Thus the blockade in differentiation of maturing T and B

cells in the Snai3-expressing HSC occurs between the c-Kit+Sca− stage and the more mature common lymphoid progenitor population. The data presented in this report indicate that the expression of Snai3 in bone GDC-0973 nmr marrow progenitors alters neither the maintenance of the stem cells nor the early stages of stem-cell differentiation but does dramatically skew the production of cells committed to the lymphoid or myeloid lineages. The Snai3 protein could alter these maturation profiles either through the repressor function of the SNAG domain of the protein, or by competing with endogenous transcriptional regulators for binding to E box sites. The identification of genes whose expression is influenced by the presence of Snai3 in these precursor populations may provide key insight into the regulation of differentiation of myeloid- and lymphoid-precursor cells. Animals were housed in the Animal Resource Center (University of Utah Health Science Center, Salt Lake City, UT) according to the guidelines of the National Institutes PS-341 chemical structure of Health. C57BL/6 and B6.SJL-Ptprc Pepc/BoyJ were obtained from The Jackson Laboratories. C57BL/6CrSlc-Tg(ACTb-EGFP)OsbC14-Y01-FM131 mice ubiquitously expressing GFP were utilized [[27]]. The pBMN-1-GFP retrovirus was obtained from Addgene (plasmid 1736). The coding sequence of Snai3

(base pair (bp) 79–942 of NM_013914.2) was cloned into the Bam HI and XhoI sites of the vector. The Snai3 encoding cDNA was obtained by RT-PCR amplification of mouse thymus cDNA using 5′-CGGATCCATGCCGCGCTCCTTCCTGGTGA and 5′-GCTCGAGCTAGGGGCCAGGACAGCAGC oligonucleotides. PCR amplification was performed using Platinum pfx (Invitrogen, Grand Island, NY, USA). PCR amplification was 55°C annealing

http://www.selleck.co.jp/products/BafilomycinA1.html (30 s), 68°C extension (2 min) and 95°C denaturing (30 s) for 40 cycles. After subcloning the sequence was confirmed to match that of the reference sequence of NM_013914.2. Plat E cells were grown in stem cell media (SCM): Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 15% FCS, P/S, 1 μg/mL puromycin (Sigma, St. Louis, MO, USA), and 10 μg/mL blasticidin (Sigma) except during virus production when antibiotics were subtracted [[28]]. Retroviral vectors were transfected into the Plat E packaging cell line using Fugene HD reagent (Roche, Pleasanton, CA, USA) at a 6:1 ratio. Cells were incubated at 37°C for 24 h then switched to 32°C for virus production in fresh media. Supernatant was collected and filtered through a 0.45 μm filter prior to use in transduction. B6.SJL were injected with 300 μL 10 mg/mL 5′fluorouracil (Sigma) in PBS [[29]]. Four days later their BM was collected and cultured with RBC in SCM with 100 ng/mL SCF (Sigma), 20 ng/mL IL-6 (Sigma), and 10 ng/mL IL-3 (Sigma) at 5–6 × 106 cell/mL for 2 days at 37°C. Stem cell cultures were collected and red blood cell (RBC) lysed with ammonium chloride potassium (ACK). Remaining cells were resuspended in 7.