Abstract
An important characteristic of chickens is that the antibody repertoire is based on a single framework, with diversity found mainly in the CDRs of the light and heavy chain variable regions. Despite this apparent limitation in the antibody repertoire, high-affinity antibodies can be raised to a wide variety of targets, including those that are highly conserved. Transgenic chickens have previously been generated that express a humanized antibody repertoire, with a single framework that incorporates diversity by the process of gene conversion, as in wild-type chickens. Here, we compare the sequences and antibodies that are generated purely by gene conversion/somatic hypermutation of a pre-rearranged heavy chain, with the diversity obtained by V(D)J rearrangement followed by gene conversion and somatic hypermutation. In a gene converting species, CDR-H3 lengths are more variable with V(D)J rearrangement, but similar levels of amino acid diversity are obtainable with gene conversion/somatic hypermutation alone.
Highlights
As in all higher vertebrates, a critical checkpoint in B cell development in chickens is in-frame V(D)J rearrangement, leading to expression of a functional B cell receptor complex at the cell surface [1,2,3]
Imperfect joins and exonucleolytic chewing back in the V-J and V-D-J junctions can generate some diversity, but chicken B cells do not express TdT [9], so there are no N-additions in CDR-H3, and in general, the immunoglobulin diversity produced by the gene rearrangement process is minimal [5, 10, 11]
To produce a diverse repertoire, chickens employ a process of gene conversion in which upstream pseudogenes in the light and heavy chain loci serve as sequence donors to mutate the expressed, functional V [8, 11,12,13]
Summary
As in all higher vertebrates, a critical checkpoint in B cell development in chickens is in-frame V(D)J rearrangement, leading to expression of a functional B cell receptor complex at the cell surface [1,2,3]. To produce a diverse repertoire, chickens employ a process of gene conversion in which upstream pseudogenes in the light and heavy chain loci serve as sequence donors to mutate the expressed, functional V [8, 11,12,13]. These pseudogenes do not contain promoters or recombination signal sequences, so they cannot be expressed themselves, but their sequences are incorporated in segments of varying length into the single functional V. Multiple rounds of overlapping gene conversion from different pseudogenes in the pool lead to a highly diverse naïve repertoire
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