No abstract

A method of gene targeting that allows the inducible inactivation of a target gene in mice is presented. The method uses an interferon-responsive promoter to control the expression of Cre recombinase. Here, Cre was used to delete a segment of the DNA polymerase beta gene flanked by IoxP recombinase recognition sites. Deletion was complete in liver and nearly complete in lymphocytes within a few days, whereas partial deletion was obtained in other tissues. This method can be used for the inducible inactivation of any other gene in vivo.

MicroRNAs are small RNA species involved in biological control at multiple levels. Using genetic deletion and transgenic approaches, we show that the evolutionarily conserved microRNA-155 (miR-155) has an important role in the mammalian immune system, specifically in regulating T helper cell differentiation and the germinal center reaction to produce an optimal T cell-dependent antibody response. miR-155 exerts this control, at least in part, by regulating cytokine production. These results also suggest that individual microRNAs can exert critical control over mammalian differentiation processes in vivo.

Of the various classes of antibodies that B lymphocytes can produce, class M (IgM) is the first to be expressed on the membrane of the developing cells. Pre-B cells, the precursors of B-lymphocytes, produce the heavy chain of IgM (mu chain), but not light chains. Recent data suggest that pre-B cells express mu chains on the membrane together with the 'surrogate' light chains lambda 5 and V pre B (refs 2-7). This complex could control pre-B-cell differentiation, in particular the rearrangement of the light-chain genes. We have now assessed the importance of the membrane form of the mu chain in B-cell development by generating mice lacking this chain. We disrupted one of the membrane exons of the gene encoding the mu-chain constant region by gene targeting in mouse embryonic stem cells. From these cells we derived mice heterozygous or homozygous for the mutation. B-cell development in the heterozygous mice seemed to be normal, but in homozygous animals B cells were absent, their development already being arrested at the stage of pre-B-cell maturation.

Each antibody-producing B cell makes antibodies of unique specificity, reflecting a series of ordered gene rearrangements which must be successfully performed if the cell is to survive. A second selection process occurs during immune responses in which a new antibody repertoire is generated through somatic hypermutation. Here only mutants binding antigen with high affinity survive to become memory cells. Cells expressing autoreactive receptors are counter-selected at both stages. This stringent positive and negative selection allows the generation and diversification of cells while rigorously controlling their specificity.

The genomic region encoding the miR-17−92 microRNA (miRNA) cluster is often amplified in lymphoma and other cancers, and miRNAs within this cluster are expressed in high amounts in cancer cells carrying this amplification. Retroviral expression of miR-17−92 accelerates cMyc-induced lymphoma development, but precisely how elevated miR-17−92 expression promotes lymphomagenesis remains unclear. Here we generated mice with elevated miR-17−92 expression in lymphocytes. These mice developed lymphoproliferative disease and autoimmunity, and died prematurely. Lymphocytes from these mice showed increased proliferation and reduced activationinduced cell death. miR-17−92 miRNAs suppressed expression of the tumor suppressor Pten and the pro-apoptotic protein Bim. This mechanism likely contributed to the lymphoproliferative disease and autoimmunity observed in miR-17−92 transgenic mice, and to lymphoma development in patients carrying amplifications of the miR-17−92 coding region.

SummaryImmunoglobulin (Ig)M ϩ IgD ϩ B cells are generally assumed to represent antigen-inexperienced, naive B cells expressing variable (V) region genes without somatic mutations. We report here that human IgM ϩ IgD ϩ peripheral blood (PB) B cells expressing the CD27 cell surface antigen carry mutated V genes, in contrast to CD27-negative IgM ϩ IgD ϩ B cells. IgM ϩ IgD ϩ CD27 ϩ B cells resemble class-switched and IgM-only memory cells in terms of cell phenotype, and comprise ‫ف‬ 15% of PB B lymphocytes in healthy adults. Moreover, a very small population ( Ͻ 1% of PB B cells) of highly mutated IgD-only B cells was detected, which likely represent the PB counterpart of IgD-only tonsillar germinal center and plasma cells. Overall, the B cell pool in the PB of adults consists of ‫ف‬ 40% mutated memory B cells and 60% unmutated, naive IgD ϩ CD27 Ϫ B cells (including CD5 ϩ B cells). In the somatically mutated B cells, V H region genes carry a two-to threefold higher load of somatic mutation than rearranged V genes. This might be due to an intrinsically lower mutation rate in light chain genes compared with heavy chain genes and/or result from light chain gene rearrangements in GC B cells. A common feature of the somatically mutated B cell subsets is the expression of the CD27 cell surface antigen which therefore may represent a general marker for memory B cells in humans.

Dicer is the enzyme that cleaves double-stranded RNA (dsRNA) into 21-25-nt-long species responsible for sequence-specific RNA-induced gene silencing at the transcriptional, post-transcriptional, or translational level. We disrupted the dicer-1 (dcr-1) gene in mouse embryonic stem (ES) cells by conditional gene targeting and generated Dicer-null ES cells. These cells were viable, despite being completely defective in RNA interference (RNAi) and the generation of microRNAs (miRNAs). However, the mutant ES cells displayed severe defects in differentiation both in vitro and in vivo. Epigenetic silencing of centromeric repeat sequences and the expression of homologous small dsRNAs were markedly reduced. Re-expression of Dicer in the knockout cells rescued these phenotypes. Our data suggest that Dicer participates in multiple, fundamental biological processes in a mammalian organism, ranging from stem cell differentiation to the maintenance of centromeric heterochromatin structure and centromeric silencing.[Keywords: RNA interference; microRNA; heterochromatin silencing; DNA methylation] Supplemental material is available at http://www.genesdev.org.