The product of V-D-J joining and N addition, H chain complementarity determining region 3 (HCDR3) contains the majority of the diversity of the pre-immune antibody repertoire. The D gene segments at the center of HCDR3 can be read in each of six different reading frames. Although DH sequences 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, the latter often encoding pathogenic antibodies, e.g. anti-DNA antibodies. In order to test the hypothesis that non-neutral HCDR3s are inherently deleterious, we are generating Cre-loxP gene targeted mice in which we have forced the use of alternative reading frames. In the first experiment, an inverted DSP2.2 gene segment has replaced the center of JH distal DFL16.1, forcing use of a charged reading frame. Preliminary data indicate that heterozygous mutant mice exhibit a 25% reduction in mature B cells expressing receptors derived from the targeted locus and a loss of the peritoneal Mac1+CD5- B cell population. The targeted chromosome contains an additional loxP site immediately adjacent to JH. In these mice, Cre-mediated recombination will delete the remaining DH gene segments forcing use of only the mutated DH.