+37-kb deleted mice (+42Mkb) show low Cebpa levels and develop neutropenia. (A) ChIP-seq H3K27ac in murine total bone marrow (ENCODE) shows multiple regions of open chromatin. A region located at +37 kb in mice is highly homologous (supplemental Figure 5A) to the human +42-kb enhancer and is H3K27ac marked. (B) Table showing Mendelian ratios. (C) PCR genotyping using primers flanking the gRNAs generate an amplicon of 1.65 kb on the intact/WT allele and an amplicon of 550 bp on the deleted/rearranged allele. (D-E) Flow cytometric analysis to distinguish neutrophils in peripheral blood or bone marrow of WT (blue), heterozygous (green), and homozygous (red) mice using the myeloid differentiation markers Mac1 and GR1. (F) Neutrophil absolute counts in peripheral blood and bone marrows of WT and homozygous mice. (G) Cebpa mRNA expression from total bone marrow obtained from WT (n = 6) or homozygous +42Mkb knockout mice (n = 6) is presented as fold change. (H) Cebpa mRNA expression from liver, lung, and spleen does not show significant changes. ***P < .0001; **P < .001; N.S., not significant.
