BCR-ABL rescues JAK2^−/− erythroid progenitors.

The population of GFP⁺Ter119⁻ cells encompasses all retrovirally transduced erythroid progenitors and, as detailed in the legend to Figure 4B, this population was FACS sorted from JAK2^−/− fetal liver cells transduced with either JAK2 or P210 BCR-ABL. From these, CFU-E (A) and BFU-E (B) colonies were generated. Total numbers of diaminobenzidine-positive CFU-Es cultured in the presence or absence of 2 u/mL Epo and 100 ng/mL SF (A) and BFU-Es cultured in the presence or absence of 2 u/mL Epo, 100 ng/mL SF, and 10 ng/mL IL-6 (B) were counted after 2 (A) and 9 (B) days, respectively. Results are presented as percentage of colonies formed in the JAK2-infected populations and cultured under optimum conditions (Epo and SF for CFUE [A] and Epo, SF, and IL-6 for BFU-E [B]) shown by the hatched bars. Graphs are averages from duplicate cultures of 4 (A) and 5 (B) independent experiments. Under optimum conditions, the average absolute number of JAK2-transduced colonies derived from JAK2^−/− fetal liver cells is 1248 ± 263 CFU-E and 96 ± 9 BFU-E colonies per 10⁵GFP⁺Ter119⁻ FACS-sorted cells. (C) Representative BFU-E–derived colonies from GFP⁺Ter119⁻ JAK2^−/− fetal liver cells transduced with either P210 or JAK2 and cultured in the presence of SF and IL-6, with (JAK2-transduced cells) or without (P210-transduced cells) Epo (× 100). (D) PolyA RT-PCR from single P210- or JAK2-generated BFU-E colonies of similar size were analyzed by southern blotting probed with β-major globin, GATA-1, and L-32. Shown are representative results from 3 independent experiments. Controls consist of HCD57 (10⁴ cells) in lane 1 and PCR reagents with no cells in lane 2.