(a) Fsd1 atg-morpholino efficiency was validated by western blot. (b) Fsd1 splice-morpholino efficiency was validated by RT-PCR and sequencing. The PCR product was 292 bp in control embryos and 194 bp in fsd1 sMO injected embryos. The sequencing result of the PCR product below showed deletion of exon 2 (98 bp) in fsd1 sMO injected embryos. Purple, orange and blue colors mark exon 1, 2 and 3, respectively. The red box indicates the fsd1 sMO targeted sequence. (c) Fsd1 MOs caused left-right asymmetry defects. The cmlc probe was used to label the heart tube in the whole-mount in situ hybridization at 26 hpf. Scale bar, 100 μm. n, number of fishes. (d) The target sequence of fsd1 aMO and sequence of aMO-resistant form of zebrafish fsd1 mRNA (fsd1 re-zmRNA). (e, f) Zebrafish fsd1 mRNA partially rescued the LR asymmetry defects induced by fsd1 aMO marked by spaw or cmlc. (g) Schematic diagram illustrates the difference between fsd1 re-zmRNA and fsd1-CC re-zmRNA. (h, i) Zebrafish fsd1-CC re-zmRNA could not rescue the LR asymmetry defects induced by fsd1 aMO marked by spaw or cmlc. Data are presented as mean ± s.d. of at least three independent experiments. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001. n, number of fishes.
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