Effect of enforced TAL1 expression on the differentiation of CD34⁺ cells in lymphomyeloid cultures. (A) Lentiviral construct and localization of primers designed to evaluate the transduction efficiency. (B) Transduction efficiency revealed by flow cytometry (GFP⁺ cells) or by PCR (TAL1⁺ cells). GFP-transduced cells were cultured during 3 days in lymphoid/myeloid conditions, and GFP expression was detected by flow cytometry (indicated is the percentage of GFP-expressing cells, left). TRIP-TAL1–transduced cells were plated in CFC assay; colonies were harvested after 14 days and individually subjected to PCR analysis to detect the vector integration. Shown in the right panel is a typical analysis of 11 colonies of which 9 are positive for the vector integration. Controls are Jurkat-L4 cells transduced (+) or nontransduced (-) with TRIP-TAL1 vector. (C) Western blot analysis of TAL1 and ΔbTAL1 protein expression in human CD34⁺ cells transduced with the TRIP vectors. Blots were stripped and reprobed with antiactin antibody as a control of protein loading. (D-E) In vitro production of CD19⁺ B cells and of CD14⁺/CD15⁺ myeloid cells from transduced cells. Immediately after transduction, cells were cultured in lymphoid/myeloid conditions during 3 weeks, and the proportion of CD19⁺ B cells and CD14⁺/CD15⁺ granulomonocytic cells was evaluated by FACS. Shown is a typical experiment (D) and the overall 6 independent experiments performed (E). Numbers indicate the percentage of positive cells among gated cells. Results obtained with GFP⁺ cells and TAL1⁺ cells shown (E) are linked for every experiment. Indicated is the ratio (expressed as a mean percentage) obtained by comparing results from TAL1⁺ cells and GFP⁺ cells. Each symbol represents an independent experiment.