Quantitative analysis of gene-expression levels surrounding CIS clusters in Eed^hypo/hypo and wild-type tumors. (A) Schematic representation of the retroviral insertional mutagenesis screen. Briefly, DNA was extracted from Eed mutant or wild-type MMLV-induced lymphomas, and inverse PCR was used to amplify genomic DNA flanking the retroviral integrations. Amplicons were purified, sequenced, and BLATed against the mouse genome. RIS indicates retroviral insertion site; and CIS, common insertion site. (B) Schematic representation of the strategy used to assay expression of genes surrounding a CIS. The median position for every CIS that was found in at least 3 independent MMLV-induced lymphomas was first determined. Genes encompassed in a region spanning 50 kb upstream and downstream of the median CIS (100-kb window) were then assayed for their expression levels by quantitative RT-PCR using Taqman assays. A total of 48 different genes, plus 2 other CISs unrelated to this study (Pim2, Pim3) were analyzed. (C) Color gradient representation of the ΔCt values of CIS-associated genes in nontransformed CD4⁺, CD8⁺, CD4⁺CD8⁺ (T DP), or B200⁺IgM⁺ (B) lymphoid cells from healthy mice. Phenotype and genotypes of the analyzed cells are shown on top. Genes analyzed are shown to the right of panel D. * indicates known CISs unrelated to this study. Reactions were done in triplicate, and average values were calculated. Average ΔCt values were determined using the 18S rRNA as the internal endogenous control gene to normalize the levels of target gene expression. Low ΔCt values denote high expression levels. Average Ct values for the 18S rRNA endogenous control was approximately 9. Mutant indicates Eed^hypo/hypo; WT, wild-type. (D) Relative fold differences in gene expression levels of Eed^hypo/hypo mutant and wild-type MMLV-induced lymphomas compared with nontransformed wild-type lymphoid cells. The phenotype, genotype, and ID of the analyzed tumor cells are shown on top. Genes analyzed are shown to the right. Genes are grouped by CIS cluster, indicated by a colored bar beside the gene name. Reactions were done in triplicate, and average values were calculated. Relative fold differences were determined using the ΔΔCt method. The corresponding wild-type nontransformed cells were used as the reference calibrators to assess relative fold differences in expression levels in tumors. Differences of less than −5-fold or more than 5-fold were considered significant. Tumor tissues taken for quantitative RT-PCR contained 75% to 100% lymphoid blasts. T DP indicates CD4⁺CD8⁺; B, B220⁺IgM⁺; Mutant, Eed^hypo/hypo; and WT, wild-type.