Construction of retroviral vector and target HSC for gene transduction. (A) Flow cytometric analysis of murine hematopoietic stem cells in adult bone marrow. Staining profile of lineage marker versus CD34 for lineage-depleted cells (left) and Sca-1 versus c-Kit in lineage^-/CD34^-/low-gated populations (right) in bone marrow is shown. (B) Schematic representation of the retroviral vector, pMY/WT SCL and pMY/DN SCL, encoding WT SCL and DN SCL linked by IRES to a cDNA encoding enhanced GFP (EGFP). (C) Expression of WT SCL and DN SCL proteins in retrovirally transduced 32D cells. Arrowheads indicate approximately 37 kDa (lane WT SCL) and 29 kDa (lane DN SCL). The other bands commonly seen in all the lanes, including the Mock control lane, represent nonspecific reaction of the antibody. (D) Expression of SCL mRNA in retrovirally transduced HSCs. Arrowheads indicate 308-base pair (bp) (lane WT SCL) and 170-bp (lane DN SCL) transcripts, the latter of which resulted from the deletion of a 138-bp sequence corresponding to the bHLH domain.