Inhibition of zebrafish fgf8 Pre-mRNA splicing with morpholino oligos: A quantifiable method for gene knockdown

摘要

Antisense morpholino oligonucleotides (MO) have been used successfully in zebrafish and Xenopus to knock down gene function by gene-specific inhibition of mRNA translation (Ekker, 2000). In addition to their ability to block cytosolic processes, MO can enter the nucleus (Partridge et al., 1996) and have been shown to be effective inhibitors of pre-mRNA splicing in mammalian tissue-culture cell lines (Schmajuk et aL, 1999). We show here that MO efficiently block pre-mRNA splicing in zebrafish embryos. Splice-blocking MO have the advantages that the efficacy of gene knockdown can be quantiffed without the use of antibodies, and that they specifically target zygotic, and not maternal, transcripts. We targeted the fgf8 gene (Furthauer et al., 1997; Reifers et aL, 1998) with splice-blocking MO. An ENU induced mutation in fgf8, acerebellar (ace; referred to here as fgf8~(it282)), has previously been described (Reifers et al., 1998). fgf8~(it282) is a splice donor mutation that results in the production of an aberrantly spliced mRNA (Reifers et al., 1998). Hence, blocking the same splicing event using MO should result in a phenotype similar to fgf8~(it282). We determined the fgf8 exon/intron structure and designed two 25-mer MO complementary to the exon 2 and exon 3 splice donor sites (designated E2I2 and E3I3, respectively; Fig. la). Both MO span the exon/intron junction, including the most conserved residues of the splice donor consensus sequence (Fig. 1 legend).