Abstract
The ecotropic viral integration site-1 (Evi-1) was first identified as a gene located at the integration site of an ecotropic retrovirus leading to murine myeloid leukemia. Since its identification, Evi-1 has been recognized as one of the dominant oncogenes associated with human myeloid leukemia and myelodysplastic syndrome. Evi-1 is a member of the SET/PR domain family of transcription factors and it contains two separated zinc-finger DNA-binding domains, which recognize cognate DNA sequences respectively. Recently, it was shown that Evi-1 is predominantly expressed in hematopoietic stem cells (HSCs), and HSC activity is significantly impaired in Evi-1-deficient embryos, which succumb to the multiple organ defects before birth. In order to investigate a role of Evi-1 in adult hematopoiesis, we generated the mutant mice harboring Cre recombinase recognition sites (loxP sites) flanking exon 4 of the Evi-1 gene (Evi1flox/flox), together with the mice in which the same region was completely deleted (Evi-1−/−). Evi-1−/− mice died around embryonic day 13–16. In Evi-1−/− embryos, Lineage−Sca1+cKit+ cells (LSK cells) and colony forming cells were severely reduced. Furthermore, Evi-1−/− fetal liver cells could not reconstitute hematopoiesis of sublethally irradiated mice in the transplantation experiments, indicating an apparent defect in hematopietic stem/progenitor cells. In contrast, more committed cells was unaffected in Evi-1−/− fetal liver. Then, we ablated Evi-1 by breeding Evi-1flox/flox mice with mice expressing Cre-recombinase under the control of the Tie2 promoter and enhancer, a marker of endothelial and hematopoietic stem cells. These mice virtually phenocopied Evi-1−/− mice, demonstrating that Evi-1 function is distinctly required in Tie2+ cells to establish HSCs. Next, we assessed the requirement of Evi-1 in adult hematopoiesis using an interferon (IFN) -inducible Mx-Cre gene-targeting method, in which floxed alleles can be effectively deleted in hematopoietic cells upon injection of IFN-inducer pIpC. Immediately upon injection of pIpC, platelet counts modestly declined in Evi-1flox/flox/Mx-Cre mice, while white blood cell counts or hemoglobin levels were not affected. Platelet recovery after 5FU-treatment is also delayed in Evi-1-deficient mice compared to the control mice. Furthermore, two weeks after pIpC infection, LSK cells in Evi-1-deficient mice decreased to about half the number of those in the control mice. By 10 weeks, however, LSK cells in these mice began to recover. At this time point, a majority of hematopoietic cells in the bone marrow retained the intact Evi-1 alleles, suggesting that HSCs can not maintain hematopoiesis in the absence of Evi-1 and are outcompeted by a fraction of HSCs that escaped Cre-mediated Evi-1 exicision. Together, these results show that Evi-1 plays an indispensable role not only in the establishment of HSCs during embryogenesis but also in the maintenance of HSCs in adults.
Long-Term Vector Integration Site Analysis Following Retroviral Mediated Gene Transfer to Hematopoietic Stem Cells for the Treatment of HIV Infection
