Efficient transduction of c-kit⁺ primary human hematopoietic cells by SCF-eco retrovirus. (A) The c-kit staining of CD34⁺ normal bone marrow. Normal human bone marrow-derived CD34⁺ cells were isolated by immunomagnetic separation and stained for CD34 with FITC-conjugated monoclonal antibody and for c-kit with a phycoerythrin (PE) conjugate. The proportion of doubly stained cells is indicated in the top right quadrant. (B) Targeted transduction of CD34⁺ bone marrow cells. CD34⁺ cells were transduced with retroviral supernatants and assayed for expression of EGFP by flow cytometry. Transduction efficiency is shown relative to the results obtained with ampho virus and data are derived as for Figure 3 but from 2 independent marrows transduced in duplicate. (C) Targeted transduction of CFU-GM. CD34⁺ cells were cocultured with retroviral producers using Transwells. The transduced cells were plated in semisolid media containing G418 to select for colonies expressing the retrovirally encoded neo gene, and resistant colonies were counted. The data (mean ± SEM) are derived from 3 independent marrows transduced in duplicate. (D) Nested PCR analysis of colonies surviving in semisolid media supplemented with G418 using primers that detect the neo gene. The figure shows data from a representative sample of the colonies analyzed. Lane 1, 100-bp ladder size marker; lanes 2-7, cells transduced with ampho virus; lanes 8-17, cells transduced with SCF-eco virus; lane18/-, semisolid media only; lane 19/+, plasmid DNA containing retroviral genome. (E) Replating assay on G418-resistant colonies. Colonies surviving in semisolid media containing G418 were replated to assay expression of the CDK4 gene encoded by the retroviral genome. Replating activity, expressed as area under the curve (AUC), was compared with that obtained from an identical retroviral vector transducing only the neo resistance marker. (F) Effect of polycations on targeted transduction. CD34⁺ progenitors were transduced as in panel C but in the presence (+PS) or absence (-PS) of 4 μg/mL protamine sulfate; transduction was determined, as before, from the percentage of colonies resistant to G418. The data shown are a representative example of the experiment that was performed 3 times.