Abstract
Rabbits have been used extensively as a model system for the elucidation of the mechanism of immunoglobulin diversification and for the production of antibodies. We employed Next Generation Sequencing to analyze Ig germline V and J gene usage, CDR3 length and amino acid composition, and gene conversion frequencies within the functional (transcribed) IgG repertoire of the New Zealand white rabbit (Oryctolagus cuniculus). Several previously unannotated rabbit heavy chain variable (VH) and light chain variable (VL) germline elements were deduced bioinformatically using multidimensional scaling and k-means clustering methods. We estimated the gene conversion frequency in the rabbit at 23% of IgG sequences with a mean gene conversion tract length of 59±36 bp. Sequencing and gene conversion analysis of the chicken, human, and mouse repertoires revealed that gene conversion occurs much more extensively in the chicken (frequency 70%, tract length 79±57 bp), was observed to a small, yet statistically significant extent in humans, but was virtually absent in mice.
Highlights
B cell development and repertoire diversification vary significantly among vertebrate species [1]
Total RNA was isolated from bone marrow (BM) plasma cells (PCs) and total PBMCs of three adult New Zealand white (NZW) rabbits
IgG heavy chain and Igk/Igl light chain cDNAs were amplified by 59 RACE using primers that annealed respectively to the CH1 or CK/Cl constant region directly 39 of the J segment (Table S1), and the resulting amplicons were sequenced by Roche 454 sequencing. 172,126 high quality reads corresponding to 88,830 unique heavy chain sequences across the three rabbits were obtained (Table 1)
Summary
B cell development and repertoire diversification vary significantly among vertebrate species [1]. Compared to humans and mice, which use a diverse assortment of germline VH gene segments during VDJ recombination of the heavy chain, the rabbit IgH repertoire displays highly restricted VH gene segment usage. The large number of previously unannotated VH-like sequences identified within the a1/a2 Thorbecke rabbit, as well as previously identified sequences from latent heavy chain allotypes [2,8], clearly demonstrate the complexity of the germline Ig repertoire. Because the sequenced Thorbecke rabbit was heterozygous at the IgH locus (a1/a2 based on mapping of the VH1 gene), the actual number of distinct VH gene elements in the haploid genome is unclear
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