Generation of human models of CG2 leukemia. (A) Experimental procedure used to establish xenograft models of CG2 AMKL (mCG2 AMKL) using independent lentiviral transduction in CB CD34⁺ cells (CB CD34⁺, pool of 6 CB units) and transplantation in recipient NSG mice. (B) Schematic representation of 6 CG2 leukemia models (mCG2) describing initial gene transfer (GT; %GFP) at the time of transplantation and leukemia latency in primary recipient mice. Mouse identification shown in brackets; 1 of 10 mice was not available for analysis. (C) Detection of CG2 fusion transcript expression by reverse transcription polymerase chain reaction (RT-PCR) with RNA isolated from leukemic blasts, as indicated. M07e and normal lineage-depleted CB (CB LIN⁻) cells were used as the positive and negative control, respectively. ABL1 was used as housekeeping gene. (D) Percentage of infiltrating human blasts (hCD45⁺ GFP⁺) and (E) CD41⁺ cells (of hCD45⁺ GFP⁺ population) in the BM and spleen of CG2 primary recipient mice (color code indicates distinct primary mice). (F) Hematoxylin phloxine saffron–stained longitudinal sections of tibia bones harvested from primary recipient mice that received transplantation with control CB CD34⁺ cells transduced with empty vector (top panel, 47 weeks after transplantation) and from a CG2-1 AMKL model (secondary recipient mice that received transplantation with 1 × 10⁶ CG2-1 AMKL xenograft cells and were euthanized 11.1 weeks after transplantation) (bottom). (G) Survival curves of primary recipient mice that received transplantation with CB CD34⁺ cells transduced with CG2 (black line) or empty vector (gray dashes), and mice that serially received transplantation up to 3 times with CG2-1 AMKL (red lines). Giemsa-stained cytospin and flow cytometry profiles of leukemic BM cells from representative (H-I) primary (1^ary) and (J-K) secondary (2^ary) CG2-1 AMKL recipient mice (2^ary recipient mice that received transplantation with 1.4 × 10⁶ CG2-1 AMKL xenograft cells and that were euthanized 6.9 weeks after transplantation). Detailed characteristics of other CG2 leukemia xenografts in recipient mice that serially received transplantation are described in supplemental Table 5 and supplemental Figure 1. (L) Survival curves of recipient NSG mice that received transplantation with CG2-1 AMKL xenograft cells in a limiting-dilution cell transplantation assay and (M) estimation of LIC frequency and 95% confidence interval (red dashes) using the extreme limiting dilution analysis software (http://bioinf.wehi.edu.au/software/elda/).