Rac2 is important during development of MA9 leukemia. (A) Survival curve comparing mice transplanted with Cre^Tg+/WT (WT/WT), Cre^Tg+/ Rac1^flox/flox (FL/WT), and Cre^Tg+ /Rac2^−/− (WT/KO) LDBM cells expressing REW-EGFP-MA9 via retroviral transduction. Rac1 floxed genomic sequences were deleted by treating mice with PIPC (KO/WT). WT/WT and KO/WT mice uniformly developed leukemia and died with median latencies of 3 to 5 months. Recipients of WT/KO LDBM cells expressing MA9 survived significantly longer (P < .001), in some cases > 350 days after transplantation. (B) Percentage of EGFP⁺ cells in the PB was monitored monthly by FACS analysis beginning 15 days after transplantation. All groups showed sustained engraftment of MA9-expressing cells throughout the study. An abrupt rise in EGFP percentage was uniformly seen at time of death from leukemia in all groups (not plotted). (C) Each genotype gave rise to identical end-stage AML. Peripheral blasts demonstrate myelomonocytic morphology on Wright-Giemsa staining. Scale bars represent 10 μm. EGFP⁺ cells in BM, spleen, and PB were Gr1⁺/Mac1⁺/CD3⁻/B220⁻/Sca1⁻/cKit⁺. Shown is representative FACS analysis of a WT/WT BM specimen. (D) Survival curve comparing secondary recipients of MA9 leukemia cells from each group showed similar latency. (E) PCR genotyping confirmed deletion of Rac1 or Rac2. Genotyping of sublethally-irradiated secondary recipients of KO/WT MA9 tumors (KO/WT 2°) reveals the Rac1 deletion band in addition to host WT cells, but no residual FL/WT MA9 cells. Each lane represents a different tumor. (F) Knockout of Rac1 or Rac2 was also confirmed by immunoblot. KO/WT tumors demonstrated a compensatory increase in Rac2 expression. Each lane represents a distinct tumor. Images were taken from the same membrane under identical exposure conditions.