Figure 3
Validation of FACS analysis by fluorescence-based microscopy. Co-transfection of COL7A1-MG together with high-efficiency RTM (RTM28) (a) into HEK293 cells resulted in the expression of GFP upon accurate 3′ RNA trans-splicing. Similar results were achieved after RTM28 (b) transfection into the COL7A1-MG-CL-expressing cell line. GFP expression was undetectable in HEK293 cells exclusively transfected with RTM28 (a, upper panel) and the COL7A1-MG-CL-expressing cell line (b, upper panel). HEK293 cells transfected with RTM28 containing dsRED and the 3′ half of GFP (a, upper panel) and stably expressing COL7A1-MG cells (COL7A1-MG-CL) containing mRuby (b, upper panel) showed red fluorescence upon reporter molecule expression. Overlay of the different flurochromes in trans-spliced cells resulted in a yellow to orange colour, depending on the strength of the merged green GFP expression and the red target expression (a, b, lower panel). Scale bars: 50 μm.
