Mmessage mmachine t7 kit

The psen2WT-EGFP and psen2S4Ter-EGFP expression vector constructs were restricted with XbaI before transcription using the mMessage mMachine T7 kit (Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA) to generate mRNA. All mRNAs were precipitated with LiCl and then redissolved in water for injection of 2–5 nL at a concentration of 400 ng/μL. No obvious developmental abnormalities were seen after injection of these mRNAs into zebrafish embryos. At ~24 hpf, both the psen2WT-EGFP mRNA-injected and the psen2S4Ter-EGFP mRNA-injected embryos showed weak EGFP fluorescence (as visualized by fluorescence microscopy). For mRNA-injected and non-injected embryos (as negative controls) 15 embryos were collected for subsequent western immunoblot analysis.

gRNAs were synthesized and microinjected as described previously [21] (link). Human codon-optimized Cas9 (hCas9) and sgRNA cloning vector a gift from George Church (Addgene plasmid #41815 and #41824, respectively) [20] (link). Essentially, sequences that recognize Dmrt1 and Dmrt3 target sites were introduced into gRNA cloning vector through inverse PCR. gRNA sequences were PCR amplified from the plasmids and served as templates for the in vitro transcription by mMessage mMachine T7 kit (Thermo Fisher Scientific, Waltham, MA, USA). Transcribed gRNAs were purified with Megaclear (Thermo Fisher Scientific) and ethanol precipitation and microinjected into the cytoplasm of the fertilized eggs obtained from intercross of F1 hybrid (C57BL/6×DBA/2) BDF1 (Sankyo Labo Service Corporation). The concentrations of the RNAs are as follows: Dmrt1 gRNA, 166 ng/μl; Dmrt3 gRNA, 166 ng/μl; and hCas9 mRNA, 166 ng/μl. Primer sequences used for the cloning and template amplification of the gRNAs are listed in Supplementary table 1. All animal protocols were approved by the Animal Care and Use Committee of the National Research Institute for Child Health and Development, Tokyo, Japan.

Cas9 cDNA in the pCS2+ vector was obtained from Thermo Fisher Scientific Inc. (Yokohama, Japan). The DNA construct was linearized with NotI and transcribed with the mMessage mMachine SP6 Kit (Thermo Fisher Scientific Inc.) to produce capped Cas9 mRNA, which was then purified with the RNeasy Mini Kit according to the RNA clean protocol (QIAGEN). To create an engineered single guide RNA (gRNA) expression vector, we placed a T7 promoter followed by two BsaI sites upstream of the recently described gRNA scaffold [13 (link)]. The gRNA was designed to target protospacer sequences in the AR gene of interest with the form 5’-CC-(N)20-GGG-3’. The GGG was the protospacer-adjacent motif (PAM). The resulting construct was digested with DraI and transcribed using the mMessage mMachine T7 Kit (Thermo Fisher Scientific Inc.). The gRNA was purified using the RNeasy Mini Kit (QIAGEN). We produced KD ZW frogs using the CRISPR/Cas9 system by which we have successfully disrupted the NTD, DBD and LBD of the AR gene by frame-shift.

Human GluN1a-wt (hGluN1a), human GluN2B-wt (hGluN2Bwt) and human GluN2B-G689C (hGluN2B-G689C) cloned in pCl-Neo were obtained from Dr. Garin-Shkolnik T (produced by the Traynelis S. Lab). Human GluN2B-G689S (hGluN2B-G689S; c.G2065A) was generated by us using site-directed mutagenesis (primers: sense- 5’-CCGCTTTGGGACCGTGCCCAACAGCAGCACAGAGAGAAATATTCG-3’, antisense 5’-CGAATATTTCTCTCTGTGCTGCTGTTGGGCACGGTCCCAAAGCGG-3’) and verified by full DNA sequencing (Faculty of Medicine, Biomedical Core Facility- Technion). For mRNA preparation, DNA plasmids were linearized by restriction enzymes (NotI), followed by in vitro mRNA transcription using mMessage-mMachine T7 kit (Thermo Scientific, Cat.#AM1344), as previously described (Berlin et al., 2010 (link)). mRNA concentrations were determined using a spectrophotometer. Oocytes were injected with 5–16 ng/oocyte mRNA of each subunit at 1:1 ratio in all of the experiments. For dominant-negative experiments, we co-injected hGluN1a, hGluN2Bwt and hGluN2B-G689C or −2B-G689S with the following mRNA quantities (ng/oocyte): 5:16.6:1, 5:5:5: and 5:1:16.6 yielding GluN2B-wt high (wt_H), even (wt_E), and low (wt_L), respectively.

According to the principle of CRISPR/Cas9, sgRNAs against the il7r gene (ENSDARG00000078970) were designed using a CRISPR design tool (http://crispr.mit.edu/)^{43 (link)}. The sgRNA target sequences for il7r were as follows: ACTCCACTCACTCCAGTCACCGG. sgRNA was generated with a pX330 vector template (BioVector NTCC Inc. Beijing, China) and transcribed using a MAXIscript T7 kit (Thermo Fisher Scientific, Waltham, MA, USA). The pGH-T7-zCas9 plasmid was linearised by XbaI and then transcribed in vitro to generate Cas9 mRNA using a mMESSAGE mMACHINE T7 kit (Thermo Fisher Scientific). Zebrafish embryos were injected with 1 nL mixed solution containing 50 ng/mL sgRNA and 250 ng/mL Cas9 mRNA^{44 (link)}. Embryos were then incubated with sterile E3 medium and raised at 28.5 °C.

For expression of the human homomeric ASIC1a (hASIC1a) and human heteromeric ASIC1a/α-ENAC/γ-ENAC (hASIC1a/α-ENAC/γ-ENAC) channels in Xenopus laevis oocytes, the linearized plasmids were transcribed using the T7 mMESSAGE-mMACHINE Transcription Kit (Thermo Fischer Scientific). Oocytes were defolliculated and injected with 2.5–10 ng of mRNA. mRNA transcripts of hASIC1a, hENACα, and hENACγ were synthesized by the mMESSAGE-mMACHINE T7 kit (Cat# AM1344, Thermo Fisher Scientific) according to the protocol for capped transcripts supplied by the manufacturer. For hASIC1a/ENACα/ENACγ expression, the corresponding mRNAs were mixed at the 1:1:1 molar ratio. The preparation of Xenopus oocytes at defolliculated stages V–VI was done as previously described (29 (link)). Non-injected defolliculated oocytes were used as the control for an absence of endogenous ASIC expression. After injection, the oocytes were kept for 2–3 days at 19°C and then up to 7 days at 15°C in ND-96 medium supplemented with gentamycin (Cat# G1264, Merck, Darmstadt, Germany) (50 μg/ml) and containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl₂, 1 MgCl₂, and 10 HEPES, pH 7.4. Plasmids bearing hASIC1a, hα-ENAC, and hγ-ENAC were kindly provided by Dr. Alexander Staruschenko (University of South Florida, Tampa, FL, USA).