During the 2009 fiscal year we accomplished the following: 1. Continued studies of sterile transcription and epigenetic state of unrearranged VH gene segments in pro-B cells from RAG2-deficient bone marrow. We mapped the position and distribution of L1 retrotransposous in the VH locus, and found one striking cluster of 4 L1 elements that separates the 5-prime most 5% VH gene segments from the rest of the VH genes. We propose that this cluster of L1s may serve as a boundary element to delineate independently regulated VH domains. 2. Designed additional primer pairs to score for transcription and chromatin modifications in the sub-telomeric region of mouse chromosome 12. Standardization of these newly-designed primers is ongoing. 3. Constructed a targeting vector to replace endogenous E with multimerized Gal4 and Tet-operator sequences. Used BAC recombineering technology to construct targeting vectors to introduce Gal4, Tet0, and Lac0 sequences at different locations of the VH locus. These vectors will be used to alter the IgH locus in order to introduce a tag that can be used to visualize chromosome movements during antigen receptor gene rearrangements.
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