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5 protocols using mmessage t7 ultra transcription kit

1

CRISPR-Cas9-Mediated Genome Editing in Newts

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Cas9 mRNA and gRNA were synthesized using mMESSAGE T7 ULTRA Transcription Kit (Invitrogen) and GeneArt Precision gRNA Synthesis Kit (Invitrogen), respectively. Genomic DNA was extracted with Qiagen DNAeasy kit. The sgRNA sequence is: GAGATGGCAAAGACCCCTTC. The IVT was performed according to the manufacturer's instructions. The sgRNAs concentration was adjusted to 50–100 ng/μl and sgRNAs were kept at −80°C until use.
Single‐cell fertilized eggs were obtained by either natural or induced breeding and injected according to previously published protocols with modifications.33 Briefly, 250 pg Cas9 RNA and 50 pg gRNA were mixed into 5 nl and injected into freshly laid single‐cell‐stage embryos. The screening of F0 mosaic animals was done by both genotyping and phenotype characterization, including immunohistochemistry of limbs/tails. The Yap−/− F1 animals were produced by crossing between adult F0 animals. The PCR primers for genotyping were PL: CTAGGTGCTGTCTCTCCCG PR: GTTGTGTGGGTTTTGTGAGCA. Before surgery, larval and adult newts were anaesthetized with 0.01% and 0.2% of ethyl‐p‐aminobenzoate (Sigma‐Aldrich), respectively.
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2

CRISPR-Cas9 Mediated Genome Editing in Newts

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Cas9 mRNA and gRNA were synthesized using mMESSAGE T7 ULTRA Transcription Kit (Invitrogen) and MEGAshortscript T7 Transcription Kit (Invitrogen), respectively. Genomic DNA was extracted with Qiagen DNAeasy kit. The PCR products were cloned into TOPO cloning vector (Invitrogen). Individual clones were sequenced with T7 primer (Supplementary Table 9).
Single-cell fertilized eggs were obtained by either natural or induced breeding and injected according to previously published protocols with modifications60 (link), 61 (link). Briefly, 500 pg Cas9 RNA and 100 pg gRNA were mixed into 5 nl and injected into freshly laid single-cell-stage embryos. The animals were raised according to60 (link). The screening of F0 animals was done by both genotyping (see above) and phenotype characterization, including pigmentation analysis and immunohistochemistry of limbs/tails, according to39 (link), 40 (link). The Pax7−/− and Tyr−/− F1 animals were produced by crossing between adult F0 animals. The larvae or post-metamorphic newts were anaesthetized within 0.01% or 0.1% ethyl-p-aminobenzoate (benzocaine; Sigma) prior to imaging and tissue collection. Newts utilized for this study were processed according to Swedish Board of Agriculture animal ethical regulations (N429/12).
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Expanded Papilloma T Cell Reactivity

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T lymphocyte cultures were initiated from fresh pretreatment and post-treatment papilloma fragments using interleukin-2 containing media. Autologous B lymphocytes were isolated from the Peripheral blood mononuclear cells (PBMC) using negative magnetic selection (StemCell) and expanded in the presence of irradiated (6000 rad) NIH3T3-CD40L feeder cells (1:1 ratio) and rhIL-4 (200 U/mL). Expanded papilloma T lymphocytes were assayed for reactivity against autologous B lymphocytes (1:2 ratio) electroporated with in vitro transcribed full-length mRNA from HPV 6 or 11 E2, E6 or E7 (mMESSAGE T7 ULTRA Transcription Kit, Thermo). T lymphocyte reactivity against these products or PMA/Ionomycin (positive control) was measured via ELISpot assay (R&D Systems). Spot counts were measured on an Immunospot ELISpot reader (Cellular Technology).
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Cloning and Mutagenesis of spHCN Channel

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The full-length spHCN cDNA (a gift from U. B. Kaupp, Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany; GenBank: Y16880) was subcloned into a modified pcDNA3.1 vector (Invitrogen, Carlsbad, CA) that contained a carboxy-terminal eYFP, a T7 promoter, and 5′- and 3′-untranslated regions of a Xenopus β-globin gene. For the ACCuRET experiments, the background cysteine-depleted spHCN construct contained mutations: C211A, C224A, C369A, and C373A. Quickchange II XL Site-Directed Mutagenesis kit (Agilent technologies, Santa Clara, CA) or overlapping PCR was used for mutagenesis. Fluorescence-based DNA sequencing was carried out by Genewiz LLC, Seattle, WA. All oligo primers used for mutagenesis, including the names and sequences, are listed in the Supplementary Table 2. The in vitro mRNA synthesis was done using the HiScribe T7 ARCA mRNA Kit (New England Biolabs, Ipswich, MA) or the mMESSAGE T7 ULTRA Transcription Kit (ThermoFisher, Waltham, MA).
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5

Engineered spHCN channel for ACCuRET experiments

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The full-length spHCN cDNA (a gift from U. B. Kaupp, Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany; GenBank: Y16880) was subcloned into a modified pcDNA3.1 vector (Invitrogen, Carlsbad, CA) that contained a carboxy-terminal eYFP, a T7 promoter, and 5'-and 3'-untranslated regions of a Xenopus β-globin gene. For the ACCuRET experiments, the background cysteine-depleted spHCN construct contained mutations: C211A, C224A, C369A, C373A. Quickchange II XL Site-Directed Mutagenesis kit (Agilent technologies, Santa Clara, CA) was used for all mutagenesis. Fluorescencebased DNA sequencing was carried out by Genewiz LLC, Seattle, WA. The in vitro mRNA synthesis was done using the HiScribe T7 ARCA mRNA Kit (New England Biolabs, Ipswich, MA) or the mMESSAGE T7 ULTRA Transcription Kit (ThermoFisher, Waltham, MA).
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