Figure 1
Transduction of human cell lines HTB-11 and U937 as well as primary hMDM by lentiviral vectors HR-Hutat2 expressing anti-HIV-1 Hutat2:Fc and EGFP. HTB-11 cells (5 × 105) were transduced in a T25 flask in the presence of 8 μg/mL polybrene for 2 h (multiplicity of infection, MOI = 10). U937 cells (1 × 105) were transduced twice by spin-infection at 1,500 × g for 90 minutes (MOI = 100). Human MDM were infected with HR-Hutat2 vectors (MOI = 50 or MOI = 10) for 1.5 h on days 7 and 8 in vitro (DIV 7 and DIV 8), respectively. The transduction efficiencies were evaluated by calculating the percentage of GFP+ cells from five randomly selected microscopic fields under a fluorescence microscope on day 3 post-transduction for HTB-11, as well as on day 8 post-transduction for U937 and hMDM, respectively. HTB-11, Non-transduced HTB-11 cells; HTB-Hutat2, HR-Hutat2 transduced HTB-11 cells; U937, Non-transduced U937 cells; U937-Hutat2, HR-Hutat2 transduced U937 cells; EGFP, Enhanced green fluorescent protein; hMDM-Hutat2 MOI = 50, HR-Hutat2 transduced hMDM at the MOI of 50; hMDM-Hutat2 MOI = 10, HR-Hutat2 transduced hMDM at the MOI of 10. (A) Expression of EGFP in HR-Hutat2 transduced HTB-11 and U937 cells. (B) Co-location of the Hutat2:Fc and EGFP expression in HR-Hutat2 transduced HTB-11. Nuclei were counterstained with DAPI (blue). The Hutat2:Fc proteins (red) were expressed in the cytoplasm while EGFP proteins (green) were expressed both in the nuclei and cytoplasm. (C) Expression of EGFP in transduced hMDM. Fluorescently-labeled cells were visualized with an epi-microscope (Nikon Eclipse TE2000-U) using a numerical aperture lens (0.30 or 0.45) and a digital camera attachment. The pictures were overlaid using ImageJ software (Version 1.48, National Institutes of Health, USA). Data represent means ± s.e.m. of three independent experiments. Scale bar = 100 μm.