Somatic activation of Nras G12D/G12D but not Nras G12D/+ leads to acute myeloproliferative disease. Five- to 6-week-old mice were injected with pI-pC as described in “Mice.” Two days after the second pI-pC injection, different tissues were isolated and analyzed. Nras G12D/+ and Nras G12D/G12D refer to pI-pC treated compound mice expressing monoallelic and biallelic oncogenic Nras, respectively, as described in “Somatic activation of Nras G12D/G12D but not Nras G12D/+ leads to an acute myeloproliferative disease.” (A) Genotyping analysis of genomic DNA to detect WT allele, LSL allele, and recombined LSL allele (1 LoxP allele). (B) Total RNA was extracted from bone marrow cells. Direct sequencing of RT-PCR amplified Nras gene using a reverse primer to confirm the sequences at the codon 12. Arrows indicate the WT and mutated nucleotides at the codon 12. (C) Flow cytometric analysis of peripheral blood (PB), spleen and bone marrow (BM) cells isolated from control (n = 5) and Nras G12D/G12D (n = 5) mice using myeloid lineage specific markers. Debris and unlysed red blood cells (low forward scatter) and dead cells (propidium iodide positive) were excluded from analysis. Data are presented as averages + SDs. (D) Splenomegaly in Nras G12/G12D mice. Results are presented as the average of spleen weights + SD. *P < .01. (E, F) 5 × 10⁴ bone marrow cells isolated from control, Nras G12D/+, and Nras G12D/G12D mice were plated in duplicate in semisolid medium with or without GM-CSF (E) or IL-3 (F). The data are presented as average percentages (from multiple mice of each group) of maximum number of colonies formed in culture with 0.2 ng/mL of GM-CSF or 10 ng/mL of IL-3. Student t test was performed. Error bars show SD. (E) Crosses indicate P < .01. (F) *P < .05.