Morpholino-Mediated Knockdown and Rescue in Zebrafish

Morpholino oligonucleotides (Gene Tools) were re-suspended in water to a stock concentration of 0.5 mM. Final morpholino concentrations were injected into 1–2 cell stage embryos in 3 nl amounts and embryos were maintained at 28.5°C. The DUOX morpholino was used as published (Yoo et al., 2012 (link)). The vimentin and Larp6 splice blocking morpholinos used were as follows:
vim MO1 targeting Exon 1- Intron 1 (80 µm):
5’-GTAATAGTGCCAGAACAGACCTTCTC-3’
vim MO2 targeting Intron 1-Exon 2 (110 µm):
5’-TCTTGAAGTCTGGAAATGAGATGCA-3’
larp6 MO targeting Exon 2-Intron 2 (80 µm):
5’-GGGTGTTGGTCTTACCTTCTTGAA-3’ control MO
5’-CCTCTTACCTCAGTTACAATTTATA-3’
To analyze knockdowns, RT-PCR was performed on RNA isolated from single larvae at 2 dpf. The following primers were used to assess knockdown:
Vim MO 1 and 2:
F-5’-ACCGGGGAAAAGAGCAAAGT-3’ R-5’-CGAGCCAGAGAGGCGTTATC-3’
Larp6 MO:
F-5’-CAAACTGGGCTTCGTCAGTG-3’ R-5’-TCCGTTGTTGGAATCTCCGC-3’
Rescue experiments were performed using zebrafish vim RNA. In short, a gene block (IDT) for vimentin was used as the template. The sequence was cloned into the PCS2 +vector from which in vitro transcription was performed (Ambion). RNA was purified with the miRvana RNA purification kit (Ambion). RNA was injected at 125 ng/µl at the 1–2 cell stage in 3 nl amounts.