Isolation of a rearranged human immunoglobulin gene from a chimeric mouse and recombinant production of the encoded immunoglobulin

Abstract

The specification relates to a method for producing a chimeric non-human animal, which comprises preparing a microcell containing a foreign chromosome(s) or a fragment(s) thereof and transferring the foreign chromosome(s) or fragment(s) thereof into a pluripotent cell by fusion with the microcell; a chimeric non-human animal which can be produced by the above method and its progeny; tissues and cells derived therefrom; and a method for using the same. Further, a pluripotent cell containing a foreign chromosome(s) or a fragment(s) thereof, a method for producing the same, and a method for using the same are also provided. Moreover, a pluripotent cell in which at least two endogenous genes are disrupted, and a method for producing the same by homologous recombination are provided.

What is claimed is:  
     
         1 . A method for producing a chimeric non-human animal, which comprises preparing a microcell containing a foreign chromosome(s) or a fragment(s) thereof and transferring the foreign chromosome(s) or fragment(s) into a pluripotent cell by fusion with the microcell.  
     
     
         2 . A method for producing a pluripotent cell containing a foreign chromosome(s) or a fragment(s) thereof, which comprises preparing a microcell containing a foreign chromosome(s) or a fragment(s) thereof and transferring the foreign chromosome(s) or fragment(s) thereof into a pluripotent cell by fusion with the microcell.  
     
     
         3 . The method of  claim 1  or  2 , wherein the foreign chromosome(s) or fragment(s) thereof is(are) larger than 670 kb.  
     
     
         4 . The method of  claim 3 , wherein the foreign chromosome(s) or fragment(s) thereof is(are) at least 1 Mb in length.  
     
     
         5 . The method of  claim 1  or  2 , wherein the foreign chromosome or fragment thereof contains a region encoding an antibody.  
     
     
         6 . The method of  claim 1  or  2 , wherein the microcell containing a foreign chromosome(s) or a fragment(s) thereof is induced from a hybrid cell prepared by the fusion of a cell from which the foreign chromosome(s) or fragment(s) thereof is(are) derived, with a cell having a high ability to form a microcell.  
     
     
         7 . The method of  claim 6 , wherein the microcell containing a foreign chromosome(s) or a fragment(s) thereof is induced from a cell prepared by a further fusion of the microcell induced from said hybrid cell with a cell having a high ability to form a microcell.  
     
     
         8 . The method of  claim 6 , wherein the cell from which the foreign chromosome(s) or fragment(s) thereof is(are) derived is a human normal diploid cell.  
     
     
         9 . The method of any one of claims  6 - 8 , wherein the cell having a high ability to form a microcell is a mouse A9 cell.  
     
     
         10 . The method of  claim 1  or  2 , wherein the pluripotent cell is one selected from embryonal carcinoma cells, embryonic stem cells, embryonic germ cells and mutants thereof.  
     
     
         11 . The method of  claim 1  or  2 , wherein the foreign chromosome or fragment thereof contains a gene of interest and the pluripotent cell has a disrupted gene identical with or homologous to said gene of interest on the foreign chromosome or fragment thereof.  
     
     
         12 . The method of  claim 11 , wherein the foreign chromosome or fragment thereof contains at least two genes of interest and the pluripotent cell has disrupted genes identical with or homologous to said genes of interest on the foreign chromosome or fragment thereof.  
     
     
         13 . The method of  claim 11 , wherein one or both alleles of a gene identical with or homologous to the gene of interest on the foreign chromosome or fragment thereof are disrupted in the pluripotent cell.  
     
     
         14 . The method of  claim 11 , wherein the gene of interest is an antibody gene.  
     
     
         15 . The method of  claim 14 , wherein the antibody gene is one or more sets of antibody heavy-chain and light-chain genes.  
     
     
         16 . The method of  claim 1 , wherein the foreign chromosome or fragment thereof contains a gene of interest and said foreign chromosome or fragment thereof is transferred into a pluripotent cell having a disrupted gene identical with or homologous to said gene of interest and then, a chimera is produced from the pluripotent cell by using an embryo of a non-human animal in a strain deficient in an endogenous gene identical with or homologous to said gene of interest.  
     
     
         17 . The method of  claim 16 , wherein the non-human animal in a strain deficient in an endogenous gene identical with or homologous to the gene of interest is produced by homologous recombination in gene targeting.  
     
     
         18 . The method of  claim 1 , wherein the chimeric non-human animal retains the foreign chromosome(s) or fragment(s) thereof, expresses the gene(s) on the foreign chromosome(s) or fragment(s) thereof, and can transmit the foreign chromosome(s) or fragment(s) thereof to its progeny.  
     
     
         19 . The method of  claim 1 , wherein the chimeric non-human animal is a mammal.  
     
