Spop knockdown in zebrafish using morpholinos results in a developmental phenotype.
(A) Multiple protein sequence alignment of SPOP from Homo sapiens and zebrafish (Danio rerio) reveals 97.3% identity between humans and zebrafish on the amino acid level. (B) Schematic representation of zebrafish SPOP mRNA. Colored boxes indicate exons. MO5 and MO7 indicate the targeted sites of the SPOP-specific morpholinos. F1 and R5 indicate the sites were the primers bind, which were used to amplify exons 1–5 for molecular validation of morpholino efficiency. (C) Image of an agarose gel used to analyze zebrafish SPOP splice variants after morpholino treatment. (D) Propidium iodide-based cell cycle analyses of cells from zebrafish embryos (24 hr) treated with or without SPOP-specific morpholino. (E) Quantification of TUNEL-positive cells in the midbrain area of zebrafish embryos (24 hpf) treated with SPOP morpholino or in combination with WT-SPOP mRNA as shown in Figure 2C. (F) Example of zebrafish embryos (48 hpf) injected with a second splice-blocking morpholino (MO7). Rescue of the effects of MO7 on zebrafish development by coinjecting WT-SPOP mRNA. (G) qPCR-based validation of differentially expressed transcripts identified by RNA-seq.