An antisense morpholino oligomer targeting the exon 3 and exon 4 splice region is sufficient to completely knockdown fxn expression in zebrafish. (A) A schematic of the fxn gene with annotations showing the morpholino and primer locations. (B) After cDNA synthesis using RT-PCR, analysis of the products through gel electrophoresis revealed that morphant animals produced a shorter fxn transcript and a wild-type product was absent. (C) Sanger sequencing data revealed that the fxn morphant transcript had a complete deletion of exon 3, which encoded an in-frame premature stop codon (underlined). (D) Three-dimensional protein folding predictions suggested a highly disrupted Fxn protein structure based on the altered mRNA sequence caused by splice interference due to the fxn morpholino. The endogenous Fxn consists of three alpha helices and five beta sheets, and the altered Fxn consists of an alpha helix and an interdomain loop. The orange refers to the mitochondrial trafficking sequence, an area of the protein that has low confidence for the folding prediction because of the lack of conservation between species in this specific domain. These folding predictions strongly suggest that significant regions of the Fxn protein are abrogated in the morphants.
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