Amplicap sp6 high yield message maker kit

Two nckap1l splice MOs to prevent premRNA splicing and a standard control MO were synthesized by Gene Tools (Gene Tools LLC): MO1, 5′-TCT​GAA​ACA​GTT​GAT​GAG​CAC​AGG​T-3′; MO2, 5′-AGA​CGC​TGA​CGG​ACT​GAC​CTT​AG-3′, and control MO, 5′-CCT​CTT​ACC​TCA​GTT​ACA​ATT​TAT​A-3′. MO1 binds to the acceptor site of exon 5, while MO2 targets the donor site of exon 6. Embryos were injected, fixed, and validated as previously described (Carapito et al., 2017 (link)). RT-PCR to validate splice modification was performed using the following primer pairs that span the respective regions: 5′-CTC​AGA​CCC​AAA​GCG​AAG​AC-3′ and 5′-GAC​CGA​ACT​CCT​CAG​ACA​GC-3′. Real-time quantitative PCR was performed as described (Konantz et al., 2016 (link)) on control and morphant fish using primer pairs for nckap1l (5′-GTG​ACG​GAG​GCT​GTT​CTC​TC-3′ and 5′-TCT​GAG​AGT​TTG​CGT​TGG​TG-3′) and gapdh. For rescue experiments, zebrafish WT nckap1l cDNA cloned into the pCS2^– expression vector (RZPD) was used. Capped mRNA was then synthesized using the AmpliCap SP6 High Yield Message Maker Kit (Cellscript). 100 pg of capped mRNA was injected into the yolk of single-cell-stage embryos together with the nckap1l MO to rescue the MO phenotype.

The bmp2b, bmp1a, tll1, and chrd mRNAs were generated from pCS2 + kzbmp2, pCS2 + bmp1a [11 (link)], pCS2 + tll1 [52 (link)], and pCS2 + chrd-myc [48 (link)] by restriction digestion with NotI and AmpliCap SP6 High Yield Message Maker Kit (CellScript, Madison, WI, USA). The synthesized capped mRNA was purified using a SigmaSpin™ PostReaction Clean-Up Column (Merck) and stored at −80 °C. The generated mRNA was injected into zebrafish embryos at the one-cell stage using an IM300 microinjector (NARISHIGE, Tokyo, Japan). In the rescue experiment, the mRNA injected embryos were exposed to the compounds at 3–24 hpf as described above, and the phenotype was observed.

Fragments of TBSV P19 (GenBank: AJ288942.1) and the derivative (R75G R78G), TCV P38 (GenBank: HQ589261.1) and a derivative (W26A W274A), CMV 2b (strain Y, GenBank: D12538.1) or 2b (strain Q, GenBank: Z21863.1), PVY Hc-Pro (GenBank: AFR11765.1), and CVYV P1b (GenBank: DQ496114.1) were amplified by PCR. Fragments of Cardamine chlorotic fleck virus (CCFV) P38 (NC_001600.1), Pelargonium flower break virus (PFBV) P38 (GenBank: DQ443018.1) were synthesized by GeneArt Strings (Thermo Fisher Scientific). DNA fragments encoding HYL1, DRB4, RDR1, RDR2, or RDR6 were amplified by PCR using cDNA prepared from RNA of A. thaliana Col-0. All the fragments were cloned into pSP64-poly(A) vector (Promega) using appropriate restriction sites. Substitution of nucleotides or addition of epitope sequences on genes was performed by PCR using plasmid clones as templates and primers containing relevant sequences. Oligonucleotides are listed in Supplemental Table S1. Each messenger RNA was prepared from the linearized plasmids using AmpliCap SP6 High Yield Message Maker Kit (Cellscript).

To generate a template for the generation of an antisense in situ probe, a 921 bp fragment of the Danio rerio adsl open-reading frame was cloned into pCRII via TOPO TA cloning (Invitrogen). To have a template for the generation of capped mRNA using the AmpliCap SP6 High Yield Message Maker Kit (Cellscript), the whole open-reading frame of zebrafish adsl was cloned with an N-terminal Flag-tag into pCS2+ using EcoRI and XhoI.

pCS2-HiFi Cas9 was constructed by inserting a HiFi Cas9-encoding DNA fragment from pX330-Flag-HiFi SpCas9 (a gift from Ervin Welker, Addgene plasmid #126778) into the pCS2 vector. pCS2-HiFi Cas9 was linearized with NotI and used as a template for mRNA synthesis using AmpliCap SP6 High Yield Message Maker Kit (CELLSCRIPT). mRNA was then purified with RNA clean & concentrator (Zymo Research). HiFi Cas9 mRNA solution (200 ng/μl) was combined with the same volume of the 3 μM gRNA solution and used for microinjection.