During FY15, we accomplished the following: 1) We followed up live-cell imaging studies using Site 4 mice. (This mouse strain carries 128 copies of Tet operator sequences embedded at the 3 end of the IgH locus). We used fluorescent in situ hybridization (FISH) and Southern blot analyses to ensure that TetO sequencers were not deleted during pro-B cell expansion ex vivo. - We explored the use of spinning disk confocal microscopy at the Johns Hopkins Microscopy facility for live cell imaging. - We tested use of RFP and GFP for these studies. - We prepared TetDBD-Halo fusion proteins to study their efficacy for these studies 2) We generated 30 new retroviral constructs to express guide RNAs (gRNAs) targeted to the VH locus, with the goal of directing Halo-tagged mutated Cas9 to this part of the IgH locus. 3) We completed the first phase of 5C studies of IgH locus conformation. Three new sites, labeled I, II and III, were identified that compact the 2.5 Mb VH locus in pro-B cells. Interaction between sites II and III was found to be Pax5-dependent but E-independent. These observations provide the structural basis for VH locus compaction. A manuscript describing these studies has been submitted for publication.
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