Increased EGFP marking in mouse hematopoietic lineages arising from rapamycin-treated and LV-transduced Lin⁻ cells transplanted into mice. Congenically marked mouse bone marrow Lin⁻ cells were transduced for 16 hours with RRL-MND-GFP LV, MOI = 2.5, in the presence of 5 μg/mL rapamycin or DMSO. Cells were washed, and 1 × 10⁶ cells per mouse were injected retro-orbitally into lethally irradiated congenically disparate recipients. Mice were sacrificed 11 (gray triangles or open red circles) or 16 (black triangles or closed red circles) weeks posttransplant. (A) Percent donor cell engraftment in bone marrow and spleen. (B) EGFP marking, assessed by flow cytometry, in total donor bone marrow cells and bone marrow HSCs (Lin⁻Sca-1⁺c-Kit⁺). (C) LV copy number per cell in Lin⁻ and Lin⁺ bone marrow cells as determined by qPCR (described in “Methods”). (D) EGFP marking in splenic subsets analyzed with the following markers: B220⁺ B cells, B220⁻CD4⁺ or CD8⁺ T cells, B220⁻CD4⁻CD8⁻CD11b⁺GR1^lo/neg monocytes, and B220⁻CD4⁻CD8⁻CD11b⁺GR1^hi neutrophils. Each point represents 1 mouse. Cells were pregated on donor-derived cells for all subset assessments. For all panels, lines represent group mean and error bars represent standard deviation. *P < .05, **P < .01, and ***P < .001 from a parametric 2-tailed unpaired Student t test.