Effect of CYT387 on JAK2 V617F⁺ allelic burden in MPN in vivo. Balb/c mice were subjected to bone marrow transplantation with bone marrow donor cells retrovirally transduced to express JAK2^V617F. Thirty-four days after transplantation, mice exhibited symptoms of MPN as measured by elevated white blood cell counts and hematocrit. Mice were divided into 3 groups and initiated on twice daily oral gavage administration of vehicle control, 25 mg/kg CYT387, or 50 mg/kg CYT387 (n = 12 per group). At day 83 after bone marrow transplantation, all mice were sacrificed and genomic DNA was isolated from splenocytes. A qPCR assay was developed to assess the relative level of GFP, normalized to the genomic GAPDH locus. (A) Parental Ba/F3 (GFP-negative) were mixed at varying ratios with Ba/F3-JAK2^V617F cells (GFP-positive). Genomic DNA was isolated and subjected to qPCR using primers specific for GFP or the GAPDH genomic locus. All values were normalized to GAPDH and then to the highest expressing well of genomic DNA from 100% GFP-positive cells. This value was set at 100%. Values represent mean ± SEM. (B) Genomic DNA from splenocytes of mice treated with vehicle control, 25 mg/kg CYT387, or 50 mg/kg CYT387 were subjected to qPCR using primers specific for GFP or the GAPDH genomic locus. The standard curve of Ba/F3 cells shown in panel A was amplified simultaneously, and all values were normalized to GAPDH and then to the highest expressing well of genomic DNA from 100% GFP-positive Ba/F3 cells. Each point represents the genomic GFP level in an individual mouse. (C) The values from panel B were averaged and presented in a bar graph. Values represent mean ± SEM and *P < .05 in a t test comparing 25 mg/kg or 50 mg/kg treatment groups with the 0 mg/kg vehicle control.