Rescue experiment of PU.1^−/− hematopoiesis by URE-driven PU.1 cDNA expression. (A) Schematic maps of the URE-based transgenic rescue constructs. A 3.4-kb fragment that included the complete URE was fused to a 2.1-kb proximal PU.1 promoter fragment and a truncated rabbit Hbb gene, providing splicing acceptor/donor sites and a polyA signal as described in “Methods.” This plasmid was equipped with the full-length murine PU.1 cDNA that was either fused to a 5′ Flag-tag or a 3′ iresEGFP reporter cassette. (B) Flow cytometry of day 18.5 prenatal fetal liver cell suspensions from the indicated genotypes derived from a PU.1iresEGFP transgenic line. CD11b was used as a pan-myeloid marker, Gr1 was used to detect granulocytes, and F4/80 was used to detect monocytes. Numbers in the upper right quadrates indicate percentages of GFP⁺ cells carrying the respective myeloid antigenes. The plots are representative of 7 mice each. (C) Real-time RT-PCR with the use of a mouse PU.1-specific primer/probe set and mRNA from c-kit⁺ fetal liver cells of the indicated mice (n = 3 each) derived from a PU.1iresEGFP transgenic line sorted day 18.5 as template. Data were normalized to the expression of β-actin. Dashed line indicates the qPCR background level detectable in URE-TG⁻PU.1^−/− fetal liver cells.