Lentiviral constructs and enforced expression of TAL1 and ΔbTAL1 in human hematopoietic cells. (A) Structure of the lentiviral vector TRIPΔU3-EF1α encoding EGFP, TAL1, and ΔbTAL1 sequences under the control of the EF1α promoter. (B) Western blot analysis of TAL1 and ΔbTAL1 protein expression in Jurkat cell lines. Protein lysates were prepared from nontransduced Jurkat (JKT) cells (NT, lane 1), or JKT-L4 cells transduced with TRIP-EGFP (EGFP), TRIP-TAL1 (TAL1), or TRIP-ΔbTAL1 (ΔbTAL1) (lanes 2 to 5). Proteins were separated by SDS-PAGE and revealed with the anti-TAL1 2TL136 monoclonal Ab. (C) Gene transfer efficiency into cord blood-derived CD34⁺ cells. Transduced cells were assayed by flow cytometric analysis (histogram indicates percentage of EFGP-expressing cells; upper panel) or by PCR analysis for the detection of integrated TRIP-TAL1 provirus into genomic DNA extracted from individual colony-forming cell (CFC)-derived colonies (lower panel) recovered after 14 days in methylcellulose culture. Eight colonies are shown, of which one was negative for the vector integration. Controls are JKT-L4 cells nontransduced (-) or transduced (+) with TRIP-TAL1. (D) TAL1 and ΔbTAL1 expression into human CD34⁺ cells transduced with the TRIP vectors. Western blot was performed as described with proteins extracted from CD34⁺ cells after transduction. Blots were stripped and reprobed with anti-ERK1 polyclonal Ab as a control of the amount of proteins loaded onto the gel. Results are representative of 2 experiments.