| CPC A01K 67/0278 (2013.01) [A01K 67/0271 (2013.01); A01K 67/0276 (2013.01); C07K 16/00 (2013.01); C12N 9/6489 (2013.01); C12N 15/8509 (2013.01); C12N 15/90 (2013.01); C12Y 301/00 (2013.01); C12Y 304/24046 (2013.01); A01K 2207/05 (2013.01); A01K 2207/12 (2013.01); A01K 2207/15 (2013.01); A01K 2217/00 (2013.01); A01K 2217/072 (2013.01); A01K 2217/075 (2013.01); A01K 2217/15 (2013.01); A01K 2217/206 (2013.01); A01K 2227/105 (2013.01); A01K 2267/01 (2013.01); A01K 2267/02 (2013.01); C07K 2317/21 (2013.01); C07K 2317/24 (2013.01); C07K 2317/33 (2013.01); C12N 2015/8518 (2013.01)] | 20 Claims |
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1. A method of generating antigen-binding proteins against a human foreign antigen of interest, comprising:
(a) making a transgenic mouse with reduced tolerance to the human foreign antigen of interest, comprising:
(i) introducing into a population of mouse one-cell stage embryos or a population of mouse embryonic stem (ES) cells:
(I) a Cas9 protein or a nucleic acid encoding a Cas9 protein;
(II) a first guide RNA or a DNA encoding the first guide RNA, wherein the first guide RNA hybridizes to a first guide RNA recognition sequence within a target genomic locus, wherein the target genomic locus comprises all or part of a gene encoding a self-antigen orthologous to the human foreign antigen of interest;
(III) a second guide RNA or a DNA encoding the second guide RNA, wherein the second guide RNA hybridizes to a second guide RNA recognition sequence within the target genomic locus;
(ii) screening the population of mouse one-cell stage embryos or the population of mouse ES cells for a modified mouse one-cell stage embryo or a modified mouse ES cell, wherein the target genomic locus is modified in a pair of corresponding first and second chromosomes to produce the modified mouse one-cell stage embryo or the modified mouse ES cell with a biallelic modification, wherein the biallelic modification comprises a biallelic deletion of all or part of the gene encoding the self-antigen, wherein expression of the self-antigen is eliminated;
(iii) producing a transgenic F0 generation mouse from the modified mouse one-cell stage embryo or the modified mouse ES cell, wherein the target genomic locus is modified in the pair of corresponding first and second chromosomes in the transgenic F0 generation mouse such that expression of the self-antigen is eliminated;
(b) immunizing the transgenic F0 generation mouse produced in step (a) with the human foreign antigen of interest,
wherein the transgenic F0 generation mouse comprises in its germline:
(i) an ectopic nucleic acid sequence encoding a mouse ADAM6 protein, wherein the mouse ADAM6 protein is functional in a male mouse;
(ii) a heavy chain locus comprising human immunoglobulin heavy chain V, D, and J gene segments, wherein the human immunoglobulin heavy chain V, D, and J gene segments are operably linked to a mouse immunoglobulin heavy chain constant region gene, wherein the mouse immunoglobulin heavy chain constant region gene is at an endogenous mouse immunoglobulin locus; and
(iii) a light chain locus comprising human immunoglobulin light chain V and J gene segments, wherein the human immunoglobulin light chain V and J gene segments are operably linked to a mouse immunoglobulin light chain constant region gene sequence,
wherein (ii) rearranges to form a heavy chain sequence comprising a human heavy chain variable region operably linked to a mouse heavy chain constant region, and (iii) rearranges to form a light chain sequence comprising a human light chain variable region operably linked to a mouse light chain constant region, and
wherein the mouse is incapable of forming an antibody that comprises a human variable region and a human constant region; and
(c) maintaining the transgenic F0 generation mouse under conditions sufficient to initiate an immune response to the human foreign antigen of interest, wherein the transgenic F0 generation mouse produces antigen-binding proteins against the human foreign antigen of interest.
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