COMPOSITION AND METHOD FOR DIVERSIFICATION OF TARGET SEQUENCES

摘要

The disclosure relates generally to the targeting of genes to, and their integration into, an innnnunoglobulin (antibody) heavy chain locus. In particular, the methods described herein contemplate replacing the single rearranged heavy chain V, D, and J genes of a B cell lymphoma such as DT40 with independently rearranged VH-D-JH genes of chicken, in a system for generating immunoglobulin diversity. Also contemplated is replacement of the chicken VH-D-JH with rearranged VH-D-JH genes of other vertebrates including human in a system for generating immunoglobulin diversity, with the exception of any substitution disclosed and claimed in PCT application WO 2009/029315 A2. Regions upstream of the rearranged VH gene, and downstream of the rearranged JH genes, are identified and incorporated into the immunoglobulin heavy chain gene-targeting vectors for efficient gene replacement. Also described is construction of a diverse chicken immunoglobulin heavy chain VDJ library in DT40 by homologous gene replacement of the single endogenous rearranged VDJ gene with a chicken VDJ repertoire using the described targeting vectors. The methods are useful for generating a diverse library of immunoglobulins that can be screened for binding to desired target antigens. Compositions include the unique targeting vectors, the diverse chicken heavy chain VDJ library in the targeting vectors, and the diverse chicken VDJ DT40 library that is created by homologous gene replacement of the single endogenous rearranged VDJ gene with a chicken VDJ repertoire, using the targeting vectors.