Functional assessment of HSCs from Ku70^−/−mice. (A) Noncompetitive serial transplants were initiated by transplanting 2 × 10⁶ whole BM pooled from 3-month-old WT (n = 3) or Ku70^−/− (n = 2) donor mice (CD45.2) into irradiated recipients (CD45.1, n = 5 per group). Secondary and tertiary transplants were performed after 16 to 24 weeks of engraftment by pooling BM from 3 to 4 reconstituted recipients to transplant 2 × 10⁶ whole BM into new groups of irradiated CD45.1 recipients. Survival of recipient mice was monitored. Shown is a representative result of 2 independent experiments. (B) Prior to transplant into tertiary recipients, BM from 5 secondary recipients of both genotypes was assayed by FACS for the frequency of LSK. Error bars indicated SD of the mean, and significance was determined by a 2-tailed Student t test. *P < .01. (C) BM from 3-month-old WT and Ku70^−/− mice (CD45.2) was harvested and mixed with WT BM (CD45.1) at a 1:1 ratio and transplanted into lethally irradiated WT mice (CD45.1, n = 5 per group). Then 16 weeks after transplantation, donor chimerism in the PB was analyzed and quantitated. Similar results were obtained in 3 independent experiments. Error bars indicate the SD, and significance was determined by a 2-tailed Student t test. *P < .005. (D) Three-month-old recipient mice (CD45.2) were transplanted with WT (CD45.1) BM cells without any ablative conditioning. Donor-derived Mac1⁺ cells in the PB were analyzed 16 weeks after transplantation from 2 separate injections (WT, n = 5; Ku70^−/−, n = 3). Then 16 to 24 weeks after transplantation, BM cells were isolated from recipient mice and chimerism of LSK population in each recipient mouse was analyzed. (E) A total of 5 × 10⁶ BM cells from WT, Ku70^−/− (CD45.2) donors were transplanted into lethally irradiated WT (CD45.1, n = 5 per group) recipients. Then 12 weeks later, the transplanted BM chimeras were challenged with 5 × 10⁶ GFP-transgenic BM cells. The percentage of donor-derived GFP⁺ Mac1⁺ cells in each mouse was determined by FACS 16 weeks later. Then 16 to 24 weeks after transplantation, chimerism of LSK population in the recipient BM was analyzed. Shown is representative result of 2 independent experiments. For Student t tests relative to WT, *P < .001. BMC, bone marrow cells.