Gene-targeting studies of D_H reading frame in V(D)J recombination

The third Complementarity Determining Region of the H chain HCDR3, which is created by direct V(D)J joining and N region addition, contains the majority of the diversity of the preimmune B cell repertoire and plays a major role in defining antigen specificity. Within HCDR3, D gene segments can encode six different peptide sequences in each of six different reading frames. Although the D sequences may vary among species, the hydropathicity signature of the different reading frames is preserved across evolution. Mammals preferentially use neutral reading frames and rarely use hydrophobic or charged reading frames. In order to test the hypothesis that highly charged HCDR3 domains are inherently deleterious, and thus selected against by evolution, we have generated mice in which the D_H locus has been forced to a charged reading frame. An inverted, frame-shifted, and thus charged, DSP2.2 gene segment was embedded within the 5′-most D_H gene segment, DFL16.1. This mutated gene, iDFL16.1, was targeted into the D_H locus of a BALB/c ES cell line wherein the 3′-most DH, DQ52, had been replaced by a loxP site. Cre-mediated recombination led to the deletion of the intervening DH gene segments, forcing use of only the mutated iDFL16.1 gene segment (ΔiDFL16.1). Preliminary data indicate that IgH allotype heterozygous ΔiDFL16.1+/- mice exhibit a 70% (bone marrow), 80% (spleen), to 90% (peritoneum) reduction in B cells that express receptors derived from the targeted locus.