Correction of aberrant splicing of HeLa EGFP and β-globin IVS2 mutants with U7 lentiviral vector.

(A) FACS analysis. EGFP-654 and EGFP-705U cells were transduced with U7.623 lentiviral vector and subjected to flow cytometry analysis after 2 weeks of transduction. (B) Fluorescence microscopy of identically treated cells. Phase contrast (left) and UV (right) images are shown. Magnification × 10. (C) RT-PCR of total RNA from HeLa β-globin IVS2-654 (lane 2), IVS2-705 (lane 5), and IVS2-745 (lane 8) cells transduced with U7.623 lentiviral vector. Untreated IVS2-654 (lane 1), IVS2-705 (lane 4), and IVS2-745 (lane 7) cells served as negative controls. Lanes 3, 6, and 9 show RNA from normal blood. Lane 10 shows IVS2-745 cells transduced with U7.623 lentiviral vector analyzed at 6 months. (D) Immunoblot analysis of total protein from HeLa IVS2-705 and IVS2-745 cells transduced as in panel C. The blots were probed with polyclonal anti–β-globin antibody. Lane 1, β-globin marker; lanes 2 and 4, untreated cells; lanes 3 and 5, lentiviral vector–transduced cells.