Fig. 4

A morpholino, MO[dlD-V], targeted to the intron-exon boundary responsible for the addition of the DeltaD terminal valine residue disrupts splicing and selectively removes the PDZ domain binding site for up to 72 hours of development. (A) The genomic structure of the zebrafish DeltaD gene surrounding the PDZ domain binding site, indicating the amino acids at the boundaries of each exon, the MO[dlDV] morpholino annealing site, and primer binding sites used to monitor the effects on splicing by RT-PCR in (B). (B) Effects on deltaD mRNA splicing, monitored by RTPCR, in embryos injected with MO[dlD-V] at the one-cell stage and left to develop until the shield stage (6 hpf). Total RNA was extracted from 20 embryos for each dose, reverse transcribed and amplified by PCR. PCR products were cloned and fully sequenced. Uninjected embryos produced a 370 bp band that corresponds to the correctly spliced transcript, coding for a protein that ends –ATEV. The amount of correctly spliced product decreases as the amount of injected morpholino increases; at a dose of 5 ng or more per embryo, no correctly spliced product is observable. Products at 469 and 269 bp were mis-spliced as shown and correspond to proteins lacking the terminal ATEV. The band at ˜330 bp was not characterized but probably represents the use of a cryptic splice donor site. The 546 bp band may result from small amounts of contaminating genomic DNA in the extracted RNA. The separate small panel below shows RT-PCR analysis of the same cDNA using oligonucleotides targeted to an upstream region of the deltaD transcript (nucleotides 389-923) that is unaffected by MO[dlD-V]. The total quantity of deltaD mRNA is not significantly altered by the MO injections. (C) Time course of the effect. Embryos were injected and allowed to develop until the indicated stages, and RT-PCR was performed as in B.