2′-deoxycytidine.  
     
     
         9 . The kit as set forth in  claim 7 , wherein the origin of replication is derived from an origin of replication of c-myc locus, dihydrofolate reductase locus, or β-globin locus.  
     
     
         10 . The kit as set forth in  claim 7 , wherein the nucleic matrix attachment region is derived from a nucleic matrix attachment region of Igκ locus, SV40 initial region, or dihydrofolate reductase locus.  
     
     
         11 . A transformant prepared by inserting into mammalian cells: 
 a first polynucleotide including an origin of replication and a nucleic matrix attachment region that function in eukaryotic cells;    a second polynucleotide encoding a protein to be expressed; and    a third polynucleotide, which has a length of 10 kbp or more.    
     
     
         12 . The method as set forth in  claim 11 , wherein the origin of replication is derived from an origin of replication of c-myc locus, dihydrofolate reductase locus, or β-globin locus.  
     
     
         13 . The method as set forth in  claim 11 , wherein the nucleic matrix attachment region is derived from a nucleic matrix attachment region of Igκ locus, SV40 initial region, or dihydrofolate reductase locus.  
     
     
         14 . The transformant as set forth in  claim 11 , wherein the mammalian cells are cells selected from the group consisting of COLO 320DM cells, COLO 320HSR cells, Hela cells, and CHO cells.  
     
     
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