Figure 1.
GFP expression in erythroid cells after in vivo HSC transduction/selection. (A) HDAd-LCR-GFP/mgmt vector for SB100x transposase-mediated integration. The GFP gene is under the control of a 4.9-kb erythroid-specific (human) β-globin “mini” LCR consisting of DNase I hypersensitivity regions (HS) 1 to 4 and the β-globin promoter. The GFP gene is linked to the β-globin 3′UTR for mRNA stabilization in erythroid cells. The ET3 and mgmt expression cassettes are separated by a chicken globin HS4 insulator. The 10.8-kb ET3/mgmt transposon is flanked by inverted repeats (IRs) that are recognized by SB100x, mediating random integration. (B) In vivo transduction of mobilized hCD46-transgenic mice. HSCs were mobilized, and animals were IV injected with a 1:1 mixture of HDAd-LCR-ET3/mgmt + HDAd-SB. At week 4, O6-BG/BCNU treatment was started and repeated every 2 weeks 4 times. With each cycle, the BCNU concentration was increased from 5 mg/kg, to 7.5 mg/kg, to 10 mg/kg. (C) Percentage of GFP-positive peripheral RBCs. Each symbol is an individual animal. (D) Representative flow cytometry analysis for GFP expression in total blood cells at week 21 after in vivo HSC transduction. (E) Peripheral blood smears were stained with May-Grünwald Giemsa (Merck) for 5 minutes (upper panel). GFP fluorescence analyzed on week 21 blood smears (lower panel). Scale bars, 20 μm. (F) Percentage of GFP-positive mononuclear cells (MNCs) measured by flow cytometry in different lineages in the bone marrow. CD3, T-lymphocytes/progenitors; CD19, B-lymphocytes/progenitors; Gr-1, granulocytes/progenitors; Ter119, erythroid cells. (G) Percentage of GFP-positive cells in different tissues measured by flow cytometry of single-cell suspensions. Note that the y-axis has 3 segments. (H) Representative kidney section demonstrating the presence of GFP-positive erythrocytes in a glomerulus. 4′,6-Diamidino-2-phenylindole (DAPI) (blue) was used to visualize cell nuclei. In vivo HSC transduced mice were kept alive without side effects for 21 weeks. Scale bars, 20 μm.

GFP expression in erythroid cells after in vivo HSC transduction/selection. (A) HDAd-LCR-GFP/mgmt vector for SB100x transposase-mediated integration. The GFP gene is under the control of a 4.9-kb erythroid-specific (human) β-globin “mini” LCR consisting of DNase I hypersensitivity regions (HS) 1 to 4 and the β-globin promoter. The GFP gene is linked to the β-globin 3′UTR for mRNA stabilization in erythroid cells. The ET3 and mgmt expression cassettes are separated by a chicken globin HS4 insulator. The 10.8-kb ET3/mgmt transposon is flanked by inverted repeats (IRs) that are recognized by SB100x, mediating random integration. (B) In vivo transduction of mobilized hCD46-transgenic mice. HSCs were mobilized, and animals were IV injected with a 1:1 mixture of HDAd-LCR-ET3/mgmt + HDAd-SB. At week 4, O6-BG/BCNU treatment was started and repeated every 2 weeks 4 times. With each cycle, the BCNU concentration was increased from 5 mg/kg, to 7.5 mg/kg, to 10 mg/kg. (C) Percentage of GFP-positive peripheral RBCs. Each symbol is an individual animal. (D) Representative flow cytometry analysis for GFP expression in total blood cells at week 21 after in vivo HSC transduction. (E) Peripheral blood smears were stained with May-Grünwald Giemsa (Merck) for 5 minutes (upper panel). GFP fluorescence analyzed on week 21 blood smears (lower panel). Scale bars, 20 μm. (F) Percentage of GFP-positive mononuclear cells (MNCs) measured by flow cytometry in different lineages in the bone marrow. CD3, T-lymphocytes/progenitors; CD19, B-lymphocytes/progenitors; Gr-1, granulocytes/progenitors; Ter119, erythroid cells. (G) Percentage of GFP-positive cells in different tissues measured by flow cytometry of single-cell suspensions. Note that the y-axis has 3 segments. (H) Representative kidney section demonstrating the presence of GFP-positive erythrocytes in a glomerulus. 4′,6-Diamidino-2-phenylindole (DAPI) (blue) was used to visualize cell nuclei. In vivo HSC transduced mice were kept alive without side effects for 21 weeks. Scale bars, 20 μm.

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