Antibody producing non-human mammals
Described are transgenic, non-human animals comprising a nucleic acid encoding an immunoglobulin light chain, whereby the immunoglobulin light chain is human, human-like, or humanized. The nucleic acid is provided with a means that renders it resistant to DNA rearrangements and/or somatic hypermutations. In one embodiment, the nucleic acid comprises an expression cassette for the expression of a desired molecule in cells during a certain stage of development in cells developing into mature B cells. Further provided is methods for producing an immunoglobulin from the transgenic, non-human animal.
1. A method of obtaining an antibody that binds to an antigen, the method comprising
(a) immunizing a transgenic mouse with the antigen, wherein the genome of the transgenic mouse comprises a transgene comprising a human immunoglobulin light chain germline V gene segment fused to a human immunoglobulin light chain germline J gene segment such that there is no mutation due to said fusion, wherein the fused human V/J gene segments encode a rearranged human immunoglobulin light chain variable region and
wherein the transgene is inserted by site-specific integration in the murine Rosa locus or wherein said transgene lacks the intronic light chain enhancer MoEκi or comprises a truncation of the transgene 3′ kappa enhancer or combination of these,
wherein the transgene comprises a murine light chain constant region gene segment or is operatively linked to an endogenous mouse light chain constant region gene segment; and
wherein if the transgene is inserted by site-specific integration in the murine Rosa locus, the transgene comprises a murine light chain constant region gene segment;
(b) obtaining a population of B cells producing antigen specific antibodies from the transgenic mouse, wherein said antibodies comprise the rearranged human light chain immunoglobulin variable region encoded by the fused human V/J gene segments and a murine constant region paired with a heavy chain variable region encoded by a rearranged and somatically hypermutated VH gene; and
wherein the population of B cells producing antigen specific antibodies from the transgenic mouse comprise a diversity of clonally unrelated rearranged immunoglobulin heavy chain variable regions that bind to the antigen;
(c) isolating nucleic acid encoding a rearranged immunoglobulin heavy chain variable region from a B cell in said population;
(d) expressing nucleic acids encoding the rearranged immunoglobulin heavy chain variable region and at least the rearranged human immunoglobulin light chain variable region in a host cell; and
(e) thus obtaining an antibody which binds to the antigen.
2. A method of obtaining an antibody that binds to an antigen, the method comprising
(a) immunizing a transgenic mouse with the antigen, wherein the genome of the transgenic mouse comprises a transgene comprising a human immunoglobulin light chain germline V gene segment fused to a human immunoglobulin light chain germline J gene segment such that there is no mutation due to said fusion, wherein the fused human V/J gene segments encode a rearranged human immunoglobulin light chain variable region and
wherein the transgene is inserted by site-specific integration in the murine Rosa locus or wherein said transgene lacks the intronic light chain enhancer MoEκi or comprises a truncation of the transgene 3′ kappa enhancer or combination of these; and
wherein the transgene comprises a murine light chain constant region gene segment or is operatively linked to an endogenous mouse light chain constant region gene segment; and
wherein if the transgene is inserted by site-specific integration in the murine Rosa locus, the transgene comprises a murine light chain constant region gene segment;
wherein said transgenic mouse, in response to said antigen, produces mature B cells that produce antibodies with immunoglobulin light chains comprising said encoded rearranged human light chain variable region and a murine light chain constant region, paired with a diversity of clonally unrelated rearranged immunoglobulin heavy chain variable regions, which bind said antigen; and
(b) isolating nucleic acid encoding a rearranged immunoglobulin heavy chain variable region from a B cell that produces one of said antibodies;
(c) expressing said nucleic acid encoding said rearranged immunoglobulin heavy chain variable region in a host cell together with nucleic acid encoding at least said rearranged human light chain variable region; and
(d) obtaining an antibody that binds to the antigen.
3. A method of obtaining an antibody that binds to an antigen, the method comprising
(a) isolating nucleic acid encoding a rearranged immunoglobulin heavy chain variable region from a B cell that produces an antibody that comprises a rearranged human light chain immunoglobulin variable region encoded by fused human V/J gene segments and comprises a murine constant region paired with a heavy chain variable region encoded by a rearranged and somatically hypermutated VH gene that encodes said antibody;
(b) expressing said nucleic acid encoding said rearranged immunoglobulin heavy chain variable region in a host cell together with nucleic acid encoding at least a rearranged human light chain immunoglobulin variable region encoded by fused human V/J gene segments; and
(c) obtaining an antibody which binds to the antigen,
wherein said B cell is obtained by immunizing a transgenic mouse with the antigen, wherein the genome of the transgenic mouse comprises a transgene comprising a human immunoglobulin light chain germline V gene segment fused to a human immunoglobulin light chain germline J gene segment such that there is no mutation due to said fusion, wherein the fused human V/J gene segments encode a rearranged human immunoglobulin light chain variable region and
wherein the transgene is inserted by site-specific integration in the murine Rosa locus or wherein said transgene lacks the intronic light chain enhancer MoEκi or comprises a truncation of the transgene 3′ kappa enhancer or combination of these; and
wherein the transgene comprises a murine light chain constant region gene segment or is operatively linked to an endogenous mouse light chain constant region gene segment;
wherein if the transgene is inserted by site-specific integration in the murine Rosa locus, the transgene comprises a murine light chain constant region gene segment;
wherein said transgenic mouse, in response to said antigen, produces mature B cells that secrete antibodies with immunoglobulin light chains comprising said encoded rearranged human light chain variable region and a murine light chain constant region, paired with a diversity of clonally unrelated rearranged immunoglobulin heavy chain variable regions, which bind said antigen.