Replating analysis of AML1-ETO–expressing progenitor cells.

(A) The effect of AML-1/ETO expression on hematopoietic progenitors. Bone marrow was harvested from femurs of mice positive for MMTV-tTA (♦) alone or MMTV-tTA/AML1-ETO (▴), and 1 × 10⁴cells were plated in MethoCult in the absence of tetracycline. Bulk cultures were harvested after 7 to 10 days in culture, and 1 × 10⁴ cells were replated for each sample. Each point represents the number of colonies generated per 10⁴ cells seeded. After 9 (G9, ✖) and 11 (G11, ▪) generations, cells derived from MMTV-tTA/AML1-ETO mice were replated in MethoCult in the presence or absence of tetracycline. (B) Northern analysis of AML1-ETO expression in colonies harvested at various generations in methocellulose. RNA was harvested from bulk populations of colonies grown for 7 to 10 days in culture. Total RNA (5 μg) was loaded on each lane. The RNA was then transferred to a nylon membrane and hybridized with ETO cDNA. The ethidium bromide staining of the 18S ribosomal RNA is presented to show the loading of the RNA samples. tTA⁺ indicates RNA prepared from MMTV-tTA bone marrow cell culture; ⁺/⁺, RNA prepared from MMTV-tTA/AML1-ETO bone marrow cell culture. (C) Morphology of cells obtained from colonies grown in the presence or absence of tetracycline. After 9 generations, cells were cultured in the presence or absence of tetracycline. Cytospins of colonies replated in the presence or absence of tetracycline after culture for 9 generations without tetracycline were stained with Wright-Giemsa solution.