Figure 6

GAK-MO1 specifically disrupts zGAK splicing. (A) RT-PCR analysis of total RNA extracted from uninjected and GAK-MO1 morphant embryos. From normally spliced zGAK mRNA, a reaction with primers complementary to exon 2 and 4 (2/4) would yield a band of 178 bp, and a reaction with primers complementary to exon 18 and 20 (18/20) would yield a band of 275 bp. While the 275 bp band seemed unaffected by GAK-MO1 injection, the level of 178 bp band was reduced and a new band of 118 bp appeared in GAK morphants. Sequence analysis of this 118 bp band showed that exon 2 was spliced into exon 4, resulting in an in-frame deletion in the kinase domain. (B) The target sequences of GAK-MO1 and GAK-MO1C were placed in front of mCherry and cloned into pCS2. In vitro transcribed mRNAs were co-injected with morpholino into one-cell stage embryos. Bright field and fluorescent images of injected embryos at 6 hpf are shown. Scale Bar, 200 μm.