Therapeutic β^AS3-globin expression levels in murine erythrocytes following lentiviral gene delivery. (A) IEF of hemolysates from recipient mice at 5 to 7 months after transplantation. The first 4 lanes are human HbS, murine hemoglobin, human HbAS3, and mock-transduced controls. The next 5 lanes are 5 primary lenti/β^AS3 mice reconstituted with more than 99% donor cells. The final lane is a representative secondary transplant at 4 months after transplantation. (B) Single-cell expression analysis of lenti/β^AS3 reticulocytes. Following thiazole orange staining, reticulocytes were sorted by flow cytometry. mRNA was isolated from individual lysed cells, reverse transcribed, PCR amplified, radioactively labeled by primer extension, and digested with Bsu36I, which digests β^AS3 but not β^S. Data are from 13 representative reticulocytes. Population controls of 100 cells from the same transplant (TP), an HbAS3 knockout-transgenic (AS3), or a donor knockout-transgenic sickle mouse (S) are also shown. (C) Lentiviral vector copy number determination in bone marrow G/M, erythroid progenitors, and WBM from secondary recipients at 7 months after transplantation. G/M is granulocyte/macrophage (Gr1⁺ and CD11b/Mac1+), T is erythroid progenitor (Ter119+), and WBM is whole bone marrow.