Transgenic expression of the human PU.1 BAC. (A) Map of the genomic BAC clone containing base pairs 47 499 973 to 47 321 096 of the human chromosome 11. Shown are the positions and orientations (open arrows) of all genes included on the BAC and positions of all PU.1 exons (gray boxes). (B-C) Comparison of endogenous mouse (top) and transgenic human (bottom) PU.1 transcripts in indicated tissues (B) and hematopoietic cell populations (C) isolated from BAC⁻PU.1^+/+ and BAC⁺PU.1^−/− mice derived from line #55. Shown are quantitative real-time reverse transcription PCR results that represent the mean ± SD of 3 mice per genotype. Data were normalized to the expression of β-actin. BM indicates bone marrow Kid, kidney; Sp, spleen; Thy, thymus. Granulocytes (Gra; Gr1⁺CD11b⁺CD19⁻CD3⁻), macrophages (Mac; CD11b⁺Gr1⁻CD19⁻CD3⁻), T cells (CD3⁺CD19⁻Gr1⁻CD11b⁻), and B cells (CD19⁺CD3⁻Gr1⁻CD11b⁻) were sorted from spleens, LSK cells (Lin⁻Sca-1⁺c-kit⁺), MEPs (megakaryocyte erythrocyte progenitors; lin⁻Sca-1⁻c-kit⁺CD34⁻FcγRII/III⁻), CMPs (common myeloid progenitors; lin⁻Sca-1⁻c-kit⁺CD34⁺FcγRII/III^low), and GMPs (granulocyte monocyte progenitors; lin⁻Sca-1⁻c-kit⁺CD34⁺FcγRII/III⁺) were sorted from bone marrow. (D) Western blot analysis showing transgenic human PU.1 protein expression in extracts of total bone marrow (BM) cells (upper left), Gr1⁺-enriched bone marrow granulocytes (upper right), CD19⁺-enriched splenic B cells (lower left) and BM-derived macrophages (BMMs; lower right) from BAC⁺PU.1^−/− mice of the indicated lines. Total extracts from 10⁶ cells were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, blotted onto a nitrocellulose membrane, and probed with the indicated antibodies.