Fig. S2

Pk1b splice-blocking morpholinos alter pk1b splicing. (A) Genomic organization of zebrafish pk1b; exons are numbered. Locations of the splice morpholinos Pk1b-MO-SA-I3E4 and Pk1b-MO-SD-E6I6, and locations of primers used to amplify the regions surrounding exon 4 (P1/P2) and exon 6 (P3/P4) are shown. As in Supplemental Fig. 1A, the region encoding the PET domain is colored red, and the region encoding the LIM domains is colored green. (B) RT–PCR analysis of pk1b mRNA in control uninjected and Pk1b-splice-MO-injected embryos. PCR primer combinations are shown above lanes. The ladder in the left lane includes bands of 1018 bp, 517 bp, and 506 bp. The region surrounding exon 4 was amplified from control embryos (cont) using P1 and P2, generating a PCR product of the expected size (582 bp). Amplification of the region surrounding exon 4 from Pk1b-MO-SA-I3E4-injected embryos (I3E4) generated two different splice variants (arrows, I3E4 lane). In one variant, the PCR product increased by the predicted size of intron 3 (515 bp). In the other variant, the PCR product increased in size, but by less than the predicted size of intron 3, suggesting the existence of a cryptic splice site within intron 3. The region surrounding exon 6 was amplified from control embryos (cont) using P3 and P4, generating a PCR product of the expected size (476 bp). Amplification of the region surrounding exon 6 from Pk1b-MO-SD-E6I6-injected embryos (E6I6) generated two different splice variants (arrows, E6I6 lane). In one variant, the PCR product increased by the predicted size of intron 6 (438 bp). In the other variant, the PCR product increased, but by less than the predicted size of intron 6, suggesting the existence of a cryptic splice site within intron 6. Overall, these data suggest that each splice-blocking morpholino blocks normal splicing, resulting in inappropriate inclusion of all or part of the adjacent intron. Aberrant translation of either intron 3 or intron 6 would result in a truncated protein due to a stop codon 5–6 amino acids into each intron. These results confirm the efficacy and specificity of both Pk1b-splice-blocking morpholinos (see Materials and methods for primer sequences).