Figure 5. Serial colony assays of mouse bone marrow progenitor cells (BMPCs) transduced with retroviral constructs.

A: Expression of various fusion protein constructs (in MSCVneo vector) at the protein level in transiently transfected Phoenix cells detected by IP-western blot. B: Expression of the various fusion genes in the MSCVneo vector (in the same order as in A) at the mRNA level, detected by RT-PCR (+ RT) in BMPC after primary plating on methylcellulose medium supplemented with IL-3, IL-6, GM-CSF, SCF and G418. The primers used in the PCR reaction amplify the junction region between 5’ MLL and the ENL portion as shown in the diagram. C: Number of primary, secondary, tertiary and quaternary colonies generated per 10⁴ plated cells following methylcellulose culture of BMPCs transduced with MLL-ENL and its PHD finger insertion mutants. The values are the mean of three independent assays and the error bars represent standard deviations. D: The colonies that appeared on the methylcellulose plates after the tertiary plating were photographed. Cells collected after the tertiary plating were prepared by cytospinnning, and stained with Wright-Giemsa (Hema3 Stain Set, Fisher Scientific).