E) kit (Roche Diagnostics K.K., Tokyo, Japan) as outlined by the manufacturer’s instructions. Relative quantity of cell development was calculated as a ratio of number of cells treated with mitogens to number of untreated cells, and is shown as mean 6 SD of six wells for cells isolated from 5 mice. In vitro colony-forming assays had been performed in duplicate by plating 500 bone marrow cells with 1 ml of MethoCultTM M3434 medium (StemCell Technologies) in 35 mm Petri dishes. Colonies were counted soon after four days by May-Grunwald Giemsa staining. Supplies and Procedures Animal modelMice were kept beneath pathogen-free conditions in animal facilities at Saga University and Hiroshima University in line with institutional recommendations. The protocol was approved by the Committee around the Ethics of Animal Experiments with the Saga University (Permit Quantity:15-005-1) and Hiroshima University (Permit Quantity: 2225). All surgery was performed under sodium pentobarbital anesthesia, and all efforts had been created to reduce suffering. The transgenic mouse carries a chimeric gene like a fragment in the promoter region in the CMV gene and all coding sequences of human HIF1A gene fused using the FLAG gene at the 59 end in the HIF1A gene (HIF1A TG mice; Fig. 1A). Six strains of transgenic mice have been obtained and backcrossed onto the BALB/c genetic background mice for far more than ten generations. To ascertain the presence of the transgene in the backcrossed mice, we performed PCR evaluation of genomic DNA obtained from tails in the age of 4 weeks working with a primer set distinct towards the FLAG tag: 59-ATG GAC TAC AAA GAC GAT GAC GAC AAG-39 (FLAG5), and an additional set precise to the human HIF1A gene: 59-ATT CTG AGA AAA AAG CTT CGC TGT GTG-39 (HINDHIF3) (Fig. 1A). Transgene copy quantity was estimated by real-time PCR, following which similarities of HIF1A mRNA expression level and phenotype have been confirmed making use of one particular line of transgenic founder mice.Fluorescence-activated cell sorter (FACS) analysisThymocytes, spleen cells, and lymphocytes from Peyer’s patches obtained from 1- to 12-month-old mice were counted and stained with fluorochrome-conjugated monoclonal antibodies making use of standard procedures. Acquisition was performed having a FACSCaliburH (BD Biosciences) and final results had been analyzed by FlowJo softwear (Tomy Digital Biology Co., Ltd, Tokyo, Japan). Antibodies have been anti-mouse against CD3e, CD4, CD8, CD19, B220, CD25, CD44, IgM (eBiosciences).Blood countPeripheral blood was collected and analyzed on an automated blood cell counter, KX-21 (Sysmex), as outlined by the manufacturer’s instructions.Lacutamab PLOS One particular | www.Bepridil hydrochloride plosone.PMID:24605203 orgDevelopment of Lymphoma by HIF-1alphaFigure 1. The construct of transgene and expression of human HIF-1alpha inside the transgenic mouse tissues. (A) The construct of chimeric gene including human HIF1A cDNA fused together with the FLAG gene in the 59 end in the HIF1A gene. (B) Activation of human HIF-1alpha protein in murine cells was confirmed by luciferase activity in BALB/3T3 cells stably transfected together with the transgenic construct. (C) Ectopic expression of human HIF1A mRNA was determined by real-time RT-PCR as described in Materials and Solutions. (D) Sequential analyses of expression levels by real-time RTPCR in several tissues of transgenic mice. Relative expression of human HIF1A mRNA was assessed by the amount of Actb mRNA. (E) Expression of HIF1alpha protein in several organs was determined by western blotting with use of both anti-HIF-1alpha and anti-FLAG antibodies. doi:10.1371/journal.p.
