ADAM6 mice
Mice are provided that comprise a reduction or deletion of ADAM6 activity from an endogenous ADAM6 locus, or that lack an endogenous locus encoding a mouse ADAM6 protein, wherein the mice comprise a sequence encoding an ADAM6 or ortholog or homolog or fragment thereof that is functional in a male mouse. In one embodiment, the sequence is an ectopic ADAM6 sequence or a sequence that confers upon a male mouse the ability to generate offspring by mating. Mice and cells with genetically modified immunoglobulin heavy chain loci that comprise an ectopic nucleotide sequence encoding a mouse ADAM6 or functional fragment or homolog or ortholog thereof are also provided.
1. A method for producing a human heavy or light chain variable region sequence comprising the steps of:
(a) immunizing a genetically modified mouse with an antigen, wherein the mouse:
(i) has a genome that comprises:
(A) one or more human V H gene segments, one or more human D H gene segments, and one or more human J H gene segments operably linked to a mouse heavy chain constant region at an endogenous mouse immunoglobulin heavy chain locus,
(B) one or more human V L gene segments and one or more human J L gene segments operably linked to a light chain constant region, and
(C) a nucleotide sequence that encodes a mouse a disintegrin and metalloproteinase 6 (ADAM6) protein, which nucleotide sequence is integrated in the genome of the mouse, wherein the mouse ADAM6 protein is expressed and functional in the genetically modified mouse when the genetically modified mouse is male;
(ii) generates antibodies when immunized with the antigen, wherein the antibodies each comprise a heavy chain including a human heavy chain variable domain operably linked to a mouse heavy chain constant domain and a light chain including a human light chain variable domain operably linked to a light chain constant domain; and
(iii) is fertile;
(b) allowing the genetically modified mouse to mount an immune response to the antigen,
(c) isolating a B cell from the genetically modified mouse that expresses an antibody that specifically binds the antigen,
(d) amplifying a human heavy or light chain variable region sequence that encodes a human heavy or light chain variable domain, respectively, of an antibody that specifically binds the antigen and that was expressed by the isolated B cell of the genetically modified mouse, thereby producing the human heavy or light chain variable region sequence.
2. The method of claim 1 , wherein the one or more human V H gene segments are selected from the group consisting of V H 6-1, V H 1-2, V H 1-3, V H 2-5, V H 3-7, V H 1-8, V H 3-9, V H 3-11, V H 3-13, V H 3-15, V H 3-16, V H 1-18, V H 3-20, V H 3-21, V H 3-23, V H 1-24, V H 2-26, V H 4-28, V H 3-30, V H 4-31, V H 3-33, V H 4-34, V H 3-35, V H 3-38, V H 4-39, V H 3-43, V H 1-45, V H 1-46, V H 3-48, V H 3-49, V H 5-51, V H 3-53, V H 1-58, V H 4-59, V H 4-61, V H 3-64, V H 3-66, V H 1-69, V H 2-70, V H 3-72, V H 3-73, V H 3-74, and a combination thereof.
3. The method of claim 1 , wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to a mouse light chain constant region at an endogenous mouse light chain locus.
4. The method of claim 1 , wherein the one or more human V L gene segments are selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-16, Vκ1-17, Vκ1-27, Vκ1-33, Vκ1-37, Vκ1-39, Vκ2-24, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-40, Vκ3-11, Vκ3-15, Vκ3-20, Vκ6-21, and a combination thereof.
5. The method of claim 1 , wherein the nucleotide sequence encoding the mouse ADAM6 protein:
(a) is present on the same chromosome as the endogenous mouse immunoglobulin heavy chain locus;
(b) is present at an ectopic position, or
(c) a combination thereof.
6. The method of claim 1 , wherein the mouse ADAM6 protein is a mouse ADAM6a protein or a mouse ADAM6b protein.
7. The method of claim 1 , wherein the nucleotide sequence that encodes the mouse ADAM6 protein is contiguous with the one or more human V H gene segments, one or more human D H gene segments, or one or more human J H gene segments.
8. The method of claim 1 , wherein the one or more human V L gene segments and one or more human J L gene segments are one or more human Vκ gene segments and one or more human Jκ gene segments.
9. The method of claim 8 , wherein the one or more human Vκ gene segments and one or more human Jκ gene segments are operably linked to a mouse κ light chain constant region at an endogenous mouse κ light chain locus.
10. The method of claim 1 , wherein the one or more human V L gene segments and one or more human J L gene segments are one or more human Vλ gene segments and one or more human Jλ gene segments.
11. The method of claim 10 , wherein the one or more human Vλ gene segments and one or more human Jλ gene segments are operably linked to a mouse λ light chain constant region at an endogenous mouse λ light chain locus.
12. The method of claim 1 , wherein the one or more human V H gene segments includes at least 18 human V H gene segments.
13. The method of claim 1 , wherein the mouse heavy chain constant region gene comprises a CH1, a hinge, a CH2, a CH3, a CH4, or a combination thereof.