Gene targeting of Rac1 or Rac1/Rac2 in HSCs and the effect on peripheral blood and bone marrow cells. (A) Rac1 gene–targeted allele. The conditional Rac1 allele was generated by sandwiching exon 1 of Rac1 gene with 2 loxP sites. (B) Generation of Rac1 or Rac1/Rac2 knockout HSCs in mice. To produce Rac1- or Rac1/Rac2-deficient hematopoietic cells in mice, Rac1^loxp/loxp or Rac1^loxp/loxp/Rac2^−/− mice were crossbred with Mx-Cre transgenic mice. Four to 5 doses of poly I:C injection of the mice resulted in efficient deletion of Rac1 gene in HSCs. (C) Expression of Rac1 in bone marrow, thymus, and spleen of Rac1 or Rac1/Rac2 Mx-Cre–targeted mice. Bone marrow cells, thymocytes, and splenocytes from WT-, Rac1-, or Rac1/Rac2-targeted mice were probed for Rac1 protein by anti-Rac1 blotting. Levels of β-actin were used as loading controls. (D) Deletion of Rac1/Rac2 but not Rac1 alone led to increased white blood cells (WBCs) and neutrophils (NEs) without apparent effect on lymphocyte numbers (LY) in peripheral blood. PB of various genotypes was analyzed by blood counting on a hematology analyzer. (E) PB of various genotypes was stained by biotinylated anti-CD3 or -B220 antibody followed by Streptavidin-Percp and analyzed by flow cytometry. (F) Deletion of Rac1 or Rac1/Rac2 reduced CLP numbers in bone marrow. Bone marrow cells were counted and stained for lineage markers with biotinylated antibodies against B220, CD3, CD4, CD8, Gr1, CD11b, and TER119. Subsequently, cells were stained with Streptavidin-Percp, anti–IL7R-APC-Cy7, anti–c-kit-APC, and anti–Sca1-PE. CLP was defined by the Lin⁻IL-7Rα⁺Sca1^medc-kit^med-high phenotype. The number of CLP was calculated based on the total number of bone marrow cells and the percentage of CLP. n = 5 in each panel. *P < .05; **P < .01. Error bars represent SD.