Assessment of the effects of HOXA9 suppression in vivo using bioluminescent imaging. (A) Before transplantation, SEMK2 cells were transduced with either 1F3-HOXA9 shRNA or GFP control shRNA and injected intravenously 24 hours after transduction. Mice were imaged 2, 24, and 72 hours after injection and then weekly. (B) There was no significant difference in total body luminescence 2 hours after injection confirming that an equal number of treated cells was injected (P = .101). Repetitive longitudinal in vivo bioluminescent imaging at later time points revealed a significant difference of total body luminescence as early as 24 hours after transplantation (P = .036). Fourteen days after transplantation, the differences in total body luminescence reached a significant maximum (P = .001), shortly before the GFP shRNA–treated control group died of overt leukemia. (C) The degree of leukemic organ infiltration in both groups was assessed. The percentage of hCD19⁺/mCD45⁻ SEMK2 cells was evaluated in spleens from wild-type SCID-beige mice that received 1F3-HOXA9–transduced or GFP-control–transduced cells. (D) The total number of human cells/spleen is graphed for both groups of mice. There is a 40-fold difference in total number of SEMK2 between groups (P = .001). (E) Spleen weights for are shown for mice that received either control or 1F3-HOXA9-transduced SEMK2 cells. (F) Analysis of HOXA9 expression in SEMK2 cells at the day of transplantation (day 0) confirmed efficient HOXA9 suppression (80%) in the 1F3-HOXA9 shRNA-treated group compared with control shRNA-transduced cells. Human cells were isolated from mice that received either control or 1F3-HOXA9 shRNA-transduced SEMK2 cells 14 days after injection, and the HOXA9 expression level was compared with SEMK2 cells growing in culture. The HOXA9 expression level at this time point was similar in the sorted SEMK2 cells from the 1F3-HOXA9shRNA group as in those sorted from the GFP shRNA control group (P = .244).