Pen enhances erythropoiesis in EMX C.1. (A) A Wright-Giemsa–stained cytospin preparation of EMX C.1. The asterisks denote megakaryocytes. Arrows point to 2 basophilic erythroblasts. Also visible are neutrophils with donut-shaped nuclei, monocytes, and mast cells. The genotyping result is at the top. (B) Northern analyses of the expression of Pen, GATA-1, β^major-globin, and β-actin in EMX C.1, EMX/EGFP, and EMX/Pen without and with EPO stimulation. FLDS-19 serves as a positive control and MPRO as a negative control for GATA-1 and β^major-globin. The same blot was sequentially hybridized to different probes. EMX C. 1 (lane 3) and EMX/EGFP (lane 4) did not express the 4.0 kb retroviral message harboring the mPen coding sequence (labeled “Transgene”) while EMX/pen did (lane 5, top panel). The 2.1 kb endogenous mPen message in FLDS-19 (lane 1) is indicated. ΔPen is a shortened Pen message transcribed from the KO allele. The level of β^major-globin message roughly correlated with the numbers of erythroblasts. (C) Relative EPO-R expression in EMX/EGFP versus EMX/pen without exogenous EPO. Quantitative real-time RT-PCR was performed in triplicate as described in “Materials and methods,” and the data were normalized according to the levels of β-actin expression. The level of EPO-R expression in EMX/EGFP was taken as 1.0. Data represent means ± SD. (D) Relative β^major-globin expression in EMX/EGFP versus EMX/pen without exogenous EPO. The data were normalized according to the levels of β-actin expression. The level of β^major-globin expression in EMX/EGFP was taken as 1.0. The expression of β^major-globin was likely stimulated by the small amount of EPO present in FBS. Means ± SD. (E) Relative β^major-globin expression in EMX/EGFP versus EMX/pen stimulated with EPO (4 U/mL) for 0, 6, and 10 days. The data were normalized according to the levels of β-actin expression. The level of β^major-globin expression in EMX/EGFP on day 0 was taken as 1.0. Data represent means ± SD. (F) A representative field of Wright-Giemsa–stained cytospin preparation of EMX/EGFP stimulated with EPO (4 U/mL) for 8 days. Arrowheads indicate 2 large erythroid progenitors. The rest are myeloid cells. Bar = 40 μM. (G) A representative field of Wright-Giemsa–stained cytospin preparation of EMX/pen stimulated with EPO (4 U/mL) for 8 days. Arrowheads indicate a large cluster of basophilic erythroblasts; arrow, an enucleated RBC.