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 of producing a fully human antibody specific against an antigen comprising the steps of:
(a) immunizing a genetically modified mouse with the antigen, wherein the mouse:
(i) has a genome that comprises:
(1) 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 sequence at an endogenous mouse immunoglobulin heavy chain locus,
(2) 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 sequence, and
(3) 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 in the genetically modified mouse;
(ii) generates antibodies when immunized with the antigen, wherein the antibodies each comprise a human heavy chain variable domain operably linked to a mouse heavy chain constant domain and a human light chain variable domain operably linked to a light chain constant domain; and
(iii) is fertile;
(b) determining:
(i) a human heavy chain variable domain sequence of an antibody that specifically binds the antigen and that was generated by the genetically modified mouse, and
(ii) a human light chain variable domain sequence of an antibody that specifically binds the antigen and that was generated by the genetically modified mouse; and
(c) producing a fully human antibody by expressing in a host cell:
(i) a nucleotide sequence that encodes the human heavy chain variable domain sequence of (b) and a human heavy chain constant domain sequence, and
(ii) a nucleotide sequence that encodes the human light chain variable domain sequence of (b) and a human light chain constant domain 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 sequence 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 mouse heavy chain constant region sequence is a gamma constant region sequence.
9. The method of claim 1 , wherein the human heavy chain constant region sequence is a gamma constant region sequence.
10. The method of claim 1 , wherein the human heavy chain variable domain sequence is encoded by a human V H 6-1, V H 1-2, V H 3-23, V H 1-69, or somatically hypermutated version thereof.
11. The method of claim 1 , wherein the genetically modified mouse comprises sperm having on its surface an ADAM protein complex including the mouse ADAM6 protein.
12. The method of claim 1 , wherein the genetically modified mouse comprises sperm capable of traversing from a uterus and through an oviduct of a female mouse as measured by a sperm migration assay.
13. The method of claim 1 , wherein the genetically modified mouse comprises sperm capable of crossing a uterine-tubule junction into an oviduct of a female mouse.
14. The method of claim 1 , wherein the genome of the genetically modified mouse is homozygous for the one or more human V H gene segments, one or more human at gene segments, and one or more human J H gene segments operably linked to the mouse heavy chain constant region sequence at the endogenous mouse immunoglobulin heavy chain locus.
15. The method of claim 1 , wherein the genome of the genetically modified mouse is homozygous for the one or more human V L gene segments and one or more human J L gene segments operably linked to the light chain constant region sequence.
16. The method of claim 1 , wherein the genome of the genetically modified mouse comprises a Cμ enhancer.