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. 2016 Jun 6;6:27315. doi: 10.1038/srep27315

Figure 3. Synthetic SINEUP-cox7B rescues MLS phenotype in medakafish.

Figure 3

(a) Structure of the medakafish cox7B transcript (exon/intron gene organization is displayed) and binding sites of cox7B-MO and SINEUP-cox7B. The cox7B-MO was designed against the acceptor splice site of exon 2 and its injection causes the skipping of exon 2. The BD of SINEUP-cox7B is designed against exon 1 in position −40 in the 5′-UTR (before ATG) to position +32 in the CDS. (b) Representative st30 embryos injected with control-MO, SINEUP-cox7B, cox7B-MO with or without SINEUP-cox7B or SINEUP-SCR. Scale bars 100 μm. SINEUPs rescue microphthalmia and microcephaly in 50% of cox7B-morphants (c). (n ≥ 300 embryos/conditions, ***p < 0,001, One-way ANOVA; Error bars are SEM). (d) RT-PCR of cox7B transcripts on total RNA extracted from embryos injected with control-MO, cox7B-MO and embryos co-injected with cox7B-MO and cox7B-SINEUP. Black arrow indicates WT mRNA whereas the red arrow indicates the mutated mRNA generated by cox7B-MO-driven aberrant splicing. (e) Real Time PCR on total RNA using specific primers to amplify WT cox7B mRNA. (n = 3 independent biological samples, ***p < 0,001, **p < 0,01, two-tailed Student’s t-test; Error bars are SEM). (f) WB analysis of cox-IV in st30 embryos injected with control-MO and cox7B-MO with or without SINEUP-cox7B. (g) Quantification of cox-IV protein levels (n = 3, **p < 0,01 one-tailed Student’s t-test; Error bars are SEM).