Generation and analysis of transgenetic mice expressing catalytically inactive recombination activating gene-1
The proteins encoded by the recombination activating genes (RAG1 and RAG2) are lymphoid cell-specific endonucleases that initiate V(D)J recombination; the mechanism by which the exons that encode the variable portion of both B and T cell receptors are assembled somatically from several variable (V), joining (J) and sometimes diversity (D) germline gene segments. The enzymatic activity of the RAG proteins has been attributed to three acidic residues (Asp600, Asp708, and Glu962) within RAG1. These residues constitute the DDE motif which functions as the active site for the RAG proteins by coordinating metal ions required for catalysis of DNA cleavage. Mutation of any of these residues abrogates V(D)J recombination without affecting the DNA binding activity of the RAG protein complex. Accordingly, we have speculated that ectopic expression of a catalytically inactive form of RAG1 in vivo may suppress the activity of endogenous RAG1 and V(D)J recombination in a dominant negative pattern. To test this hypothesis we have generated transgenic mice overexpressing a catalytically deficient form of RAG1 in which the three residues comprising the DDE motif have been mutated to alanine. We show here that transgenic mice, but not non- transgenic littermates, accumulate an unusual population of B cells that are characterized by low expression of the pan B cell marker, CD45R (B220), referring to them as B220k> B cells. Extensive phenotypic characterization using How cytometery shows that the B22010 B cells are phenotypically resemble the immature transitional- and Bl- B cell. Total splenocytes and sorted splenic B22010 B cells of transgenic mice showed significant reduction in their proliferative capacity in response to B cell receptor cross-linking. Moreover, serum levels of immunoglobulins (IgM and IgG) are significantly reduced in transgenic mice when compared to non-transgenic littermates. Taken together, these findings are consistent with significant impairment in B cell maturation due to suppression of secondary rearrangement mediated by the RAG proteins to edit receptor specificity in self-reactive B cells. This model may provide an opportunity to dissect the mechanisms of tolerance and autoimmunity in peripheral B cells
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