Knockdown of GATA-2 in wild-type GMPs by lentivirus-mediated RNA interference impairs function in vitro and in vivo. A schematic representation of the lentilox (llx) construct used to knock down GATA-2 is shown (A). Western blot analysis of GFP⁺ BAF-3 cells shows knockdown of GATA-2 (B). Bone marrow–nucleated cells from wild-type animals were sorted for CMP and GMP and transduced with either control vector (llx) or GATA-2–llx. After transduction, CMP and GMP from llx or GATA-2–llx were subjected to a reverse-transcription reaction and Q-PCR to determine the extent of GATA-2 knockdown (C). GFP⁺ cells from llx or GATA-2-llx–transduced CMPs and GMPs were plated in colony-forming medium containing myeloid, megakaryocyte, and erythroid growth factors. Individual colony types were tallied on day 10, and granulocyte-macrophage progenitors are depicted (D) (CMP, n = 4, P = .28; GMP, n = 4, P = .01). To assess in vivo functionality, irradiated B6SJL-CD45.1 mice were transplanted with C57BL/6-CD45.2 GMPs transduced with either llx or GATA-2–llx along with competing C57BL/6-CD45.1 Lin-Sca-1⁺CD117⁺CD34⁻ stem cells. A proportion of transduced cells from each group were cultured for a further 2 days to allow a retrospective analysis of GFP⁺ cells transplanted initially. Eight days after transplantation, the peripheral blood of recipient mice were analyzed for the contribution of donor CD45.2 and GFP positivity within the Gr-1⁺Mac-1⁺ compartment by flow cytometry The cumulative data of multiple recipients (n = 3; P = ND) from 2 separate experiments is shown (E). Error bars indicate SEM. ** indicates statistical significance. Statistical analysis was performed by using the paired Student t test.