Antisense morpholino oligonucleotide knockdown

Antisense MO were injected into the yolk of one-to-two cell stage embryos. Dok-7 embryos were injected with 15 ng of Dok-7 MO. MuSK embryos were injected with 5 ng of zMuSK MO. In addition, control MOs were injected at the same concentration for each injection experiment. MOs were purchased from Gene Tools LLC (Pilomath, OR). A splice-blocking MO was used to target Dok-7 transcripts at exon 2: 5′-ATTTATAGGATTTACCTGCTACCGG. This splice-blocking MO causes skipping of the exon and premature translation termination through targeting of the splice donor site of exon 2 (21). For MuSK experiments, a MO targeting the unplugged splice transcript variant 1 (unplugged/SV1) was used (19,22): 5′-GTAGAGGATTACCGTATTGCCGTT. This causes skipping of exon 2, frameshift and a premature stop codon after 24 cryptic residues. The Gene Tools standard control MO (5′-CCTCTTACCTCAGTTACAATTTATA-3′) targeting a human beta-haemoglobin gene was used as a negative control. MOs were suspended in 1× Danieau buffer (58 mm NaCl, 0.7 mm KCl, 0.4 mm MgSO4, 0.6 mm Ca(NO₃)₂, 5 mm HEPES; pH 7.6) with phenol red as an injection indicator. At least two independent MO injection experiments were performed for each MO and 200–800 injected embryos were evaluated for each treatment regimen.