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. 2014 Apr 8;22(6):1176–1187. doi: 10.1038/mt.2014.35

Figure 2.

Figure 2

The titin and dysferlin minigenes are trans-spliced in vitro. (a) RT-PCR performed 48 hours after transfection on HER911 cells using specific oligonucleotides for titin trans-splicing (see Figure 1 and Supplementary Table S1). The presence of the minigene and pre-trans-splicing molecule (PTM) transcripts were detected in the appropriated samples using the 1–2 and 3–4 polymerase chain reaction (PCR), respectively. The presence of a trans-spliced RNA was observed for all the minigene:PTM(s) (Mex6, upper panel or Mex4-6, lower panel) cotransfections using the 1–4 or 1′-4 PCR. A control (0:12) was performed with PTM only at the quantity corresponding to the 1:12 ratio and showed no corresponding trans-splicing product as expected. (b) RT-PCR performed 48 hours after transfection on HER911 cells using specific oligonucleotides for dysferlin TS (see Figure 1 and Supplementary Table S1). The presence of a trans-spliced RNA was observed (arrow) in the case of minigene:PTM dysf cotransfection using the a-d PCR. A control (0:9) was performed with minigene only and PTM without binding domain (BD) showed no corresponding trans-splicing product as expected. (c) The presence of the FINmaj mutation (in red) on the minigene product (left panel) was verified by direct sequencing using the V5R oligonucleotide (Supplementary Table S1). The trans-spliced product was verified by direct sequencing using the Flag oligonucleotide. The resulting sequence showed the correct WT hMex6 sequence (in red) both for the PTM Mex6 and the PTM Mex4-6 experiment (middle and right panels). Black arrows indicate the orientation of the coding strand. (d) Western blot (WB) performed on the cellular extracts transfected with titin PTM, showed the presence of the titin miniprotein at 69 kDa in red (V5 epitope), and the presence of the trans-spliced product in green (Flag epitope) from all ratios of transfection. A-actin was used as control. Quantification of trans-spliced protein/miniprotein is represented in arbitrary unit (AU). (e) WB was performed on HER911 extracts cotransfected with the dysferlin minigene and the PTM Dysf. Labeling using an anti-V5 antibody showed the translation of the minigene into a 32 kDa protein in the corresponding samples (in red). Trans-spliced product was detected using the Flag antibody with all minigene:PTM ratios (in green, arrow). A-actin was used as a loading control. Quantification of trans-spliced protein/miniprotein is represented in AU. Images were cropped to focus on the proteins of interest. RT-PCR, reverse transcription-PCR.