FIG. 1.

GFP target sequence amenable to silencing by specific siRNAs. A. HeLa cells were transfected with 0.2 μg of pEBS-GFP with either 1 μg of pSuper or pSuper-siGFP. Sixty hours posttransfection the cells were collected and washed with PBS. Cellular extracts were prepared for Western blot analysis using anti-GFP antibody. Fifteen micrograms of total protein (Bradford) was loaded per lane (duplicate samples). B. A549 cells were transduced with the lentivirus vectors expressing anti-GFP-siRNAs and NGFR or NGFR alone as indicated in Materials and Methods. Transduced cells were selected twice in a row by flow cytometry using anti-NGFR antibodies coupled to phycoerythrin (see Materials and Methods). The panels show the results of the second selection process, in which more than 95% of the cells were found positive for the NGFR marker. C. A549-LV-NGFR and A549-LV-siGFP were infected with a recombinant SeV-GFP (MOI = 20). Forty hours later, GFP expression was observed by microscopy under regular (left) or UV light (right; microscope Nikon Eclipse TE 300), and pictures were taken with a Nikon Coolpix digital camera 950.