Figure 2

Effects of insertion of an aberrantly spliced intron and SSOs on adenovirus vector. A. Expression of luciferase in HeLa cells infected by adenovirus vectors containing wt (AdenoLucWT) or aberrantly spliced (AdenoLuc705 and AdenoLuc654+705) intron in the presence of ONINV (100 nM, open columns) or a mixture of ON654 and ON705 (100 nM, black columns) at 24 h postinfection. The luciferase activities from both series (ONINV and ON654+ON705 transfected cells) were normalized to the activities obtained for AdenoLucWT infected cells, which were defined as 100%, and are represented on the vertical axis. Error bars represent standard deviations; experiments were repeated twice with similar results. B. Schematic presentation of luciferase reporter with recombinant intron and the RT-PCR analysis of splicing products. SF, splice forward primer; SR, splice reverse primer. Positions of thalassemic mutations and common cryptic splice site (CSS) activated by these mutations are shown by open arrows. The aberrant and correct splicing possibilities and mRNAs resulting from these processes are shown, and the length of RT-PCR product is indicated for each splicing variant. C. Agarose-gel electrophoresis analysis of RT-PCR products. SSOs are indicated above the panel; the sizes of expected DNA fragments are shown at the left.