Rescue of PU.1^−/− hematopoiesis by the PU.1 BAC. (A) Flow cytometric analysis of splenocytes from indicated mice derived from line #55. Numbers indicate the gated cell means ± SDs in percentage, based on 6 mice per genotype. (B) Total numbers of T cells, B cells, macrophages (Mac), and granulocytes (Gra) in spleens of the mice as analyzed in panel A; nd = not defined. (C) Multiple color flow cytometry showing normal frequencies of GMPs, CMPs, and MEPs. Numbers indicate the gated cell means ± SDs in percentage, based on 3 mice per genotype. A URE^−/− mouse is shown for comparison. (D) Myeloid progenitor assay. Total bone marrow cells (1 × 10⁴) from indicated mice were plated in methylcellulose supplemented with M-CSF or granulocyte-macrophage colony-stimulating factor (GM-CSF). Bar graphs show colony numbers scored after 7 days. Cells from URE^−/− mice served as a PU.1 low control. (E) Shape of M-CSF–stimulated colonies after 7 days in methylcellulose are shown as phase-contrast images. Cellular morphology is shown by May-Grünwald-Giemsa stains. (F) Real-time reverse transcription PCR, showing expression of indicated PU.1 target genes in bone marrow cells. mRNA from the bone marrow of URE^−/− mice was used as a PU.1 low control; n.s. indicates not significant.