Figure 5.

Designer PPR proteins induced exon skipping of CHK1 mRNA and the retardation of cell growth. (a) Exon–intron structure of CHK1 pre-mRNA. The target position was designed in exon 3 of the CHK1 gene. (b) RT-PCR analysis of exon skipping. The amount of the splicing variant with or without exon 3 (+Ex3 or −Ex3) was analyzed using Dox-induced expression of PPR^chk1a. PPR^sp6 was used as a negative control. The primer position used for RT-PCR is shown in (a). The skipping ratio was estimated by calculating the signal intensity of [−Ex3]/([+Ex3] + [−Ex3]). (c) Analysis of cell number with or without the induction of the designer PPR genes (PPR^chk1a or PPR^sp6). Cell number was analyzed using trypan blue staining with the cells two days after dox induction (N = 3). (d) Off-target RNA analysis. Two mismatched chk1a sequences located in MDM2 exon 9. The exon was confirmed using RT-PCR with Ex3–Ex12, Ex6–Ex12, and Ex7–Ex12 primer sets. Primer positions are indicated by blue triangles.