Figure 2.

Sequential construction of a double trans-splicing RTM. Since 3′ and 5′ trans-splicing have to work in synchrony, we believe that screening for the most efficient BDs for double trans-splicing should be done in the context of a double trans-splicing RTM. A random 3′ BD library targeting COL17A1 intron 51–exon 52 was constructed and cloned into the double trans-splicing RTM vector (version 1) harboring the initial working 5′ BD identified using the method of Wally et al. (A). To further improve the efficiency of the double trans-splicing RTM (version 2), a new 5′ BD screen was performed using the efficient 3′ BD identified in the 3′ BD screen (A) and a variable 5′ BD specific for the COL17A1 exon/intron 52 target region (B).