Mmessage t7 kit

The coding region of her9 was amplified by RT-PCR and the cDNA was cloned into a pGEMT-easy plasmid (Promega, Madison, WI). Capped mRNA was synthesized using the mMessage T7 kit (Ambion, Austin, TX) according to the manufacturer's instructions and purified by phenol–chloroform extraction and ethanol precipitation. The mRNA was injected at a volume of 4.18 nl/embryo (1.5 ng) in buffered solution with 0.025% Dextran red into the yolk of 1-cell stage zebrafish.

We selected one 23 bp sgRNA to target the OfAbd-A genome locus on the third exon of the common region in the four spliced variants, and two 23 bp sgRNAs targeting the OfUbx genome locus in exon 1 of the common region for the two spliced variants. The sgRNA was subcloned into a 500 bp linearized CloneJet PJET1.2-T vector (Thermo Fisher, Waltham, MA, USA), upstream of the protospacer adjacent motif (PAM) sequence to allow sgRNA expression under the control of the T7 promoter. The sgRNA was synthesized in vitro with a MEGAScript T7 kit (Ambion, Austin, TX, USA), according to the manufacturer’s instructions. Cas9 mRNA was synthesized in vitro using the mMESSAGE T7 Kit (Ambion, Austin, TX, USA) and a PTD1-T7-Cas9 vector as the template, according to the manufacturer’s instructions.

Two 23-bp sgRNAs were selected to target SlSer2. Each sgRNA was sub-cloned into the 500-bp linearized CloneJet PJET1.2-T vector (Thermo Fisher, Waltham, MA, United States) upstream of the protospacer adjacent motif (PAM) sequence to allow sgRNA expression under the control of the T7 promoter. The sgRNA was synthesized in vitro with a MEGAScript T7 kit (Ambion, Austin, TX, United States) according to the manufacturer’s instructions. Cas9 mRNA was synthesized in vitro using an mMESSAGE T7 Kit (Ambion, Austin, TX, United States) with a PTD1-T7-Cas9 vector as the template (Wang et al., 2013 (link)) according to the manufacturer’s instructions.

son cDNA was subcloned into pcDNA3.1^+/- and linearized with Sca1. son mRNA was generated using a mMessage T7 kit (Ambion, Austin, TX). 1nL of son MO (at 6.25μM) and 0.5nL of son mRNA (at 1μg/μL) was injected into single-cell zebrafish embryos, resulting in a total injection of 6.25ng of the MO and 500ng mRNA into each individual. son mRNA was visualized after being electrophoresed on a 1% w/v agaose gel with TAE and 1% bleach added (S1 Fig).

The antisense MO Hem1 (5′-TCTCTTTCTCCAACGGGTCTTCCAT-3′), which targets the first 25 base pairs of the zebrafish Hemogen open reading frame, was designed according to the manufacturer's instructions (Gene Tools LLC, Philomath, USA). The control MO (Hem1mm; 5′-TCTgTTTgTCCAtCGGcTCTTCgAT-3′) targeted the same sequence but contained five mismatched bases to prevent efficient binding to Hemogen mRNA. MOs were labeled with lissamine or fluorescein so that the quality of injections could be monitored by fluorescence microscopy. MOs were injected (2-8 ng) into embryos at the single-cell stage using a PLI-100 Picoinjector (Medical Systems Corporation, Greenvale, USA; 65-0001) and a micromanipulator (Narishige, Amityville, USA; MN-151). Injected embryos were sampled from 0 to 6 dpf for subsequent analyses.
Rescue of the morphant phenotype was tested by co-injection of the Hem1 MO with 500 pg synthetic zebrafish Hemogen mRNA transcribed from a zebrafish Hemogen cDNA cloned into pGem-T Easy (Promega). Primers (Table S1) introduced five silent mutations within the MO target site. The clone was digested with Spe1 and mRNA was transcribed, capped and polyadenylated in vitro using the mMessage T7 kit (Ambion; AM1340) and the Poly(A) Tailing Kit (Ambion; AM1350). mRNA was purified with the MEGAclear kit (Ambion).

We selected two 23-bp sgRNAs targeting OfMasc and one sgRNA targeting Ofdsx. The sgRNAs were sub-cloned into the 500-bp linearized CloneJet PJET1.2-T vector (Thermo Fisher Scientific) upstream of the protospacer adjacent motif (PAM) sequence, to allow sgRNA expression under the control of the T7 promoter. The sgRNAs were synthesized in vitro using a MEGAScript T7 kit (Ambion), according to the manufacturer’s instructions. Cas9 mRNA was synthesized in vitro using the mMESSAGE T7 Kit (Ambion) and a PTD1-T7-Cas9 vector as the template [35 (link)], according to the manufacturer’s instructions.