RT-PCR analysis of the expression of erythroid(β-globin and EpoR), megakaryocytic (GpIIb,AchE, and Mpl), and myeloid (Mpo) genes in single colonies derived from early erythroid, megakaryocytic, and myeloid (BFU-E, CFU-Mk, and CFU-GM, respectively) or late erythroid (CFU-E) progenitor cells.
RNA was prepared from individual colonies (lanes 1-4), reverse transcribed, and PCR-amplified for either 24 (β-globin) or 35 (all the other genes) cycles. Actin-specific fragments were amplified from all the PCR reactions presented (not shown). Lane C shows the data obtained using complementary DNA (cDNA) from normal bone marrow (positive control). The results obtained with 4 representative colonies are shown in each panel. The ratio on the right of each panel specifies the actual number of single colonies that were positive for that particular gene as a function of the total number of colonies analyzed. The cloning efficiency was 32 ± 10 BFU-E–, 6 ± 2 CFU-Mk–, 10 ± 4 CFU-E–, and 55 ± 20 CFU-GM–derived colonies per 105 normal bone marrow cells, and the number of colonies analyzed corresponds to the colonies detected in 1.5, 4, 3, and 0.5 dishes, respectively. BFU-E = burst-forming unit erythroid; CFU-E = colony-forming unit–erythroid; CFU-GM = colony-forming unit–granulocyte macrophage; CFU-Mk = colony-forming unit–megakaryocyte.
