This is a second submission of a renewal project to study B-cell differentiation in immunoglobulin-transgenic scid mice. In preliminary experiments recently published, the investigator has found that in scid mice transgenic for Ig heavy chain genes, B lineage cells reach the pre-B-cell stage of development and that RAG1/2 levels are downregulated at the pro-B-cell stage. In Ig heavy and light chain gene transgenics, scid B lineage cells can reach the B-cell stage of development and RAG1/2 levels are down-regulated both at the pro-B and the pre-B-cell stages. Data from scid/+ and scid transgenics suggest that the heavy chain gene, alone, can prevent V-DJ joining events (but not DJ joining nor V-Jkappa joining events), while heavy and light chain genes prevent V-DJ joining and recombination at the kappa light chain locus, as well. The goals of the present application are to: 1) determine how an assembled heavy chain gene down-regulates RAG expression (and V-DJ joining) in pro-B-cells. It will be determined whether pseudo-light chain plays a role in this signaling and attempts will be made to clone genes involved in the signaling. 2) Determine why some mu/kappa gene combinations work to promote scid B-cell development while others do not. An hypothesis to be tested is whether this is a function of the mu,kappa combination's being able to arrest DNA recombination events which lead to unresolved breaks in scid cell DNA. 3) Explore the notion that the antigen-specificity of the mu,kappa combination determines whether it will down-regulate DNA recombination (and RAG expression). 4) Determine whether B-cell development is accelerated in Ig transgenic mice. 5) Assess the functional activity of scid B-cells in transgenic animals. Notably, these animals lack T-cells, like RAG-/- mice, but they remain """"""""leaky,"""""""" allowing low-frequency Ig gene rearrangements to take place. Questions will be asked about the nature of Ig-secreting cells that arise in these mice (e.g., express kappa transgene or endogenous kappa genes?) and whether B-cells with anti-self Ig transgenes (dsDNA-specificity) are able to develop in these animals. 6) Differences among mu,kappa transgenic scid mice have already been observed: the B-cells in some are responsive to mitogen while the B-cells in others are not. Attempts will be made to clone the genes that, when differentially expressed, allow for mitogen-responsiveness or mitogen-unresponsiveness, respectively.
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