Partial rescue of Tbx5a-MO4 morphant phenotype by (− 3) and (− 7) binding site mutations. (A) Embryos injected with Tbx5a-MO4 display a range of tbx5a loss of function phenotypes including pectoral fin malformation or absence and severe cardiac edema. Black arrow denotes cardiac edema, black arrowheads denote pectoral fin loss, red arrowheads denote pectoral fin defects. (PFA + E, pectoral fins absent with edema, PFD + E, pectoral fin defect with edema, PFD, pectoral fin defect only) (B) Two sgRNAs, tbx5adeMO1 and tbx5adeMO2 targeting the tbx5a-MO4 binding site (MO sequence shown above in purple, PAM sites are highlighted in magenta, coding sequence is highlighted in green. Tbx5adeMO2 overlaps a RsaI restriction enzyme site used for genotyping. (C) Sequence confirmation of the (− 3) and (− 7) deletion alleles. (D) Titration of Tbx5a-MO4 in wild type (TFL) embryos. Numbers indicate percentages of embryos displaying designated morphant phenotypes. WT is wild-type, E is edema only. (E) Suppression of cardiac and/or pectoral fin phenotypes by the (− 3) and (− 7) binding site deletions at different doses of MO. Adults heterozygous for either the (− 3) or (− 7) deletion were outcrossed and embryos were injected with either 2 ng or 8 ng of tbx5a-MO4. (F) Tbx5a-MO4 is able to at least partly block the translation of mRNAs containing (− 3) and (− 7) binding site mutations. In vitro transcribed mRNAs from each eGFP construct was injected along with mRFP mRNA as a control, and half of the mRNA-injected embryos were then injected with tbx5a-MO4. At 1dpf, embryos displaying similar levels of mRFP expression were photographed. T3, T3 transcription start site, Xβg 5′ UTR and Xβg 3′ UTR, Xenopus β-globin 5′ and 3′ untranslated regions, respectively.
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