5–7 Co-expression of RAG1 bearing mutations in the DDE motif (one, two or three residues) inhibits wild-type RAG1 activity in a dose-dependent manner in a cell-culture-based plasmid V(D)J recombination assay.8 These data led us to hypothesize that over-expressing a catalytically inactive form of RAG1 in vivo could interfere with the ability of the endogenous RAG proteins to

mediate primary or secondary rearrangements through a dominant negative effect. To test this hypothesis, we generated transgenic mice expressing a full-length form of RAG1 Panobinostat containing a fully alanine-substituted DDE motif using an H-2Kb promoter and an IgH-μ enhancer construct9 to preferentially drive transgene expression in lymphocytes (dnRAG1 mice). Interestingly, we obtained two independently derived founder lines that reproducibly accumulate a clonally diverse, yet repertoire-restricted, B220lo CD19+ B-cell population. These cells display phenotypic and functional properties similar to the splenic B1 B cell, including the expression of CD5. The dnRAG1 mice show no apparent defects in T-cell development or in early B-cell development, but B-cell progression past the transitional T1 stage in the spleen is impaired, which correlates with the selective over-expression

of the dnRAG1 transgene (relative to endogenous RAG1) in the spleen compared with bone marrow or thymus. The dnRAG1 mice exhibit a moderate deficiency in serum IgM and IgG levels, and impaired immune responses to thymus-independent antigens. selleck chemical Notably, when receptor specificity is enforced in dnRAG1 mice by the expression of a functionally rearranged heavy chain transgene reactive to dsDNA that is normally subjected to receptor

editing in the bone marrow, B1-like B-cell accumulation and B-cell progression through the immature and T1 stages of development are substantially impaired, and are associated with expansion of the marginal zone B-cell compartment. Taken together, these data support a model in which peripheral over-expression of catalytically inactive RAG1 impairs receptor editing during the immature/transitional T1 stage, resulting in abnormal progression to a B1-like B-cell. A cDNA encoding untagged, full-length Thalidomide murine RAG1 containing alanine substitutions in all three residues of the DDE motif (dnRAG1) was derived by subcloning DNA fragments from published mutant RAG1 expression constructs generated using recombination PCR mutagenesis10 into the mammalian RAG1 expression construct pcRAG1.11 Diagnostic restriction sites have been engineered into the DNA sequence for each corresponding alanine substitution (D600A, FspI; D708A, AgeI; E962A, NsiI). A BamHI fragment containing the dnRAG1 cDNA sequence was subcloned into the BamHI site of the vector pHSE3’9, placing the transgene under the transcriptional control of an H-2Kb promoter and the Eμ enhancer (see Fig. 1a).