Sequences associated with the mouse Smu switch region are important for immunoglobulin heavy chain transgene expression in B cell development.

Analyses of H-chain transgenes have indicated that sequences situated between the mu intronic enhancer and the Cmu exons are important for mu gene expression. We have analyzed several variant mu transgenes and find that a sequence element located within or just upstream of Smu is important for mu transgene expression in both immature and mature B cells. This Smu -associated element appears to be required for functional mu expression in small, resting pre-B cells but not in proliferating pre-B cells. Our results also indicate that this element is responsible for previously reported differential transgene expression in resting and activated/proliferating mature B cells. However, our studies of knockout mice show that deletion of the Smu -associated element from the endogenous IgH locus does not alter early B cell maturation. This indicates that other elements within the H-chain locus can replace the function of the Smu -associated element at least to the mature B cell stage. Surprisingly, we also find that Smu deletion in the IgH locus does not affect levels of the sterile germ-line mu transcripts that are involved in B cell class switching, even though S-region sequences have been indicated to be important for the production of analogous germ-line transcripts for other isotypes.

Gene conversion and homologous recombination in murine B cells.

Gene conversion has been found to be important in the diversification of antibody genes in chickens and in rabbits. In other species, however, it is not clear whether gene conversion plays any role in antibody diversity. Analysis of an H-chain antibody gene construct that was designed to optimize the detection of gene conversion events in transgenic mice has shown that sequence transfers that resemble gene conversion events can occur in murine B cells and are associated with somatic hypermutation. This raises the possibility that an error-prone gene conversion mechanism might play a role in murine somatic hypermutation.

Regulatory regions 3' of the immunoglobulin heavy chain intronic enhancer differentially affect expression of a heavy chain transgene in resting and activated B cells.

We have compared the expression patterns of three Ig heavy chain transgenes. The three constructs differ only by deletion of J-C intron sequences located downstream of the Emu enhancer region. When stably transfected into a myeloma cell line, all three constructs are expressed at comparable levels. However, transgenic mice carrying each construct show dramatic differences in transgene expression. Our results indicate that, in addition to the Emu enhancer, at least two regions, RegA and RegS, within the J-C intron influence transgene expression. RegA, located directly downstream of the core Emu enhancer, is involved in up-regulation of transgene expression after LPS activation of splenocytes. RegS, located within or downstream of the Smu switch region, is important for normal levels of transgene expression in splenocytes of heavy chain transgenic mice.