SUMMARY B cell development is a tightly regulated process dependent on sequential rearrangements of immunoglobulin loci that encode the antigen receptor. To elucidate the role of microRNAs (miRNAs) in the orchestration of B cell development, we ablated all miRNAs at the earliest stage of B cell development by conditionally targeting the enzymes critical for RNAi in early B cell precursors. Absence of any one of these enzymes led to a block at the pro- to pre-B cell transition due to increased apoptosis and a failure of pre-B cells to proliferate. Expression of a Bcl2 transgene allowed for partial rescue of B cell development, however, the majority of the rescued B cells had low surface immunoglobulin expression with evidence of ongoing light chain editing. Our analysis revealed that miRNAs are critical for the regulation of the PTEN-AKT-FOXO1 pathway that in turn controls Rag expression during B cell development.

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Phenotypic differences among substrains of laboratory mice due to spontaneous mutations or pre-existing genetic variation confound the interpretation of targeted mutagenesis experiments and contribute to challenges with reproducibility across institutions. Notably, C57BL/6 Hsd mice and gene-targeted mice that have been backcrossed to this substrain have been reported to harbor a duplication in exons 28 and 29 of In this study, we demonstrate the presence of this variant in the widely used mice. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is a cytosolic innate immune receptor associated with inflammatory bowel disease susceptibility. Consistent with a role of NOD2 in an immunological disorder, mice bred at our institution displayed multiple B cell defects including deficiencies in recirculating B cells, marginal zone B cells, and B1a cells in vivo, as well as defects in class switch recombination in vitro. However, we found that these effects are due to the variant and are independent of deletion. Despite originating from the same gene-targeted founder mice, mice from another source did not harbor the variant or B cell defects. Finally, we show that mice display the same B cell defects as mice harboring the variant, confirming that the variant is a loss-of-function mutation and is sufficient to explain the alterations to the B cell compartment observed in mice. Our findings highlight the effects of confounding mutations from widely used inbred strains on gene-targeted mice and reveal new functions of DOCK2 in B cells.

microRNAs (miRNAs) are evolutionarily conserved, small non-coding RNAs (ncRNAs) that are crucial in the regulation of a myriad of cellular functions via RNA interference (RNAi). In hematopoietic cell lines, multiple studies describe the impact of miRNAs on lineage determination and maturation. 1,2 Likewise, because miRNAs regulate many important biological pathways in lymphocytes, their deregulation has been implicated in malignant disease and autoimmunity. 3 Transcribed in the nucleus by RNA polymerase II, miRNAs undergo processing by the microprocessor (Drosha and DGCR8) to cleave the double-stranded hairpin primary miRNA into shorter stem-loop precursor miRNAs (pre-miRNAs). In the cytoplasm, Dicer, an RNaseIII enzyme that cleaves double-stranded RNA, further cuts the pre-miRNAs into 20-23 nucleotide duplexes. Mature miRNAs are incorporated into the RNA-induced silencing complex and guide the translational repression or degradation of mRNA target genes via miRNA-dependent recognition of 3′untranslated regions (3′UTRs). 1,2,4,5 1.2 | B cell development B lymphocyte development and maturation is a tightly orchestrated process during which progenitor cells generate unique antigen

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