Rescue of erythroid progenitor defects by PU.1 reexpression. (A) Diagrams of the retroviral constructs used to transduce erythroid progenitor cells. Mig-NGFR is an MSCV-based retrovirus with an internal ribosomal entry site (IRES) and NGFR sequence insert.24 Mig-PU.1 encodes the entire PU.1 protein. (B) Expansion of transduced erythroid progenitors. The 12.5- or 13.5-dpc FL cells (2 × 106 to 4 × 106) were plated in erythroid medium for 2 days, transduced or not with retrovirus (day 0), and cell numbers counted each following day. The initial burst of proliferation in PU.1G/G FL cultures (Figure 2A) occurred in the first 2 days before transduction and thus is not represented in this graph. (C) PU.1 blocks erythroid differentiation. Two days after transduction, NGFR-negative and NGFR-positive cells from WT and PU.1G/G cultures were analyzed for their expression of TER119 and CD71. The gray histogram indicates background staining of noninfected cells. Numbers denote the percentage of cells in each quadrant. Similar results were obtained 5 days after transduction. (D) PU.1hi-expressing cells rapidly die in culture. NGFR expression was monitored over time in erythroid cultures. The gray histograms represent background staining of noninfected cells. The NGFR mean fluorescence intensity (MFI = x) for each gated population is indicated in the histograms. The MFI of noninfected cells is 0.3 for WT and 1.3 for G/G. These results represent 1 of 3 experiments. ND indicates not done.