Fig. 3. fgf24 splice-blocking morpholino oligonucleotides knock down functional fgf24 mRNA. (A) Genomic structure of the fgf24 gene. Translation initiation and termination codons are indicated. Exons are shown as boxes and intron sizes are not to scale. Splice donor site targeted by the fgf24-E3I3 morpholino oligo is shown. The colored line indicates the major splice variant observed following fgf24-E3I3 injection. Primers used for RT-PCR analysis in B are shown as arrows. (B) In addition to wild type (upper band in 5 ng lane), RT-PCR analysis detects two splice variants (arrows). (C) cDNA sequence comparison reveals that the major splice variant (bottom band in B) caused by fgf24-E3I3 results from aberrant splicing to an upstream cryptic slice donor site (underlined in wild-type sequence) that is present in exon 3. The sequences derived from exon 4 in the aberrantly spliced form are italicized. Note that use of the cryptic splice site results in a coding frame shift. (D) Position of the fgf24 antisense RNA probe (bold horizontal line) that was used for RNase protection assays in E is indicated relative to the wild-type fgf24 mRNA splice junctions (vertical lines). (E) The amount of wild-type fgf24 mRNA in MO injected and control embryos was determined by RNase protection, using odc levels as an internal control. The amount of MO injected per embryo is indicated above lane. (F) Relative levels of wild-type fgf24 mRNA in E was determined after amounts were normalized using the odc control.

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