Femtojet micromanipulator 5171

Supplementary Table S1 reports the sequences of the morpholinos applied in the experimental procedures. We prepared the injection solution by diluting the morpholino stock solution (1 mM) with 10% phenol red and water to obtain the desired final morpholino concentration. We injected 4 nl of the final solution (0.3 pmol/embryo) into 1–2 cell stage embryos by an Eppendorf FemtoJet Micromanipulator 5171. Embryos were then transferred in fish water containing 0.003% 1-Phenyl-2-thiourea at 28°C to stop the pigmentation process, and development was evaluated at 24 and 48 hpf. Images were captured using Zeiss Axio Zoom V16 equipped with Zeiss Axiocam 503 color digital camera and processed using Zen Pro software from Zeiss.

Injections were carried out on one-cell-stage embryos (with Eppendorf FemtoJet Micromanipulator 5171); the dye tracer phenol-red was co-injected as a control. After microinjection, embryos were incubated in fish water at 28 °C. Embryo development was evaluated at 24 h post fertilization (hpf), 48 hpf, and 72 hpf.

Injections were carried out on one/two-cell-stage embryos (with Eppendorf FemtoJet Micromanipulator 5171); the dye tracer phenol-red was co-injected as a control. To repress coasy mRNA translation we designed a splicing-inhibiting morpholino oligo (coasy-MO, Table S2); as a negative control we injected a standard control morpholino oligo (ST-MO) (Gene Tools) which targets a human beta-globin intron (Supplementary Table S2). Morpholinos were injected in Danieau buffer (pH 7.6) as previously described61 (link). After microinjection, embryos were incubated in egg water supplemented with 0.003% PTU at 28 °C to prevent pigmentation processes. Embryo development was evaluated at 24 and 48 hpf. RT-PCR experiments were performed on RNA extracted from coasy-MO- or ST-MO-injected and wild type embryos at 24 hpf and 48 hpf, with specific primers (P5 and P6, Supplementary Table S2). Control RT-PCR amplification on the same RNAs was carried out with actin beta 1 primers (A1 and A2, Supplementary Table S2). When indicated, the comparison between morphants and controls was performed by selecting morphants with the mildest phenotype (embryos injected with 1.2 pmol of CoA-MO) and no evident delay in development (head-trunk angle).