Generation of animals transplanted with CBFβ-SMMHC/FLT3-ITD-expressing cells. (A) Structure of the MSCV (murine stem cell virus) retroviral constructs. LTR indicates long terminal repeat; IRES, internal ribosome entry site, BEX, blue-excited GFP; VEX, violet-excited GFP. (B) Western blot analysis showing expression of CBFβ-SMMHC and FLT3-ITD in 2 × 106 bone marrow cells FACS–sorted from a 2-month posttransplantation CBFβ-SMMHC/FLT3-ITD animal (lane 2). An equivalent number of bone marrow cells sorted from a Bex/Vex-control animal was used for lane 1. (C) Representative FACS analysis of peripheral blood from Bex/Vex control or CBFβ-SMMHC/FLT3-ITD–reconstituted mice at 2.5 weeks after transplant. Frequencies for double-transduced cells are shown and were variable between experiments. (D) In vitro transduction efficiencies of highly purified KLSF cells used to reconstitute lethally irradiated recipient mice and changes in chimerism of doubly transduced cells in peripheral blood over time in vivo (n = 5 for each of control and CBFβ-SMMHC/FLT3-ITD–reconstituted mice). The same animal was analyzed at 5 and 8 weeks after transplant. Numbers on plots are percentages of total cells.(E) Kaplan-Meier survival curves of mice transplanted with cells expressing CBFβ-SMMHC (n = 9), FLT3-ITD (n = 5), CBFβ-SMMHC/FLT3-ITD (n = 17), or control Bex/Vex (n = 23) retroviruses.
