Figure 3.

FLT3/ITD mutations in the bone marrow cells of engrafted mice. CD34⁺/CD38^- cells from 6 different patient samples were isolated by immunomagnetic sorting (sorting results displayed in Figure 1). Mice were injected with either 1 × 10⁶ unsorted cells or 5 × 10³ CD34⁺/CD38^- cells. After 14 weeks, the mice were killed and bone marrow cells were harvested. (A) For each of the 6 samples (numbers correspond to those in Figure 1), genomic DNA was isolated from the unsorted cells (a), the marrow cells of mice injected with 1 × 10⁶ unsorted cells (b), or those mice injected with 3 × 10⁵ CD34⁺/CD38^- cells (c). PCR was performed using primers flanking the juxtamembrane region of human FLT3 (along with actin controls). The characteristic doublet of the FLT3/ITD mutation is easily visualized after electrophoresis in 2.5% agarose with ethidium bromide staining. Genomic DNA from HL60 cells (HL) was used as a control. (B) The sequences of the FLT3/ITD mutations from the 3 samples that engrafted (1, 3, and 4) are shown. These sequences, obtained from the DNA of the engrafted mice, matched the sequences of the mutations present in the preinjection samples. bp indicates base pair. Boldface sequence refers to the inserted, duplicated material.