Biologic effects of Bcr-Abl transduction in SHP-2^Δ/Δ cells are decreased. WT and SHP-2^Δ/Δ hematopoietic cell lines were transduced with Bcr-Abl or the vector control by retroviral-mediated gene transfer. Transduced cells were sorted by FACS, based on expression of GFP. (A) Sorted cells were seeded into 96-well plates (1 × 10⁴ cells/well) in RPMI-1640 medium with 10% FBS. Cells were quantitated on day 0 and day 3 by the MTS assay, a colorimetric method for detecting viable cells. Cell growth rates (fold changes) were determined by OD₄₉₀ values and normalized to day 0. (B) WT/Bcr-Abl–, SHP-2^Δ/Δ/Bcr-Abl–, and the vector control–transduced cells were plated into 0.9% RPMI-1640 methylcellulose medium containing 15% FBS (1000 cells/mL). After 7 days of incubation in a humidified 5% CO₂ incubator, colonies consisting of more than 50 cells were scored under an inverted microscope. (C) Bcr-Abl– or vector-transduced WT and SHP-2^Δ/Δ cells were examined by migration assay as described in “Materials and methods.” Migrated cells adhering to the lower side of trans-membranes were stained and counted under a microscope. (D) Bcr-Abl– and the vector control–transduced WT or SHP-2^Δ/Δ cells were cultured in IL-3–free RPMI-1640 medium for 24 hours. Cells were then harvested and processed for cell-cycle analyses. Fold changes of the percentages of S-phase in Bcr-Abl–transduced cells over those of the vector–transduced cells were then determined. (E) Embryonic day 9.0 to 9.5 yolk sacs were dissected from timed pregnant SHP-2^+/Δ female mice mated with SHP-2^+/Δ male mice. Embryonic tissues were used for genomic DNA extraction and subsequent PCR genotyping. Yolk sac cells were dissociated, prestimulated, and transduced with Bcr-Abl or vector as described in “Materials and methods.” Transduced cells were sorted by FACS according to GFP expression. Sorted cells were seeded into 96-well plates in IL-3–free medium and were assayed for cell growth rates as described in Figure 1A. Two to 3 independent experiments were performed, and similar results were obtained in each. Results shown are the mean ± SD of triplicates from one experiment.