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Fig. 4. vps33b morpholino targeting and altered splice product from IE18.
(A,B) Schematics showing exons 4 and 5 and intervening intron 4 (A), and exons
17 and 18 and intervening intron 17 (B) of the vps33b gene. The
sequences targeted by the IE5 and IE18 morpholinos are depicted in green and
red, respectively. The wild-type and morpholino-targeted mRNA transcripts are
depicted below the genomic region. Targeting by the IE18 morpholino generates
a novel cDNA that uses a cryptic splice acceptor site within exon 18. The site
of splicing induced by the IE5 morpholino could not be determined. (C,D)
RT-PCR shows altered vps33b expression induced by both the IE5 and
IE18 morpholinos. For these experiments, cDNA derived from 24 hpf wild-type
and morpholino-injected larvae was amplified using primers flanking exons 5 or
18. There is decreased amplification of the wild-type vps33b fragment
relative to tbp, quantified below, in both sets of
morpholino-injected larvae. Wild-type (blue) and truncated (red) fragments are
amplified from IE18 morpholino-injected larvae. (E) The shorter cDNA fragment
amplified from IE18 morpholino injected larvae has an in-frame deletion of 15
amino acids includes R438 (red), which is mutated in some individuals with
ARC.
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