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. 2017 Oct 10;11:544. doi: 10.3389/fnins.2017.00544

Figure 1.

Figure 1

Trans-splicing in HEK293 cells. (A) Schematic of PTM targeting. The PTM is driven by the CMV promoter and contains 21 CAG repeats within HTT exon 1. The PTM contains intronic splice enhancers (ISE), a branch point (BP), and a “tether” that targets the PTM to specific sequences within the HTT or mini-gene pre-mRNA. The HTT mini-gene is driven by the CMV promoter and consists of exon1-2-3 with intervening introns and a polyA signal. (B) Relative position of the PTM tethering elements for the PTMs. T7 and T8 target the 5′ end of intron 1; T4, T9, and T11 target the 3′ end of intron 1. (C) Mini-gene trans-splicing in HEK293 cells. RT-PCR of total RNA isolated from HEK293 cells transiently transfected with the indicated PTMs and the HTT mini-gene. PTMs contained a splicing branch point (+branch point) or had the branch point removed with flanking restriction sites (−branch point). trans-splicing RNA, PTM expression, mini-gene expression, and endogenous GAPDH expression is shown. (D) Endogenous trans-splicing in HEK293 cells. RT-PCR of total RNA isolated from HEK293 cells transiently transfected with the indicated PTMs. trans-splicing RNA, PTM expression, and endogenous GAPDH expression is shown.