     
         20 . The method of  claim 19 , wherein the mammal is a mouse.  
     
     
         21 . A pluripotent cell containing a foreign chromosome(s) or a fragment(s) thereof.  
     
     
         22 . The cell of  claim 21 , wherein the foreign chromosome(s) or fragment(s) thereof is(are) larger than 670 kb.  
     
     
         23 . The cell of  claim 21 , wherein the foreign chromosome or fragment thereof contains a gene of interest and the pluripotent cell has a disrupted endogenous gene identical with or homologous to said gene of interest on the foreign chromosome or a fragment thereof.  
     
     
         24 . The cell of  claim 23 , wherein the foreign chromosome or fragment thereof contains at least two genes of interest and the pluripotent cell has disrupted endogenous genes identical with or homologous to said genes of interest on the foreign chromosome or a fragment thereof.  
     
     
         25 . The cell of  claim 23 , wherein one or both alleles of an endogenous gene identical with or homologous to the gene of interest are disrupted in the pluripotent cell.  
     
     
         26 . The cell of  claim 21 , wherein the foreign chromosome or fragment thereof contains an antibody gene.  
     
     
         27 . The cell of  claim 26 , wherein the antibody gene is one or more sets of antibody heavy-chain and light-chain genes.  
     
     
         28 . The cell of  claim 21 , wherein the pluripotent cell is one selected from embryonal carcinoma cells, embryonic stem cells, embryonic germ cells and mutants thereof.  
     
     
         29 . A chimeric non-human animal retaining a foreign chromosome(s) or a fragment(s) thereof and expressing a gene(s) on the foreign chromosome(s) or fragment(s) thereof, or its progeny retaining the foreign chromosome(s) or fragment(s) thereof and expressing the gene(s) on the foreign chromosome(s) or fragment(s) thereof.  
     
     
         30 . The chimeric non-human animal or its progeny of  claim 29 , wherein the foreign chromosome(s) or fragment(s) thereof is(are) larger than 670 kb.  
     
     
         31 . The chimeric non-human animal or its progeny of  claim 29 , wherein the foreign chromosome or fragment thereof contains a gene of interest and the animal has a disrupted endogenous gene identical with or homologous to said gene of interest.  
     
     
         32 . The chimeric non-human animal or its progeny of  claim 31 , wherein the foreign chromosome or fragment thereof contains at least two genes of interest and said animal has disrupted endogenous genes identical with or homologous to said genes of interest.  
     
     
         33 . The chimeric non-human animal or its progeny of  claim 31 , wherein one or both alleles of an endogenous gene identical with or homologous to said gene of interest are disrupted.  
     
     
         34 . The chimeric non-human animal or its progeny of  claim 31 , wherein the gene of interest is an antibody gene.  
     
     
         35 . The chimeric non-human animal or its progeny of  claim 34 , wherein the antibody gene is one or more sets of antibody heavy-chain and light-chain genes.  
     
     
         36 . A non-human animal which can be produced by mating the chimeric non-human animals or their progenies of  claim 29 , said non-human animal retaining the foreign chromosome(s) or fragment(s) thereof and expressing the gene(s) on the foreign chromosome(s) or fragment(s) thereof, or its progeny retaining the foreign chromosome(s) or fragment(s) thereof and expressing the gene(s) on the foreign chromosome(s) or fragment(s) thereof.  
     
     
         37 . A non-human animal retaining the foreign chromosome(s) or fragment(s) thereof and expressing a gene(s) on the foreign chromosome(s) or fragment(s) thereof, which can be produced by mating the chimeric non-human animal or its progeny of  claim 29 , or the non-human animal or its progeny of  claim 36 , with a non-human animal in a strain deficient in said gene(s) or a gene homologous thereto, or its progeny retaining the foreign chromosome(s) or fragment(s) thereof and expressing the gene(s) on the foreign chromosome(s) or fragment(s) thereof.  
     
     
         38 . A tissue from the chimeric non-human animal or its progeny of  claim 29  or from the non-human animal or its progeny of  claim 36  or from the non-human animal or its progeny of  claim 37 .  
     
     
         39 . A cell from the chimeric non-human animal or its progeny of  claim 29  or from the non-human animal or its progeny of  claim 36  or from the non-human animal or its progeny of  claim 37 .  
     
     
         40 . The cell of  claim 39 , which is a B cell.  
     
     
         41 . A hybridoma prepared by the fusion of the cell of  claim 40  with a myeloma cell.  
     
     
         42 . A method for producing a biologically active substance, which comprises expressing the gene(s) on the foreign chromosome(s) or fragment(s) thereof in the chimeric non-human animal or its progeny of  claim 29 , the non-human animal or its progeny of  claim 36  or the non-human animal or its progeny of  claim 37 , or a tissue or a cell thereof, and recovering the biologically active substance as an expression product.  
     
