Retroviral transduction of CD34⁺ cells from patients with MPN. (A) pMXs.IG retroviral construct to express AML1 D171N. LTR indicates long terminal repeat. The FLAG-tagged D171N mutant was inserted into a retrovirus vector plasmid that encoded IRES-GFP. (B) Transduction of the AML1 D171N mutant into CD34⁺ cells obtained from patients with MPN. CD34⁺ cells were isolated from 8 patients with PV (nos. 1 and 2), ET (no. 3), PMF (no. 4), MPN-unclassified (nos. 5 and 6), post-PV MF (no. 7), and post-ET MF (no. 8). The cells were transduced either with a control vector (pMXs.IG) or with the AML1 mutant (D171N). A typical flow cytometric profile of cells transduced with either pMXs.IG or D171N retrovirus. The efficiency of retrovirus transduction was 30% to 50%. The GFP⁺ cells shown within the gate were collected. (C) Anti-FLAG immunoblotting of sorted GFP⁺ cells to confirm the expression of FLAG-tagged AML1 D171N protein. (D) Colony number and cell proliferation from CFC replating assays. The experiment was performed twice for patient nos. 6 and 7 and once for other patients. Shown are representative data for 8 of 13 patients. After 14 days of culture in methylcellulose, plates of cells from the patients with PV, ET, and PMF (nos. 1-4) contained more than 100 colonies, whereas those from patients with MPN-unclassified and post-PV/ET MF (nos. 5-8) contained only 1 to 80 colonies. Note that the y-axis scales vary. BFU-E indicates burst-forming unit-erythroid; CFU-GM, colony-forming unit-granulocyte, macrophage; GEMM, colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte. (E) Percentage of CD34⁺ cells in total cells recovered from the first CFC plates 14 days later. (F) Percentage of CD34⁺ cells in the cells cultured on MS5 stroma cells for 5 weeks. MS5 cells were excluded by flow cytometry gating. (G) The retrovirus-transduced and sorted cells were cultured on MS5 cells for 5 weeks and subjected to subsequent CFC assays. LTC-IC assays were carried out in triplicate, and the average numbers of LTC-ICs per 10 000 original input cells and SDs are indicated.