Smad7 expression, retroviral constructs, and packaging lines. (A) Expression of Smad7 in human fetal blood (FB) and cord blood (CB) hematopoietic cell populations, including the primitive CD34⁺CD38^– cell fraction devoid of lineage commitment markers (Lin^–), mature CD33⁺ myeloid cells, CD19⁺ B lymphocytes, and CD3⁺ T lymphocytes, as determined by RT-PCR analysis. An RT-PCR reaction was performed on human fetal head cDNA as a positive control, and on a sample containing no cDNA (H₂O) as a negative control. RT-PCR for β-glucuronidase (β-gluc), a housekeeping gene expressed at a single copy per cell, was used to assess the quality of the cDNA templates. Quantitative PCR was used to measure the expression of Smad7 in (B) highly purified Lin^–CD34⁺CD38^– and Lin^–CD34⁺CD38⁺ subsets from human cord blood (summarized as relative expression in Lin^–CD34⁺CD38^– [▪] compared with Lin^–CD34⁺CD38⁺ cells [□], right) and (C) in mature human CB subsets containing the myeloid (▪) and B-cell lineages (□). *Smad7 not detectable by Q-PCR. (D) Control vector and Smad7 retroviral constructs. The gene for Smad7 was subcloned into the control vector backbone using EcoRI and BamHI cloning sites, upstream of an internal ribosomal entry site (IRES). An enhanced green fluorescent protein (EGFP) gene downstream of the IRES acts as a reporter for selection of stable cell lines and tracking of transduced cells. (E) (i) PG13 packaging cell lines transduced with vector or Smad7 retrovirus were isolated by fluorescence-activated cell sorting (FACS) based on GFP expression. (ii) Validation of the PG13 retroviral packaging cell lines by retroviral transduction of the human hematopoietic cell line MBA.1 with vector- or Smad7-containing retrovirus. Transduced cells were selected based on GFP expression and verified by fluorescence microscopy. (F) Western blot analysis of cells transduced with vector or Smad7 retrovirus, and selected based on GFP expression. Equal amounts of protein were loaded per lane in all experiments. PG13 and MBA.1 cell lines transduced with Smad7 retrovirus express a higher level of Smad7 protein than cells transduced with vector retrovirus. Values indicated are protein band intensity relative to vector control. (G) Fold increase in Smad7 mRNA expression in Smad7 compared with vector-transduced HeLa and M-O7e cells, determined by amplified RNA analysis (n = 3). (H) TGF-β growth inhibition assay. 20 000 M-O7e cells, transduced with vector (□) or Smad7 (▪), were grown with 1 ng/mL TGF-β₁ or without for 6 days. Total viable cells were enumerated at days 2, 4, and 6 of culture. Shown is the percent difference in growth of Smad7–M-O7e and vector–M-O7e at 1 ng/mL TGF-β₁ compared with no TGF-β₁ (dashed line), for triplicate samples in 2 independent experiments. Inset shows changes in cell number over time for all treatments.