Mmessage mmachine transcription kit

Xenopus laevis oocytes were collected from frogs anesthetized in water containing 0.2% ethyl 3‐aminobenzoate methanesulfonate salt (Sigma‐Aldrich, USA). The isolated oocytes were defolliculated by treatment with type I collagenase (1.0 mg/ml, Sigma‐Aldrich) in ND96 solution (96 mmol/L NaCl, 2 mmol/L KCl, 1.8 mmol/L CaCl₂, 1 mmol/L MgCl₂, 5 mmol/L HEPES, 0.1 mg/ml gentamycin, 5 mmol/L Sodium Pyruvate, pH 7.5 [adjusted with NaOH]) and then injected with 50 nl of cRNA. cRNAs were synthesized from linearized plasmid DNA using a mMESSAGE mMACHINE transcription kit (Thermo Fisher Scientific, USA). The oocytes were incubated for 1.5–2 days at 18°C in ND96.

To facilitate mutations within the 3’UTR of CHIKV-Cbn [17 (link)] and CHIKV-LR [67 (link)] infectious clones, a unique SacI restriction site was introduced downstream of the stop codon of the viral structural proteins. To this end, the PCR product generated with primers sense 94 and antisense 92, and the product of a second PCR generated with primers sense 93 and antisense 95, were fused by overlapping PCR. The XhoI- NotI fragment of CHIKV-Cbn or CHIKV-LR was replaced by the XhoI- NotI fragment of the overlapping PCR products to generate CHIKV-Cbn SacI or CHIKV-LR SacI, respectively. A 3’UTR cloning cassette between unique SacI and NotI restriction sites allowed us to exchange the wild type sequences by mutant 3’UTRs, as described in S1 Table. Nucleotide sequences of primers used for PCRs are listed in S2 Table.
The chimeric viruses were obtained digesting the infectious clones with SacI and NotI and introducing the products in the alternate lineage. The DNAs of the recombinant constructs were linearized by digestion with NotI enzyme and used as templates for transcription by SP6 polymerase in the presence of GpppG cap structure analog, using the mMessage mMachine transcription kit (Thermo Fisher) according to manufacturer’s instructions. The RNAs were gel-quantified and used for transfection in cell culture.

All electrophysiological studies were done in Xenopus oocyte. We tried expression of CiNav1a in HEK293T cells but failed to observe functional currents. Xenopus oocytes were harvested from animals anesthetized in water containing 0.2% ethyl 3-aminobenzoate methanesulfonate salt (Sigma–Aldrich). The oocytes were defolliculated by treating with type I collagenase (1.0 mg/ml; Sigma–Aldrich) in ND96 solution containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), 0.1 mg/ml gentamycin, 5 mM sodium pyruvate, pH  = 7.5 adjusted by NaOH. The defolliculated oocytes were then injected with cRNA synthesized using mMESSAGE mMACHINE Transcription Kit (Thermo Fisher Scientific).

Enhanced green fluorescent protein (eGFP) with three perfect miR430 target sites in its 3′ UTR (eGFP-3×PT-miR430b) was inserted in the pCS2⁺ plasmid (Addgene) as described previously^{24 (link)}. The coding sequence of cdk9 (ENSDART00000065859.8) was amplified from cDNA using SuperScript III (Invitrogen, catalogue number 18080085) and oligodT primers, and inserted in the pCS2⁺ plasmid (Addgene) using restriction-enzyme cloning. mRNA was synthesized using the mMessage mMACHINE Transcription Kit (ThermoFisher, catalogue number AM1340) and purified with the RNeasy MinElute Cleanup Kit (QIAGEN, catalogue number 74204). mRNA was injected into embryos at the one-cell stage in the following amounts: 50 pg of eGFP mRNA and 100 pg of red fluorescent protein (RFP) or cdk9 mRNA.

pT7T-EcadTSMod, pT7T-EcadTL, pT7T-EcadTI, pT7T-CcadTSMod were obtained by PCR amplification of EcadTSMod, EcadTL and EcadTI plasmids (a Kind gift of Dr. N. Borghi) and C-cadherin cDNA (kindly provided by Dr. BM Gumbiner), respectively. PCR products were cloned between BglII and SpeI sites into the pT7T vector using the NEB Gibson assembly kit (NEB). pT7-CcadTSMod was constructed with the NEB Gibson assembly kit following the same scheme as for EcadTSMod: the sequence coding for the 734 first amino acid of C-cadherin were inserted after the BglII site of pT7T, the TS module containing the spider silk protein flanked by mTFP1, EYFP and glycine linker at each side, is inserted just after the first fragment. The last 146 amino acid of C-cadherin were then inserted between TS module and SpeI site of pT7T. Constructs were verified by sequencing. In-vitro transcription was performed with mMessage mMachine transcription kit according to manufacturer’s instructions (Ambion) using the EcoRI site for linearization.