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. 2022 Feb 2;23(3):1732. doi: 10.3390/ijms23031732

Figure 1.

Figure 1

Fluorescence-reporter based screening for most functional RTMs. (A) Trans-splicing was evaluated either in the presence of a mini-gene target for screening of highly functional BDs, or in keratinocytes, where successful trans-splicing to the endogenous COL7A1 target pre-mRNA was investigated. In the presence of a functional BD, specific trans-splicing results in a single mRNA transcript encoding either DsRed (transfection control) and full-length AcGFP in the mini-gene reporter setting, or a 5′AcGFP/COL7A1 exons 65–118 hybrid when splicing to the endogenous transcript. (B,C) HEK293FT cells co-transfected with the mini-gene target and individual RTMrs (i,ii) and positive control (iii). Hybrid DsRed and AcGFP expression is shown by fluorescence microscopy and flow cytometry. AcGFP/DsRed ratios were calculated from the amount of GFP-positive cells (sector B2 + B4)/total amount of transfected cells (sector B1 + B2 + B4), and is given in percent (%). RTMrA showed an AcGFP/DsRed ratio of 97.65%; RTM B, 97.03%. 100% of cells transfected with the positive control expressed both DsRed and acGFP. (D,E) In keratinocytes, correct trans-splicing to COL7A1 was confirmed by RT-PCR using a GFP forward and a COL7A1 exon 67 reverse primer. The 335 bp amplification product was verified to be the GFP-COL7A1 fusion by Sanger sequencing.