Figure 1
A fluorescence-based screening system for RTM selection. (a) The screening system is built up by a COL7A1-MG, containing the 5′ portion of GFP and the selected COL7A1 pre-mRNA region of intron 46/exon 47, and an RTM, carrying a randomly cloned BD, splicing elements for efficient trans-splicing, the 3′ portion of GFP, an IRES and dsRED. Co-expression of both screening molecules in HEK293 cells leads to the restoration and expression of GFP upon accurate trans-splicing. (b – d) The transfection of the RTM library into HEK293 cells, stably expressing the COL7A1-MG (COL7A1-MG-CL) and the red fluorescence reporter molecule mRuby, results in a diverse expression of the red and green reporter molecules. This is dependent on the levels of RTM and COL7A1-MG expression and more importantly on the functionality of the introduced RTM. A high GFP expression indicates trans-splicing through highly efficient RTMs in the cell, detected by microscopic (b, c) and FACS analysis (d). Overlay of the different fluorochromes in trans-spliced cells results in a yellow to orange colour, depending on the strength of green GFP expression together with red target/RTM expression (c). The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue fluorescence). Scale bars: 50 μm (b); 20 μm (c). PPT, polypyrimidine tract.
