Fig. 4

Rescue morpholinos cause restoration of full-length protein without detectable changes in mRNA. (A) Phenotypic rescue by morpholinos i3 and i3.5 is accompanied by the restoration of the WT protein. Forty-eight hours after fertilization, lysates from embryos of the indicated genotype were run on a SDS/6% PAGE gel and blotted for zebrafish Atp7a by using an antibody generated to the zebrafish protein. β-catenin was used as a loading control. Morpholino injection restored protein to 30–45% of WT levels in this experiment. (B) qRT-PCR using primers specific for WT and mutant atp7a transcript were used to measure the amounts of each in single embryos. Normalization of WT transcript was to cal ^+/+ embryos and mutant transcript to cal^-/- embryos. Error bars indicate one standard deviation around the mean. (C) Polyacrylamide gel of RT-PCR products reveals a band containing 800 bp of intronic sequence caused by morpholino injection. This band was sequenced and represents use of a cryptic 32 splice site located within the intron. Also seen are mutant and WT transcripts in uninjected and rescued embryos. A nonspecific band was present in both uninjected and injected mutant embryos (*).