Expression of Xenogenous (Human) Immunoglobulins in cloned, transgenic ungulates

Abstract

The present invention relates to the production of a transgenic ungulate which comprises a genetic modification that results in inactivation and loss of expression of its endogenous antibodies, and the expression of xenogenous antibodies, preferably human antibodies. This is effected by inactivation of the IgM heavy chain expression and, optionally, by inactivation of the Ig light chain expression, and by the further introduction of an artificial chromosome which results in the expression of non-bovine antibodies, preferably human antibodies.

1 . A transgenic ungulate whose cells comprise a mutation in an immunoglobulin locus that reduces the expression of an endogenous antibody and whose cells further comprise one or more nucleic acids, each nucleic acid comprising one or more xenogenous immunoglobulin heavy or light chain loci that undergo rearrangement and are expressed in B cells to produce xenogenous immunoglobulin molecules in response to one or more antigens. 
     
     
         2 . The ungulate of  claim 1 , wherein said mutation reduces the expression of a functional immunoglobulin heavy chain. 
     
     
         3 . The ungulate of  claim 2 , wherein said mutation reduces the expression of functional IgM heavy chain. 
     
     
         4 . The ungulate of  claim 1 , wherein said mutation reduces the expression of functional immunoglobulin light chain. 
     
     
         5 . The ungulate of  claim 1 , wherein said mutation is a nonsense or deletion mutation. 
     
     
         6 . The ungulate of  claim 1 , wherein said immunoglobulin molecule is an antibody protein. 
     
     
         7 . The ungulate of  claim 6 , wherein said antibody protein is a human antibody protein. 
     
     
         8 . The ungulate of  claim 1 , wherein said ungulate is a pig. 
     
     
         9 . The ungulate of  claim 1 , wherein said xenogenous immunoglobulin loci comprise both a human immunoglobulin light chain locus and a human immunoglobulin heavy chain locus. 
     
     
         10 . The ungulate of  claim 1 , wherein said one or more nucleic acids comprise one or more artificial chromosomes. 
     
     
         11 . The ungulate of  claim 10 , wherein said one or more artificial chromosomes comprise a human artificial chromosome. 
     
     
         12 . The ungulate of  claim 11 , wherein said human artificial chromosome is ΔHAC or ΔΔHAC. 
     
     
         13 . The ungulate of  claim 12 , wherein said human artificial chromosome is derived from one or more of human chromosome 14, human chromosome 2, and human chromosome 22. 
     
     
         14 . The ungulate of  claim 1 , wherein said one or more nucleic acids comprise a chromosome fragment. 
     
     
         15 . The ungulate of  claim 14 , wherein said chromosome fragment is selected from the group consisting of a YAC, BAC, or cosmid vector. 
     
     
         16 . An isolated transgenic ungulate cell comprising a mutation in an immunoglobulin locus that reduces the expression of an endogenous antibody and further comprising one or more nucleic acids, each nucleic acid comprising one or more xenogenous immunoglobulin heavy or light chain loci that undergo rearrangement and are expressed in B cells to produce xenogenous immunoglobulin molecules in response to one or more antigens. 
     
     
         17 . The ungulate cell of  claim 16 , wherein said mutation reduces the expression of a functional immunoglobulin heavy chain. 
     
     
         18 . The ungulate cell of  claim 17 , wherein said mutation reduces the expression of functional IgM heavy chain. 
     
     
         19 . The ungulate cell of  claim 16 , wherein said mutation reduces the expression of a functional immunoglobulin light chain. 
     
     
         20 . The ungulate cell of  claim 16 , wherein said mutation is a nonsense or deletion mutation. 
     
     
         21 . The ungulate cell of  claim 16 , wherein said xenogenous immunoglobulin molecule is human immunoglobulin. 
     
     
         22 . The ungulate cell of  claim 16 , wherein said xenogenous immunoglobulin loci comprise both a human immunoglobulin light chain locus and a human immunoglobulin heavy chain locus. 
     
     
         23 . The ungulate cell of  claim 16 , wherein said one or more nucleic acids comprise one or more artificial chromosomes. 
     
     
         24 . The ungulate cell of  claim 23 , wherein said one or more artificial chromosomes comprise a human artificial chromosome. 
     
     
         25 . The ungulate cell of  claim 24 , wherein said human artificial chromosome is ΔHAC or tΔHAC. 
     
     
         26 . The ungulate cell of  claim 24 , wherein said human artificial chromosome is derived from one or more of human chromosome 14, human chromosome 2, and human chromosome 22. 
     
     
         27 . The ungulate cell of  claim 16 , wherein said one or more nucleic acids comprise a chromosome fragment. 
     
     
         28 . The ungulate cell of  claim 27 , wherein said chromosome fragment is selected from the group consisting of a YAC, BAC, or cosmid vector. 
     
     
         29 . The ungulate cell of  claim 16 , wherein said cell is a fetal fibroblast or a B-cell. 
     
