Methods of making a nucleic acid encoding a human variable region

A method of producing a nucleic acid encoding a human immunoglobulin heavy chain variable region, the method comprising:

• (a) providing a transgenic mouse whose genome comprises in its germline unrearranged human immunoglobulin heavy chain V, D, and J gene segments, wherein the unrearranged human heavy chain V, D, and J gene segments in situ replace mouse endogenous immunoglobulin heavy chain V, D, and J gene segments, and the unrearranged human immunoglobulin heavy chain V, D, and J gene segments are operably linked to an endogenous mouse heavy chain constant region gene, wherein the mouse heavy chain constant region gene is located at an endogenous mouse immunoglobulin heavy chain constant region locus, wherein rearrangement of the human heavy chain V, D, and J gene segments in the mouse results in a rearranged human heavy chain variable region gene linked to the mouse heavy chain constant region gene, wherein the mouse in response to an antigen produces a hybrid antibody that comprises a human heavy chain variable region encoded by the rearranged human heavy chain variable region gene and a mouse heavy chain constant region encoded by the mouse heavy chain constant region gene, wherein the mouse heavy chain constant region gene encodes a Fc region, and wherein the mouse does not produce fully human antibodies;
• (b) stimulating an immune response in the mouse by exposing the mouse to an antigen;
• (c) preparing a hybridoma expressing the hybrid antibody from the spleen of the mouse stimulated by the antigen in step (b);
• and
• (d) isolating DNA encoding the human heavy chain variable region of the hybrid antibody from the hybridoma of step (c).