Message machine kit

EcDnd (GenBank: KX881943) ORF, GFP ORF/DsRed ORF linked to Ecnanos3 3′-UTR and GFP ORF fused with zfnanos3 3′-UTR were inserted into pCS2+ vector digested with BamH1 and EcoR1, respectively. The series of 3′-truncate mutations (del^538–699, del^510–699, del^206–699) fused with GFP ORF similarly cloned into pCS2+ vector. The miR-430 binding site “GCACGTTT” in the constructed GFP-Ecnanos3 3′-UTR was replaced with “GCCAGTTT” by overlap PCR. The mutations of deleted URR1a, URR1b, URR1c, URR1d and URR1e were cloned by overlap PCR from the del^206−699 plasmid. The mutant del^44–139 where URR1 alone was deleted was cloned by overlap PCR from the WT-UTR plasmid.
For RNA synthesis, the plasmids linearized by NotI were transcribed in vitro by using Message Machine-Kit (Ambion, Austin, TX, USA). Microinjections were performed as described previously [90 (link)]. Subsequently, every embryo was observed in Leica M205FA stereomicroscope to analyze GFP expression pattern (n > 150). For measuring GFP expression in vivo, 40 embryos were collected to perform fluorophotometry detection by fluorophotometric scan (TECAN, Grödig, Austria) [84 (link)].

Xenopus laevis embryos were obtained and staged using current protocols (Nieuwkoop and Faber, 1975 ; Sive et al., 2000 ). All mRNAs were synthesized using the Message Machine kit (Ambion, Foster City, USA) and injected at the following doses: noggin (500 pg), FGF8,wnt8a (100 pg), pax3GR/zic1GR (100 pg each), hoxa2 (70 pg), hoxb2, tead1, tead2 (50 pg), vgll3 (0.25-1 ng), vgll3mis (0.5 ng) and vgll3Δhis, vgll2, ets1 (1 ng). For retinoic acid (RA) (Sigma-Aldrich) treatment, embryos were treated at stage 8 with 10⁻⁶ M to 10⁻⁸ M or with DMSO for control. Pax3GR- and zic1GR-injected embryos were cultured in 0.1× MMR with or without 10 µM dexamethasone from stage 10.5. Silencing of selected genes was performed using antisense morpholino oligonucleotides (GeneTools) (Table S2).
Synthetic mRNAs or MOs were co-injected with 250 pg β-galactosidase (lacZ staining) or gfp mRNA as a lineage tracer. Animal caps were dissected from early stage 9 embryos and cultured until appropriate stages before RNA extraction and RT-PCR analysis (Naye et al., 2007 (link); Tréguer et al., 2009 (link)). Primers are listed in Table S3. All results shown are representative of three independent experiments.

Zebrafish YY1a and PSR [31 (link)] were cloned into the pCDNA3 vector, which contains a T7 RNA polymerase promoter site. Linearized plasmid DNA was used as a template for in vitro transcription carried out using the Message Machine Kit (Ambion Inc, Austin, TX), according to the manufacturer’s instructions. For rescue of defective morphants, either 0.1 nl of 200 ng/μl mRNA encoding soluble YY1a mRNA or 0.1 nl of 80 and 40 ng/μl mRNA encoding soluble PSR mRNA and YY1-MO (0.5 μM) were injected into the one-cell stage of each embryo using a gas-driven microinjector (Medical System Corp, Greenvale, NY) [42 (link)].

Salts were obtained from Sigma-Aldrich. In vitro synthesis of TRPV1 cRNA was carried out with the Message Machine kit (Ambion) according to the manufacturer’s instructions. Xenopus laevis care, surgery, and oocyte preparation are described in detail elsewhere (González-Pérez et al., 2008 (link)).

Embryos of Xenopus laevis were staged according to Nieuwkoop and Faber (1967) and injected according to standard procedures (Sive et al., 2000 ). Capped mRNAs were synthesized with Message Machine Kit (Ambion) and injected into single blastomeres at the 2- to 4-cell stage that give rise to the dorsal ectoderm. Unless otherwise noted, the following amounts of mRNAs were injected: mGFP: 250 pg; GR–Eya1: 500 pg; GR–Six1: 500 pg. Morpholinos (see above) were injected singly or as a cocktail (1–10 ng each) into single blastomeres at the 2–4 cell stage. mGFP was coinjected as lineage tracer to identify the injected side. For activation of hormone-inducible constructs, embryos were incubated in dexamethasone (DEX; 10 μM; Sigma) from stages 16–18 onward until they reached the stage for fixation. In a previous study, we confirmed that these GR-fusion constructs function comparable to wild-type protein, that embryos injected with GR-constructs and raised in the absence of DEX showed minimal effects and that DEX treatment alone does not significantly alter gene expression (Schlosser et al., 2008 (link)) in accord with published accounts of this method (Hollenberg et al., 1993 (link); Mattioni et al., 1994 (link); Kolm and Sive, 1995 (link); de Graaf et al., 1998 (link)).

The translation-blocking MOs of DrRIG-I and splice junction MO of DrNOD2 were designed, synthesized by Gene Tools (Philomath, OR, USA), and dissolved in water (2 mM). The MO sequences used were as follows: DrRIG-I MO, 5′-GATTCTCCTTCTCCAGCTCGTACAT-3′; and DrNOD2 MO, 5′-ACCTGCCAAAAATCCAACATGGTTA-3′. The 5′UTR sequence (complement to the MO sequence) of DrRIG was amplified with DrRIG-I F1 and R1 primers (Table S1), and then cloned into the EGFP-N1 vector to evaluate the translation blocking efficiency of DrRIG-I MO. Along with DrRIG-I MO or standard control MO (4 ng/embryo), the constructed vector was injected into one-cell stage embryos (100 pg/embryo). The embryos were collected at 24 hpi, and the GFP fluorescence was visualized through an Olympus MVX10 MacroView. The splice inhibition efficiency of DrNOD2 MO was examined through RT-PCR. The one-cell stage embryos of zebrafish were injected with DrNOD2 MO (4 or 6 ng/embryo) or standard control MO (6 ng/embryo). The embryos were collected at 24 hpi for RNA isolation and cDNA reverse transcription. A forward primer in exon 1 and reverse primer in exon 2 were used to detect the deletion of exon 2 (Table S1). Capped DrRIG-I and DrNOD2 mRNA was synthesized in vitro using a Message Machine kit (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) according to the manual and then solubilized in DEPC water.

Fertilized Xenopus embryos were transferred into mesh-bottomed dishes with 3% Ficoll and injected with capped, synthetic mRNAs (made using the Ambion Message Machine kit) dissolved in water at the stages indicated. The doses per cell were KRAS^G12D [71 (link)] 40pg; Arch [69 (link)], 60pg; and ChR2^D156A [72 (link)] 50pg. Two hours after injection, embryos were transferred into 0.75 × MMR for 45 minutes before they were washed and cultured in 0.1 × MMR until desired stage was reached. Injected embryos were stimulated with the appropriate wavelength of light and irradiance before or after ITLSs fully form (stages 28–35). Embryos were scored for the presence of ITLSs using bright field microscopy as described in [3 (link), 41 (link), 42 (link)].