During FY13, we accomplished the following: 1) Used bacterial artificial chromosome (BAC) probes to validate 5C analyses of the IgH locus in pro-B cells. We identified three major interaction sites separated by approximately 1Mb that divided the VH locus into two prominent topological domains. These domains were selectively present in pro-B cells compared to non-B lineage bone marrow cells. To determine the molecular basis of this chromatin configuration, we carried out FISH studies in E- and Pax5-deficient pro-B cells expanded in culture. E-deficiency did not affect these domains, whereas the distal 1Mb domain was disrupted in Pax5-deficent pro-B cells. By incorporating these observations into our existing model, we arrived at a more refined 3D-structure of the pre-rearrangement IgH locus. 2) Two strains of newly-generated mice with modified IgH locus were bred for experimentation. Delta Emu strain replaces the intron enhancer E with short arrays of TetO and Gal4 binding sites. Site 4 strain incorporates tandem arrays of TetO sequences into a region 3 of the IgH locus. Both strains were first bred to -actin-cre mice to delete Neo gene used for targeting. Neo-deleted mmice were further bred to RAG2-deficient mice to generate Delta Emu/RAG2- and Site 4/RAG2- strains. Two independently derived lines were obtained for each genotype. Just prior to experimentation, our entire Delta Emu/RAG2- colony (both lines) was exterminated. This unfortunate incidence has put this research goal back by approproximately two years.
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