Mmessage mmachine high yield capped rna transcription kit

Capped reporter mRNAs were prepared using a mMESSAGE mMACHINE high-yield capped RNA transcription kit (Ambion) by following manufacturer's instructions. Reticulocyte lysate (Ambion) or wheat germ lysate (Progema) was used to generate shifted and non-shifted protein products. In each assay, a total of 5 μl reaction containing 50–250 ng of capped reporter mRNA, 2.5 μl of translation lysate and 0.2 μl of 10 μCi/μl ³⁵S-labeled methionine (NEN) was incubated at 30°C for 1.5–2 h. Samples were then resolved by 12% SDS-PAGE, and exposed to a phosphorimager screen for quantification after drying. Frameshifting efficiencies were calculated, by dividing the counts of the shifted product by the sum of the counts for both shifted and non-shifted products, with calibration of the methionine content in each protein product. All the radioactivity-based -1 PRF activity measurements in vitro were then performed by assuming that ribosome drop-off effect (15 (link)) was minimized for the translation of the shortened -1 frame product. As we present all of our in vitro -1 PRF results in term of relative -1 PRF activity, the ribosome drop-off effect was indeed removed by the procedure.

mMESSAGE mMACHINE High Yield Capped RNA Transcription kit was from Ambion (Austin, TX, USA). TransMessenger Transfection Reagent, RPMI-1640 medium, X-VIVO-15 serum-free medium, L-glutamine, and fetal calf serum were from Life Technologies Gibco/BRL division (Grand Island, NY, USA). Yeast poly(A) polymerase and Acl I enzyme were from United States Biochemical (Cleveland, OH, USA). Other restriction enzymes, T4 DNA ligase, and PureYield Plasmid Miniprep System were from Promega (Madison, WI, USA). DNA marker DL2000 was from Takara Biotechnology (Dalian) (Dalian, China). The other reagents were analytical grade chemicals from domestic and international companies.

In vitro synthesis of RNA was performed using the mMessage mMachine High Yield Capped RNA Transcription Kit (Ambion, AM1340) to produce SP6-promotor driven RNA from linearised plasmid DNA templates following the manufacturer’s instructions. RNA was purified using the RNA purification kit (Sangon Biotech, B511361-0100). RNAs were diluted in injection buffer (5 mM Tris, 5 mM NaCl, 0.1 mM EDTA, pH 7.4) and microinjected into presumptive zygotes as follows: Membrane-GFP, LAMP1-3xeGFP, LAMP1-mScarlet, L10A-eGFP, Emerald-Sec61β, eGFP-eIF2β, PABPC1-eGFP, ANXA11-mEmerald, mScarlet-Borcs7, RPS6-mScarlet, HTATSF1-mScarlet, 24xGCN4_v4-Cdx2-24xPP7, 24xGCN4_v4-eIF2β−24xPP7, ScFv-sfGFP at 35 ng; eGFP-MAP2c, G3PB1-eGFP, mTFP-Utrophin, BFP-Utrophin, PCP-2xmCherry, eGFP-Rab11a at 30 ng. ANXA11-R346C-mEmerald was microinjected at 90 ng. siRNAs (Qiagen) were microinjected at 400 nM described in Table S1.