Mmessage mmachine kit

Xenopus embryos were generated and cultured according to general protocols and staged according to the normal table of Nieuwkoop and Faber [55 ]. All procedures were performed according to the German animal use and care law (Tierschutzgesetz) and approved by the local authorities and committees (animal care and housing approval: I/39/EE006, Veterinäramt Erlangen; animal experiments approval: 54–2532.2–8/08, German state administration Bavaria/Regierung von Mittelfranken).
RNA for microinjection was prepared using the mMessage mMachine Kit (ThermoFisher Scientific, USA). The following amounts were injected: 500 pg for alk3–5′-gfp and alk6–5′-gfp, 50 pg of alk3-flag and alk6-flag RNA; 25 pg of pCS2 alk3-flag and pCS2 alk6-flag DNA and 100 pg of pCS2-β-galactosidase DNA. Knock-down was achieved by injection of 0.8 pmol or 1.6 pmol of ALK3 MO and ALK6 MO as indicated.
Embryos were injected and cultured until they reached the desired stage as indicated.
For subsequent in situ hybridization, single side injections were performed and traced by co-injection of pCS2-β-galactosidase DNA. The injected side was visualized by ß-galactosidase staining and in situ hybridizations were carried out, as described by Harland [104 (link)], using chordin [77 (link)], sox2 [105 (link)], twist [106 (link)], msx2, alk3, alk6 or bmpr2 as antisense probes, respectively.

5′capped and polyadenylated mRNAs encoding TFs expressed by cells in the neural plate (foxd4l1.1; Sullivan et al., 2001 (link)), neural crest (foxd3, Sasai et al., 2001 (link); msx1, Suzuki et al., 1997 (link); Tribulo et al., 2003 (link); Monsoro-Burq et al., 2005 (link); zic1, zic2, and zic3, Nakata et al., 1997 (link), Nakata et al., 1998 (link)), PPR (six1;Brugmann et al., 2004 (link)), or epidermis (dlx5, Papalopulu and Kintner, 1993 (link); Luo et al., 2001 (link)), as well as a nucleus-localized β-galactosidase (nβgal) as a lineage tracer, were synthesized in vitro (mMessage mMachine kit, ThermoFisher). Antisense RNA probes for in situ hybridization (ISH) were synthesized in vitro (MEGAscript kit; ThermoFisher) as previously described (Yan et al., 2009 (link)).

For the generation of tardbp and tardbpl knockout fish, target sequences for Cas9-mediated cleavage were searched by CRISPRscan^{63 (link)}. The target sequences CAAGACTTAAAAGACTACTTcgg and CAAGACTTAAAAGACTACTTcgg, where the protospacer adjacent motifs (PAMs) are indicated by lower cases, were chosen for the generation of tardbp-n115 and tardbpl-n94 alleles, respectively. hSpCas9 was in vitro-transcribed with mMESSAGE mMACHINE Kit (Thermo Fisher Scientific, AM1340) by using pCS2 + hSpCas9 plasmid as a template (a gift from Masato Kinoshita, Addgene plasmid # 51815). Wild type embryos were injected with 25 pg of sgRNA and 300 pg of hSpCas9 mRNA at the one-cell stage.

Channel chimeras were generated using sequential PCR with K_V2.1Δ7^{47 (link),48 (link)} (Genscript, USA) and hNa_V1.6 (NM_014191, Origene Technologies, USA) as templates. cRNA was synthesized using T7 polymerase (mMessage mMachine kit, Thermo Fisher, USA) after linearizing cDNA with appropriate restriction enzymes. This chimeric approach was previously shown to robustly indicate the binding locus of toxins^{32 (link)}. Boundaries of the chimeras were chosen as reported previously in Fig. S2 of Bosmans et al. 2011^{33 (link)} and include the transfer of hNa_V1.6 regions (MMAYITEFVNLGNVSALRTFRVLR (VSDI), GFIVSLSLMELSLADVEGLSVLRSFRLLR (VSDII), SLVSLIANALGYSELGAIKSLRTLRALR (VSDIII), SIVGMFLADIIEKYFVSPTLFRVIRLARIGRILR (VSDIV) into K_V2.1.

The infectious cDNA clone was linearized by SacI (New England Biolabs) digestion, followed by phenol/chloroform extractions. The linearized templates were in vitro transcribed with mMESSAGE mMACHINE kit from ThermoFisher Scientific (Waltham, Massachusetts, USA); and the viral RNAs thus obtained were electroporated in BHK21 (ATCC CCL-10) cells. The viral supernatants were harvested after 48 h and used to infect new BHK21 cells and constitute a stock for the experiments. All the viral stocks produced were titrated by plaque assay on Vero cells [33 (link)].

As previously described^{7 (link)}, we generated cRNA transcripts encoding human KCNE1, KCNE3, KCNQ1, KCNQ2, KCNQ3, KCNQ4 or KCNQ5 by in vitro transcription using the mMessage mMachine kit (Thermo Fisher Scientific), after vector linearization, from cDNA sub-cloned into plasmids incorporating Xenopus laevis β-globin 5′ and 3′ UTRs flanking the coding region to enhance translation and cRNA stability. We generated mutant KCNQ1, KCNQ2 and KCNQ3 cDNAs by site-directed mutagenesis with a QuikChange kit (Stratagene, San Diego, CA) and prepared the cRNAs as above. We injected defolliculated stage V and VI Xenopus laevis oocytes (Xenoocyte, Dexter, MI, US and NASCO, Fort Atkinson, WI, US) with KCNE and/or KCNQ cRNAs (2–20 ng). We incubated the oocytes at 16 °C in ND96 oocyte storage solution containing penicillin and streptomycin, with daily washing, for 2–4 days prior to TEVC recording.

PCR reactions were performed with DreamTaq (Thermo Fisher) in 50 μL reaction volume using 10 ng of a pYes plasmid construct as a template, 0.4 μM forward (5′-CGGATCGGACTACTAGCAGCTG -3′) and 0.4 μM reverse (5′-TTCATTAATGCAGGGCCGCAAATT-3′) primers that anneal upstream and downstream of the P_T7 and the TER_CYC1 elements, respectively. PCR products were purified and concentrated using type D4004 ZYMO column (ZYMO Research) and DNA concentrations were estimated spectrophotometrically. To synthetize uncapped RNA, 1 μg of PCR-generated DNA template was transcribed in the T7-polymerase reaction using TranscriptAid T7 High Yield Transcription kit (Thermo Scientific) according to the manufacturer's recommendations. Reaction mixtures were incubated for 120 min at 37 °C, followed by DNase treatment for 15 min at 37 °C. RNA transcripts were first purified by phenol/chloroform extraction, followed by EtOH precipitation; pellets were resuspended in 50 μL of H₂O and separated from unincorporated nucleotides by gel filtration on Micro Bio-Spin P-30 columns (Bio-Rad). RNA was aliquoted and stored at − 80 °C.
To generate capped RNA, purified PCR products (described above) were used as DNA templates in coupled T7 RNA polymerization–RNA capping reactions (mMESSAGE mMACHINE kit, Thermo Fisher #AM1344M), incubated for 1 h at 37 °C.