siRNA Knock down of MPL Receptor in Mouse Hematopoietic Progenitors.

We recently showed that the expression of the thrombopoietin (TPO) receptor, MPL, could have different impacts on the generation and the expansion of hematopoietic stem cells (HSC) during establishment of definitive hematopoiesis in the mouse embryo. In order to investigate further MPL/TPO signaling pathways involved in the emergence or in the amplification and maturation of HSCs as well as their downstream targets, we set up RNA interference to knock down Mpl expression in hematopoietic progenitors and HSCs. We used the retrovirus pSUPERretro for stable and efficient delivery of siRNAs in primary cells. Three siRNAs were first tested in a model of rat fibroblast overexpressing Mpl (FREmpl) which get transformed in the presence of TPO. This transformation requires pathways involved in TPO-induced proliferation and differentiation of hematopoietic cell lines (

We next introduced these siRNAs in bone marrow cells from 5-FU treated adult C57Bl6 mice, and showed that they were also able to inactivate Mpl expression in primary hematopoietic progenitors after culture of the cells for 7 days in the presence of SCF, Flt3 ligand, IL-3, IL-6 and TPO (1,1% of MPL⁺ cells instead of 23% in the control population). Further molecular analysis as well as in vivo an in vitro study of these cells is under current investigation and will be presented.