Progress during FY18: 1) Continued breeding mouse strains that carry point mutations in E. Current experimental standards suggest 5-6 backcrosses of mouse strains generated by CRISPR/Cas9 technology to minimize chances of confounding interpretations due to off-target mutations. We generated 4 such strains of mice, with 2-4 independent lines of each strain. Backcrossing to WT C57BL6 mice is almost complete for 2 strains. Preliminary developmental phenotyping shows a partial block at the pro- to pre-B transition in mice that carry E2, E5-double-mutated alleles. 2) Our working model is that inappropriate E activation directs the IgH rearrangement phenotype in DP thymocytes. To identify which enhancer motifs directed E activity in thymocytes we assayed DH recombination in D cells from E-mutated mice. We noted that the A/B-mutated enhancer conferred higher levels of recombination, suggesting protein binding to these sites may attenuate E activity in DP cells. 3) Two of the 4 enhancer-mutated strains were bred to a RAG2-deficient background to hold the IgH locus in unrearranged configuration. Bone marrow pro-B cells will be used to map the transcriptional and epigenetic state of the locus to identify the roles of individual DNA binding proteins in establishing the pre-rearrangement structure of the IgH locus. 4) continued collaboration with Stephen Desiderio's lab to study the function of the autoinhibitory domain of RAG2. these studies were published in Molecular and Cellular Biology (2018).
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