PMC

Aberrant neutrophilic maturation in PEBP2βMYH11 transgenic mice. (A and E) Samples from control mice. (B and F) Samples from PEBP2βMYH11 transgenic mice (line Tg237). (C and G) Samples from NRAS^G12D transgenic mice (line Tg7). (D and H) Samples from PEBP2βMYH11/NRAS^G12D double transgenic mice. (A–D) Peripheral blood neutrophils. (E–H) Bone marrow. (Original magnification, ×250.)

Morphology of bone marrow cells cultured in vitro. Bone marrow cells were grown in semisolid methylcellulose medium in the presence of G-CSF at 2.5 × 10⁵ cells/ml for 7 days, and cytospins of harvested cells were prepared. Numerous macrophages (arrow) and neutrophilic cells (arrowhead) were observed in the control samples. Neutrophilic cells were decreased in the samples from transgenic mice. (Insets) Neutrophil morphology. Neutrophils derived in vitro from the bone marrows of transgenic mice exhibited atypical morphology. (Original magnification, ×250.)

(A) Bone marrow differential cell counts. Differential cell counts (total of 400 cells each) were performed on bone marrow smears. Data are shown as arithmetic means ± SD. Pro, promyelocytes; Immature Neut, neutrophilic myelocytes and metamyelocytes; Mature Neut, neutrophilic band, mature ring, and polymorphonuclear forms; Eryth, nucleated erythroid cells; Lymph, lymphocytes; Eosin, eosinophilic cells. Control, n = 6; Tg259, n = 6; Tg237, n = 6. (Inset) Ratios of mature to immature neutrophilic cells. ∗ indicates a significant shift toward immature cells is present in the bone marrow of the transgenic mice, P = 0.004. (B) Density of bone marrow neutrophilic cells. Bone marrow cells were separated into low and high density fractions on a Percoll step gradient. Total numbers of cells present in each fraction were determined. Differential cell counts (total of 100 cells each) were performed on cytospins of each fraction, with cells morphologically identified as neutrophilic (neutrophilic myelocytes, metamyelocytes, ring, band, and polymorphonuclear forms) or nonneutrophilic. Total cell counts and differential counts were used to calculate the mean percentages of neutrophilic cells present in the high density fractions. Data are shown as arithmetic means ± SD. Control, n = 7; Tg259, n = 5; Tg237, n = 6. †, Control vs. Tg259, P = 0.0006. ‡, Control vs. Tg237, P = 0.00002; Tg259 vs. Tg237, P = 0.002.

(A) Map of the transgene. The PEBP2βMYH11 cDNA inserted into the BglII site of the human MRP8 promoter cassette corresponded to the most common inv(16) breakpoint. The nucleotide locations of the breakpoint within the coding sequences of PEBP2β and MYH11 are shown. White boxes represent noncoding portions of MRP8 exons. Solid lines represent 5′ and 3′ flanking sequences and intron 1 of MRP8. (B) Expression of the transgene. Whole-cell lysates were subjected to denaturing PAGE and Western blotting using a rabbit polyclonal anti-Pebp2β antiserum. The signal at 70 kDa corresponds to the Pebp2βSmmhc protein. The first two lanes are lysates of NIH 3T3 cells that contained retroviral vectors expressing either a lacZ or a PEBP2βMYH11 cDNA. The remaining lanes are lysates of tissues from control FVB/N mice or transgenic mice of lines Tg259 or Tg237. (C–F) Immunofluorescence of bone marrow neutrophilic cells. (C and D) Control. (E and F) Tg237. (C and E) Anti-Pebp2β antiserum. (D and F) Hoechst 33258. (Original magnification, ×250.)