Splenic B cells from two Enh(f/f) or Enh(f/f);Mx1-Cre mice exposed 4 wks earlier to pIpC were enriched by negative selection for CD43, followed by culture with IL-4 and either anti-CD40 or LPS. in marrow, SB-224289 hydrochloride but not in other organs that express Enh(f/f);Mx1-Cre mice using SB-224289 hydrochloride pIpC leads to reduced marrow GMP and neutropenia, with a 20% decrease in marrow B220+ B lineage cells (6). Despite little apparent change in total (B220+) B lymphoid numbers with ORF or +37 kb enhancer deletion, several prior observations suggest a role for in B lineage development. Most notably, upon 1:1 competitive transplantation of CD45.2+ enhancer-deleted marrow with CD45.1+ control marrow into lethally irradiated CD45.1+ recipients, CD45.2+ cells not only contribute minimally to blood or marrow neutrophils at 19 weeks, but also manifest 4-fold reduced contribution to B220+ B cells and increased contribution to CD3+ T cells (6). This finding suggests that compensatory homeostatic mechanisms allow ORF- or enhancer-deleted mice to retain near normal B cell numbers. In GMP, the activating H3K4me1 and H3K27Ac histone marks are prominent at the +37 kb enhancer, whereas in Megakaryocyte-Erythroid Progenitors (MEP) these are absent. Notably, these modifications are present at intermediate levels at the locus in the Common Lymphoid Progenitor (CLP), which express mRNA at levels below that of GMP but above that of MEP (5, 6). Deletion of the enhancer in Enh(f/f);Mx1-Cre mice reduces mRNA 8-fold in CLP and leads to 4-fold fewer B cell colony-forming units (B-CFU) when marrow is cultured in methylcellulose with IL-7 (6). Additionally, a human CD4 transgene under control of the enhancer/promoter (Enh/prom-hCD4) is expressed not only in 70% of GMP, but also in 36% of CLP and 40% of B220+CD43+ preproB/proB/early preB cells, with loss of expression at the preB stage (8). Expression of the transgene is undetectable in CD4+ or CD8+ T cells and their DN3 and DN4 precursors and is present in only 1% of MEP. Finally, when Enh/prom-hCD4 marrow is sorted into hCD4- and hCD4+ populations and plated with IL-7, the hCD4+ subset yields B/myeloid colony-forming units, with the CD19+ B cells from these colonies having increased expression of c-kit, a marker of immaturity (8). We have now further investigated the role of C/EBP in B lineage development, finding SB-224289 hydrochloride 2-fold reduced B220+IgM+ B cells in marrow and spleen of Enh(f/f);Mx1-Cre mice exposed to pIpC. These mice have 6-fold expanded CLP, 2-fold fewer preproB cells, and markedly reduced proB, early preB, and preB cells. is expressed at high levels in preproB cells, but is absent at the proB stage. Expression of the Enh/prom-hCD4 transgene allows division of preproB into +37 kb enhancer, and Enh(f/f);Mx1-Cre mice were previously described, as were B6 Enh/Prom-hCD4 mice harboring a transgene Rabbit Polyclonal to Akt1 (phospho-Thr450) in which the +37 kb enhancer and ?725/+125 bp promoter are linked to a human CD4 reporter (6, 8). B6 Mb1-Cre mice (9) were bred with Enh(f/f) mice to obtain Enh(f/f);Mb1-Cre mice. RAG1-GFP B6 mice were kindly provided by K. Medina (10). Pu.1(kd/kd) mice (11), kindly provided by D. Tenen, were bred into the B6 background for >10 generations. 12C20 wk male and female mice were utilized. To induce deletion of the floxed enhancer, 300 g of pIpC (Invivogen) was provided i.p. every other day for three doses, followed by analysis 4 wks later. PCR of tail clip DNA using a primer pair that spans the 5 +37 kb enhancer alleles (6). The Mb1-Cre knockin allele was genotyped using primers 5-CCCTGTGGATGCCACCTC-3 and 5-GTCCTGGCATCTGTCAGAG-3. 5-bromodeoxyuridine (BrdU) was provided at 100 g/g i.p. 3 hrs before marrow harvest. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and the protocol was approved by the Johns Hopkins University Animal Care and Use Committee. Flow cytometry Peripheral blood was obtained by lancing the facial vein and collecting drops of blood into an EDTA microtainer (Pharmingen). Marrow for analysis was obtained by flushing the femurs and tibias with PBS with 3% heat-inactivated FBS (HI-FBS), followed by red blood cell lysis using ammonium chloride and enumeration of total mononuclear cells using a hemocytometer. Marrow for cell sorting was obtained by crushing femurs, tibias, and spine using mortar and pestle, followed by passage through a 40 m cell strainer. Spleen and thymus were SB-224289 hydrochloride passed through a 40 m cell strainer to obtain single cell suspensions. Antibodies used were from Pharmingen unless otherwise indicated. Cell analysis or sorting was done using an LSR II or FACSAria II flow cytometer (BD Biosciences). Mature B cells were enumerated SB-224289 hydrochloride using anti-B220-APC (RA3C6B2, Biolegend), anti-IgM-PE (R6160.2), and anti-IgD-APC (11C26c.2a, Biolegend), neutrophils were.