Silencing of Gprasp1 or Gprasp2 enhances HSPC–in vivo hematopoietic repopulating activity. (A) Messenger RNA (mRNA) expression analysis of Gprasp1 and Gprasp2 in the hematopoietic hierarchy. (B) Schematic of shRNA vector. (C) Experimental schematic. CD45.2⁺ “test” LSK cells were transduced with control or Gprasp shRNAs and then transplanted along with CD45.1⁺ “competitor” LSK cells into lethally irradiated recipients. Recipient peripheral blood (PB) or BM was analyzed for CD45.2⁺mCherry⁺ cells. (D) Enhanced PB-repopulating activity relative to HPSCs treated with control shRNA in (i) Gprasp1-deficient HSPCs and (ii) Gprasp2-deficient HSPCs. (E) Analysis of CD45.2⁺mCherry⁺ cells in recipient BM HSPC compartments 20 weeks posttransplant. (F) Distribution of mature lineages in CD45.2⁺mCherry⁺ PB in recipients of control or Gprasp-shRNA–treated HSPCs. Data in panels D, E, and F are from 3 independent transplants with 5 recipients per condition per transplant. Data represented as mean plus or minus standard error of the mean (SEM). */#, P < .05; **/##, P < .005; ***/###, P < .001 relative to recipients of control-shRNA HSPCs. In panel D, * refers to shRNA A; # refers to shRNA B. CLP, common lymphoid progenitor; CMP, common myeloid progenitor; FSC, forward scatter; GMP, granulocyte/macrophage progenitor; Gran., granulocyte; LTR, long terminal repeat; MEP, megakaryocyte/erythrocyte progenitor; MPP, multipotent progenitor; MSCV, murine stem cell virus; PGK, phosphoglycerate kinase; shRNAmiR, microRNA-adapted short hairpin RNA; ST-HSC, short-term HSC.