Figure 1.

Transient knockdown of cln3 in zebrafish larvae.

(A) Schematic illustration of the cln3 gene showing the target sites for the translation-blocking (TB) and the two splice-blocking (SB; i2e3-MO and e2i2-MO) morpholinos (MOs) used in this study. Black arrows show the binding sites for the PCR primers used for SB-MO validation. (B, C) Microinjection of 8 ng SB- or TB-blocking MOs did not lead to any obvious morphological phenotype, and larvae displayed a normal development. The number of larvae analyzed at the indicated developmental time points: uninjected, 177; Ctrl-MO, 317; e2i2-MO, 250; i2e3-MO, 147; and TB-MO, 130. (D) Validation of MO efficiency by PCR amplification of target regions in cDNA of the cln3 morphants in comparison with uninjected (UN) and control morpholino–injected larvae. (E) Schematic representation of transcribed sequences and resulting translation products after treatment with the SB-blocking morpholinos. Microinjection of i2e3-MO resulted in exon 3 skipping, leading to an early stop codon. In contrast, e2i2-MO treatment led to two different splicing events, both also resulting in a premature stop codon. Excluded exons are marked with a red cross, included intron in green, and new amino acid sequence in light blue.