CD34⁺ cultures expressing AML1-ETO become mono- or oligoclonal. Cells were lysed and genomic DNA was isolated. DNA was digested with BamHI, run on an agarose gel, and transferred to a nitrocellulose membrane by standard protocols. The GFP cDNA was used as the probe to detect the integrated provirus in the genomic DNA. (A) Cells from culture AE.4 do not show clonal outgrowth at week 4 even though 100% of cells contain retrovirus by GFP analysis. However, at week 12, 2 dominant viral integrants were detected. Cells from culture AE.5, split into 2 independent cultures at week 6, show the outgrowth of cell populations that contain the same viral integrant at week 15. (B) Oligoclonal expansion of AML1-ETO-expressing CD34⁺ cells in cultures that were split into independent culture wells soon after viral transduction. No dominant clone is detected in these separated cultures. Cells obtained from a CAFC assay were also assessed and found to have a different clonal integrant. (C) AML1-ETO-expressing CD34⁺ cells were sorted for GFP expression immediately after transduction, remixed with the GFP-negative cells to give a final population with 50% GFP⁺ cells (to monitor the effect of AML1-ETO on the expansion of GFP⁺ cells), and plated at the cell densities indicated at the top of the figure. After 9 weeks of expansion, Southern blot analysis was performed on the cultures that continued to proliferate.