     
         43 . The method of  claim 42 , wherein the cell of the chimeric non-human animal is a B cell.  
     
     
         44 . The method of  claim 43 , wherein the B cell is immortalized by fusion with a myeloma cell.  
     
     
         45 . The method of  claim 42 , wherein the biologically active substance is an antibody.  
     
     
         46 . The method of  claim 45 , wherein the antibody is an antibody of a mammal.  
     
     
         47 . The method of  claim 46 , wherein the antibody of a mammal is a human antibody.  
     
     
         48 . A biologically active substance which can be produced by the method of  claim 42 .  
     
     
         49 . A non-human animal retaining at least one human antibody gene larger than 670 kb and expressing the gene.  
     
     
         50 . The non-human animal of  claim 49 , which retains at least one human antibody gene of at least 1 Mb and expresses the gene.  
     
     
         51 . The non-human animal of  claim 49 , wherein the human antibody gene is selected from the group consisting of human heavy-chain gene, human light-chain κ gene, human light-chain λ gene, and combinations thereof.  
     
     
         52 . The non-human animal of  claim 49 , which is deficient in a non-human animal antibody gene identical with or homologous to the human antibody gene.  
     
     
         53 . The non-human animal of  claim 52 , wherein the deficiency of said non-human animal antibody gene is caused by disrupting the non-human animal antibody gene by homologous recombination.  
     
     
         54 . A hybridoma prepared by the fusion of a spleen cell of the non-human animal of  claim 49  with a myeloma cell.  
     
     
         55 . An antibody produced by the hybridoma of  claim 54 .  
     
     
         56 . A non-human animal expressing at least one class or subclass of human antibody.  
     
     
         57 . The non-human animal of  claim 56 , which is deficient in an endogenous antibody gene identical with or homologous to the expressed class or subclass of human antibody gene.  
     
     
         58 . The non-human animal of  claim 56 , wherein the class or subclass of human antibody is selected from IgM, IgG, IgE, IgA, IgD and their subclasses, and combinations thereof.  
     
     
         59 . A non-human animal retaining a foreign DNA(s) larger than 670 kb and expressing a gene(s) on the foreign DNA(s).  
     
     
         60 . The non-human animal of  claim 59 , which is deficient in an endogenous gene identical with or homologous to the expressed gene on the foreign DNA.  
     
     
         61 . The non-human animal of  claim 59  which retains a foreign DNA(s) of at least 1 Mb and expresses the gene(s) on the foreign DNA(s).  
     
     
         62 . The non-human animal of  claim 61 , which is deficient in an endogenous gene identical with or homologous to the expressed gene on the foreign DNA.  
     
     
         63 . A method for producing a transgenic non-human animal, which comprises preparing a microcell containing a foreign chromosome(s) or a fragment(s) thereof, transferring the foreign chromosome(s) or fragment(s) into a cultured cell derived from a blastcyst by fusion with the microcell and transplanting the nucleus of the cultured cell into an enucleated unfertilized egg.  
     
     
         64 . A pluripotent cell in which at least two endogenous genes are disrupted.  
     
     
         65 . The cell of  claim 64 , in which each of the endogenous genes is disrupted in one or both alleles.  
     
     
         66 . The cell of  claim 64 , wherein the disrupted endogenous genes are antibody genes.  
     
     
         67 . The cell of  claim 66 , wherein the antibody genes are antibody heavy-chain and light-chain genes.  
     
     
         68 . The cell of  claim 64 , wherein the pluripotent cell is one selected from embryonal carcinoma cells, embryonic stem cells, embryonic germ cells and mutants thereof.  
     
     
         69 . A method of producing the cell of  claim 64  by at least two homologous recombinations.  
     
     
         70 . The method of  claim 69 , which comprises the steps of: disrupting one allele of the endogenous gene in the pluripotent cell by homologous recombination using a drug-resistant marker gene; 
 culturing the pluripotent cell in the presence of the drug to select drug-resistant cells; and    screening the selected drug-resistant cells to yield a cell in which both alleles of the endogenous gene have been disrupted.    
     
     
         71 . The method of  claim 69 , in which one allele of the endogenous gene in the pluripotent cell is disrupted by homologous recombination using a drug-resistant marker gene and the other allele of the endogenous gene is disrupted by another homologous recombination using a drug-resistant marker gene.  
     
     
         72 . The method of  claim 71 , wherein the same drug-resistant marker gene is used in the two homologous recombinations.  
     
     
         73 . The method of  claim 71 , wherein different drug-resistant marker genes are used in the two homologous recombinations.