CFTR model constructs and illustration of trans-splicing by 5′ exon replacement. (A) Detailed structure of a 5′ER PTM (CFTR-PTM11), and a mini-gene target (CFTR-T11). The PTM lacks a poly(A) signal and the target lacks a methionine initiator codon. Modified codons are engineered in exons 9 and 10 of the PTM, and exons 11 and 12 of the target to aid in distinguishing trans-spliced products from cis-spliced or endogenous CFTR products. The position of the three oligonucleotide primers (CF1, CF93, and CF111) used in RT–PCR experiments are indicated by arrows. The diagram shows the PTM binding to the 5′ splice site of intron 10 of the minigene target at the pre-mRNA level. (B) Demonstration of trans-splicing by RT–PCR. Cells were transfected with either PTM plus target, or PTM, target, or vector (pc3.1DNA) alone, or PTM plus target, but without the reverse transcription step (four different negative controls). Cis-spliced (ΔF508) products were detected with primers CF1 and CF111 (right arrow), and trans-spliced products with primers CF93 and CF111 (left arrow). (BD) Binding domain.
