Fig. 4.
Fig. 4. CD68-based retroviral vectors generate expression specifically in mφs in vivo. / (A) Peritoneal cells elicited 4 days after injection with thioglycollate were analyzed by FACS. Three distinct cellular populations could be distinguished on the basis of forward- and side-scatter properties: neutrophils (blue), macrophages (red), and lymphocytes (green). These designations were confirmed by staining with cell type–specific antibodies (data not shown). By using a gate for EGFP-positive cells, which was established using samples from mice that had not received transplants, the cell-type specificity of EGFP expression could be established. The data are from cells isolated from a single mouse and are representative of data from 10 mice per retroviral construct. (B) Immunoblot analysis of tissue lysates (50 μg total protein per lane) from mice that had received HSC transplants (16 weeks after transplantation), using antibodies recognizing macrosialin (α-mCD68) or HA epitope–tagged EGFP (α-HA). Blots are from identical exposures to allow comparisons of the relative levels of HA-EGFP expression. Data are from a single mouse per retroviral construct, and are representative of results from 2 similar mice. L.N.s indicate lymph nodes. (C-H) Immunohistochemical staining of tissues from mice that had received transplants of HSCs transduced with CD68S–HA-EGFP retrovirus. Tissue sections from mice killed 16 weeks (C-D) or 55 weeks (E-H) after transplantation were stained with antibodies recognizing F4/80 (C,F) or HA-EGFP (D-E, G-H). Serial sections of spleen (C-D, F-G) show expression of HA-EGFP predominantly in the macrophages of the red pulp, a pattern that is identical to that of the F4/80 antigen. The differences in the extent of HA-EGFP staining between tissues taken at 16 weeks and at 55 weeks after transplantation represent the relatively slow kinetics of repopulation of the spleen with macrophages of donor HSC origin. Staining of sections of liver (E) and brain (H) reveal expression of HA-EGFP in Küpffer cells and microglia, respectively. Sections stained with species-matched antibody controls showed no specific staining pattern. Original magnifications: × 10, C-D, F-G; × 40, E-H.

CD68-based retroviral vectors generate expression specifically in mφs in vivo.

(A) Peritoneal cells elicited 4 days after injection with thioglycollate were analyzed by FACS. Three distinct cellular populations could be distinguished on the basis of forward- and side-scatter properties: neutrophils (blue), macrophages (red), and lymphocytes (green). These designations were confirmed by staining with cell type–specific antibodies (data not shown). By using a gate for EGFP-positive cells, which was established using samples from mice that had not received transplants, the cell-type specificity of EGFP expression could be established. The data are from cells isolated from a single mouse and are representative of data from 10 mice per retroviral construct. (B) Immunoblot analysis of tissue lysates (50 μg total protein per lane) from mice that had received HSC transplants (16 weeks after transplantation), using antibodies recognizing macrosialin (α-mCD68) or HA epitope–tagged EGFP (α-HA). Blots are from identical exposures to allow comparisons of the relative levels of HA-EGFP expression. Data are from a single mouse per retroviral construct, and are representative of results from 2 similar mice. L.N.s indicate lymph nodes. (C-H) Immunohistochemical staining of tissues from mice that had received transplants of HSCs transduced with CD68S–HA-EGFP retrovirus. Tissue sections from mice killed 16 weeks (C-D) or 55 weeks (E-H) after transplantation were stained with antibodies recognizing F4/80 (C,F) or HA-EGFP (D-E, G-H). Serial sections of spleen (C-D, F-G) show expression of HA-EGFP predominantly in the macrophages of the red pulp, a pattern that is identical to that of the F4/80 antigen. The differences in the extent of HA-EGFP staining between tissues taken at 16 weeks and at 55 weeks after transplantation represent the relatively slow kinetics of repopulation of the spleen with macrophages of donor HSC origin. Staining of sections of liver (E) and brain (H) reveal expression of HA-EGFP in Küpffer cells and microglia, respectively. Sections stained with species-matched antibody controls showed no specific staining pattern. Original magnifications: × 10, C-D, F-G; × 40, E-H.

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