Restoration of Hoxa-9 expression corrects the in vitro proliferative defects and in vivo repopulating ability of Hoxa-9^-/- marrow cells. Marrow cells from 5-fluorouracil-treated Hoxa-9^-/- mice were transduced either with a retroviral vector expressing Hoxa-9^-/- together with GFP (HOXA9 vector) or a control vector expressing GFP alone (MIG). The transduction efficiency of the HOXA9 vector was 53% and the MIG vector 71%, as measured by the percentage of GFP-positive cells by FACS 3 days after transduction. Unsorted cells (ie, mixtures of transduced and untransduced cells) were placed in liquid cultures, as described in panel A, or transplanted into cohorts of lethally irradiated CD45.1⁺ recipients (3 animals each) together with equal numbers (500 000 cells) of unfractionated CD45.1⁺ competitor cells. (A) Cell-growth curves of cultured Hoxa-9^-/- marrow cells transduced with either the Hoxa-9 or MIG vector, compared with untransduced control wild-type and Hoxa-9^-/- cells. (B) The number of myeloid progenitors on day 8 of liquid culture as assayed in clonogenic assays in semisolid medium. An aliquot of cells was removed from the cultures on day 8 and plated directly into methylcellulose cultures and scored for colony formation. (C) FACS analysis of peripheral blood of mice that received transplants 4 weeks previously with either Hoxa-9-transduced (top panels) or MIG-transduced (bottom panels) Hoxa-9^-/- marrow cells, gated for the granulocyte (Gran, left panels) and lymphocyte (Lymph, right panels) populations. In each panel, the transduced Hoxa-9^-/- cells (CD45.2⁺GFP⁺) are represented in the top-right quadrant; untransduced Hoxa-9^-/- cells (CD45.2⁺GFP^-), in the top-left quadrant; and wild-type competitor cells (CD45.2^-GFP^-), in the bottom-left quadrant.