Model system to optimize trans-splicing efficiency. (A) Schematic diagram showing a LacZ PTM binding to a chimeric LacZ-ΔF508-CFTR target at the pre-mRNA level. (B) Trans-splicing efficiency by 5′ exon replacement is increased by binding to more than one splice site. The panels above the histogram shows the location of four different PTM-binding domains (CF-PTM42, CF-PTM43, CF-PTM44, CF-PTM45) targeted to different regions of a defective LacZ-ΔF508 target (LacZCF10m). CF-PTM45 and CF-PTM45(+) are identical, except that the latter has a poly(A) signal and the former does not. The histogram below the five panels compares the repair efficiency of the five PTMs using an in-solution β-galactosidase assay. Transfections, preparation of cell lysates, and assessment of activity was as described previously (Puttaraju et al. 2001). Values are the mean of two independent experiments (± standard error) and are expressed as units of β-galactosidase activity per milligram of total protein. PTMs and the target are cloned in pc3.1DNA(−). The vector-alone sample refers to plasmid pc3.1DNA(−) that does not contain either a PTM or a target. Each transfection was performed with 2 μg of target plasmid and 2 μg of PTM plasmid. All samples contained the same amount of plasmid DNA; PTM-alone and target-alone samples were balanced to 4 μg with vector pc3.1DNA(−). (BD) Binding domain; (SS) splice site; (+), with bGH poly(A) signal; (−), without bGH polyA signal.
