Retrovirus-mediated gene transduction of mouse bone marrow cells using vectors expressing RhoH and EGFP. (A) The bi-cistronic retroviral vectors expressing EGFP and hemagglutinin (HA)–RhoH (or HA-RhoH T36A). IRES indicates internal ribosome entry segment. LDBM cells were transduced with each of these 3 vectors. The EGFP⁺, c-Kit⁺ cells were sorted by fluorescence activated cell sorting (FACS) and used for the following assays. (B) Northern blot analysis using the RhoH cDNA probe showed levels of endogenous RhoH and exogenous RhoH-IRES-EGFP mRNAs. The ethidium bromide–stained 28S and 18S rRNAs were used as loading controls. (C) HA-RhoH and HA-RhoH T36A proteins in the transduced cells were examined by immunoblot probed for HA tag or β-actin as a control. (D) Cell expansion. Cells (1 × 10⁵) were plated in 10% serum-containing medium with 100 ng/mL SCF. Cell numbers were counted every 2 days up to 6 days during culture. (E) Myeloid progenitor colony-forming unit assay. Cells were plated in methylcellulose in the presence of SCF alone or a cocktail of SCF, G-CSF, and MGDF. Colonies were enumerated 7 days later using an inverted microscope. In panels D and E, data represent the mean ± SD; n = 3. *P < .01, RhoH-versus vector-transduced cells. The results shown in panels B-D are representatives of more than 3 experiments.