     
         30 . A method of producing xenogenous antibodies against one or more antigens, said method comprising the steps of:
 (a) administering one or more antigens to a transgenic ungulate whose cells comprise a mutation in an immunoglobulin locus that reduces the expression of an endogenous antibody and whose cells further comprise one or more nucleic acids, each nucleic acid comprising one or more xenogenous immunoglobulin heavy or light chain loci that undergo rearrangement and are expressed in B cells to produce xenogenous immunoglobulin, resulting in production of xenogenous antibodies against said one or more antigens; and   (b) recovering said xenogenous antibodies from said ungulate.   
     
     
         31 . A method of producing xenogenous antibodies against one or more antigens, said method comprising recovering xenogenous antibodies from a transgenic ungulate whose cells comprise a mutation in an immunoglobulin locus that reduces the expression of an endogenous antibody and whose cells further comprise one or more nucleic acids, each nucleic acid comprising one or more xenogenous immunoglobulin heavy or light chain loci that undergo rearrangement and are expressed in B cells, resulting in the production of xenogenous antibodies against said one or more antigens. 
     
     
         32 . The method of  claim 30  or  31 , wherein said mutation reduces the expression of a functional immunoglobulin heavy chain. 
     
     
         33 . The method of  claim 32 , wherein said mutation reduces the expression of a functional IgM heavy chain. 
     
     
         34 . The method of  claim 30  or  31 , wherein said mutation reduces the expression of a functional immunoglobulin light chain. 
     
     
         35 . The method of  claim 30  or  31 , wherein said mutation is a nonsense or deletion mutation. 
     
     
         36 . The method of  claim 30  or  31 , wherein said xenogenous antibodies are human antibodies. 
     
     
         37 . The method of  claim 30  or  31 , wherein said one or more nucleic acids comprise one or more artificial chromosomes. 
     
     
         38 . The method of  claim 37 , wherein said one or more artificial chromosomes comprise a human artificial chromosome. 
     
     
         39 . The method of  claim 38 , wherein said human artificial chromosome is ΔHAC or ΔΔHAC. 
     
     
         40 . The method of  claim 38 , wherein said human artificial chromosome is derived from one or more of human chromosome 14, human chromosome 2, and human chromosome 22. 
     
     
         41 . The method of  claim 30  or  31 , wherein said one or more nucleic acids comprise a chromosome fragment. 
     
     
         42 . The method of  claim 41 , wherein said chromosome fragment is selected from the group consisting of a YAC, BAC, or cosmid vector. 
     
     
         43 . The method of  claim 30  or  31 , wherein said antibodies are directed against a desired antigen. 
     
     
         44 . The method of  claim 30  or  31 , wherein said antibodies are polyclonal. 
     
     
         45 . The method of  claim 30  or  31 , wherein said antibodies are recovered from the milk or serum of said ungulate. 
     
     
         46 . The method of  claim 30  or  31 , wherein said ungulate is a pig. 
     
     
         47 . A method for producing a transgenic ungulate having reduced expression of an endogenous antibody, said method comprising the steps of:
 (a) incubating a permeabilized cell of  claim 16  in an extract from a mitotic somatic cell or oocyte under conditions that allow chromatin condensation and nuclear envelope breakdown in said permeabilized cell;   (b) inserting said cell formed in step (a) into a nucleated or enucleated ungulate oocyte, thereby forming a reconstituted oocyte; and   (c) transferring said reconstituted oocyte or an embryo formed from said reconstituted oocyte into the uterus of a host ungulate under conditions that allow said reconstituted oocyte or said embryo to develop into a fetus.   
     
     
         48 . The method of  claim 47 , wherein, prior to step (b), said cell is incubated under conditions that allow the membrane of said cell to reseal. 
     
     
         49 . The method of  claim 47 , wherein said cell is purified from said extract prior to insertion into said oocyte. 
     
     
         50 . The method of  claim 47 , wherein said fetus develops into a viable offspring. 
     
     
         51 . The method of  claim 50 , further comprising mating two offspring to produce a transgenic ungulate whose genome comprises mutations in both alleles of an endogenous immunoglobulin heavy chain or light chain locus. 
     
     
         52 . The method of  claim 47 , wherein said oocyte from step (b) is cultured under conditions that allow cell division and one of the resulting cells is recloned one or more times. 
     
     
         53 . The method of  claim 47 , wherein said permeabilized cell and said oocyte are from the same species. 
     
     
         54 . The method of  claim 47 , wherein said permeabilized cell is a fibroblast, epithelial cell, neural cell, epidermal cell, keratinocyte, hematopoietic cell, melanocyte, chondrocyte, macrophage, monocyte, fibroblast, muscle cell, embryonic stem cell, embryonic germ cell, fetal cell, placental cell, a cell of the female reproductive system, or embryonic cell. 
     
     
         55 . The method of  claim 47 , wherein said ungulate is a pig.