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Fig. 4 S10
Validation of transcriptional changes using splice morpholinos to target ptgs1, ptgs2a, ptger2a, and ptger4a.
Embryos were microinjected with morpholino and allowed develop until 24 hpf, where their RNA was then harvested for RT-PCR. Schematics illustrate each genetic locus, with the location targeted by the MO(s) and primers used for transcriptional analysis on 2% agarose gels. (A) ptgs1 knockdown targeted the exon one splice donor site, and caused inclusion of the 91 bp intron 1–2 that encodes a premature stop codon in ptgs1 transcripts in morpholino-injected zebrafish. (B) ptgs2a knockdown targeted the exon three splice donor site, and caused inclusion of the 111 bp intron 3–4 that encodes a premature stop codon in ptgs2a transcripts in morpholino-injected zebrafish. (C) ptger2a knockdown as well as (D) ptger4a knockdown targeted the exon one splice donor and exon two splice acceptor site in ptger2a and ptger4a, respectively. Green asterisks in these gel images indicate the complete absence of the WT PCR product in the morphant samples. We were unable to PCR across the entire intron of either gene due to their sizes. However, in contrast to WT samples, we detected ptger2a and ptger4a transcripts that included intronic sequence in the respective morphant samples. In each case, inclusion of the intron is predicted to encode a truncated peptide. (E) Embryos were microinjected at the 1-cell stage with either ptger2a MO splice (MO3/MO4 combination) or the ptger4a MO splice (MO3/MO4 combination) and allowed to developed until 24 hpf, where WISH was used to stain for the DL (slc12a3) segment. Black bars indicate segment gene expression domain. Red scale bar, 70 µm. (F) The DL domain was measured in WT controls, ptger2a MO splice (MO3/MO4 combination) or the ptger4a MO splice (MO3/MO4 combination) in microns (n = 5 for each control and experimental group). Data are represented as ± SD, ANOVA used to compare samples, *p<0.01, where n.s. indicates not significant.