Generation of a stem cell leukemia (FLA2). Overview of the experimental strategy. (A) E14.5 FL cells were isolated from C57BL/6:Pep3b (Ly5.1) mice and sorted for KLS cells. A fraction of sorted cells was plated at limiting dilution in 24-well plates containing murine stem cell virus Hoxa9-ires-Meis1a-pgk-Neo retroviral producers. Transduced cells were transplanted into congenic mice at a dose of one well per mouse. (B) Summary of CFC and transplanted CRU infection efficiency. Numbers of infected CFCs and CRUs were estimated according to progenitor infection percentage. (C) Northern blot analysis of Hoxa9 and Meis1 mRNA levels in leukemia (*) and healthy mice (B3 and B4) with 18S RNA levels were used as loading control. + indicates GP + E86 viral producers of retrovirus; and −, noninfected GP + E86 cells. (D) Leukemia colony-forming cell (L-CFC; bottom panel) and L-HSC frequencies (top panel) determined in primary recipients of FLA2, FLB2, and FLB1 cultures as measured by limiting dilution analyses. (E) Kaplan-Meier-like plot comparing survivals of secondary mice transplanted at limiting dilutions (ie, one L-HSC per recipient, corresponding to 1 cell for FLA2 and several hundred for FLB1 and FLB2) where the survival curves for FLA2 and FLB1 are significantly different (P = .0001).