Efficacy of splice-blocking morpholino oligonucletides (MO). (A) The zebrafish manf gene includes four exons. The translation start site is located in the exon 1. The targeting sites of two MOs are in red (MOsp1 and MOsp2). The primer pair used for RT-PCR is shown in blue (F1 and F2). Transcripts of 3-dpf uninjected, control and manf MOsp embryos were used to determine the MO efficiency. Band 1 in the gel electrophoresis indicates the regular manf transcript. Bands 2, 3 and 4 represent the aberrant splicing variants. All PCR products were verified by sequencing. qPCR was performed to quantify the mRNA expression level (*p<0.05, n=3, Student′s t-test). (B) The western blot analysis of MANF expression in the wild type, control MO and manf MOsp- injected groups at 3 dpf. The blot of anti-β-actin was used as an internal control. (C) Bright-field images of control, manf MOsp morphants and manf RNA rescue larvae at 3 dpf. (D) MANF immunostaining on 5-dpf ctrl MO, manf MOsp injected embryos. A higher magnification of a posterior neuromast is shown by double-labeling with MANF shown in red and acetylated tubulin shown in green. The uninjected larvae stained using MANF antibody preabsorbed with MANF blocking peptide to examine the specificity of MANF antibody. MANF positive cells were labeled in bright red. an, anterior neuromast; L, liver; pn, posterior neuromast. Scale bar=100 μm.
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