HOXB4-mediated leukemia in transplanted mice due to vector insertion site-mediated oncogene activation. (A) Immunophenotyping of BM cells from recipients displaying leukemia cases L1 and L2. The y-axis shows the indicated cell surface markers identified by antibody staining, and the x-axis shows GFP expression associated with the HOXB4 vector. Expression of monomyeloid cell surface markers (Mac-1, CD16/32, Gr-1) and also erythroid (Ter119) and B-lymphoid (B220) markers are shown. A smaller percentage of the leukemic cells also expressed the stem cell markers c-kit and Sca1. (B) Retroviral insertion sites in L2 were identified by splinkerette PCR. The location of the 2 insertion sites in the adjacent to Notch1 and within Prdm16 and shown schematically along with the orientation of the vector at the insertion site. (C) Northern blot analysis of relevant oncogenic mRNAs from control and HOXB4 leukemic cells from both the L1 and L2 cases. The control cells consisted of BM cells of mice transplanted with normal BM cells transduced with the “empty” MSCV/GFP vector (lane labeled GFP). There is a markedly increased level of N-Ras mRNA (15- to 20-fold) and a mild increase (∼twofold) of Lmo2 mRNA in L1 BM cells when compared with control BM cells. There is a markedly increased level of Prdm16 mRNA and a mild increase (∼twofold) of Notch1 mRNA in L2 BM cells compared with control BM cells. Actin serves as an internal loading control.