t7 mscript tm standard mrna production system c-msc100625  (Cellscript Inc)

Journal: bioRxiv

Article Title: Mouse fertilization triggers a conserved transcription program in one-cell embryos

doi: 10.1101/2020.09.15.298018

Figure Lengend Snippet: ( A ) Upstream transcription regulators inferred for each iEGA time-point (INGENUITY). ( B ) Hoffman micrographs of embryos produced by in vitro fertilization (IVF) and incubated in c-Myc inhibitor, 10058-F4 (0, 1 or 2 μM) after six (upper row) or 24 h. Scale bar, 100 μm. ( C ) Quantification of developmental rates (percentage of embryos surviving IVF) of embryos of ( B ) for n =3 independent biological replicates. 2-cell, two-cell embryo (24 h after IVF); 4-cell, four-cell embryo (∼48 h); eB, expanded blastocyst (∼96 h). ( D ) Histogram plots of transcript levels determined by qPCR using intron-flanking primers for random-primed cDNA derived from metaphase II (mII) oocytes (open bars) and embryos 6 h after in vitro fertilization. Intron-exon primer pairs gave products for genomic DNA but not cDNA. ( E ) qPCR analysis with intra-exonic primers and primers flanking exon-exon junctions. ( F ) Spliceosome component and guanylyltransferase ( Rngtt ) transcript levels determined by qPCR in germinal vesicle (GV) oocytes, mII oocytes (0) and one-cell embryos at the times shown (h) after sperm injection. ( G ) Injection of mCherry cRNA (mCh, top left: orf, mCherry open reading frame; c, cytoplasmic polyadenylation element; t, mRNA cleavage/polyadenylation signal). Fluorescence intensity quantification (lower left) at the times shown after injection of mCherry cRNA (0.6 ng/μl) polyadenylated in vitro (pA + ) or not (pA - ). Fluorescence micrographs show representative oocytes with corresponding bright field images (insets, upper left) and a non-injected control (neg, inset upper right). Bars, 100 μm. Values in ( C - G ) are ± s.e.m. Unpaired t -tests show p -values <0.05.

Article Snippet: 5’-capped cRNA was synthesized in vitro from linearized plasmid template DNA in a T7 mScript(tm) Standard mRNA Production System (Cellscript, USA) according to the recommendations of the manufacturer, as previously described , , .

Techniques: Produced, In Vitro, Incubation, Random Primed, Derivative Assay, Injection, Fluorescence, Control

Journal: Molecular cell

Article Title: Fragile X Mental Retardation Protein Regulates Translation by Binding Directly to the Ribosome

doi: 10.1016/j.molcel.2014.03.023

Figure Lengend Snippet: (A) Domain organization of dFMRP (1-681 amino acids) and NT-dFMRP (220-681 amino acids). NLS, nuclear localization signal; KH, K-homology domain; NES, nuclear export signal; RGG, motif rich in arginine and glycine. (B) Time course of luciferase mRNA translation. Red trace, control translation without NT-dFMRP; light blue trace, translation with 1.2 μM bovine serum albumin (BSA); cyan trace, translation with 1.2 μM RNA-binding MS2 coat protein; blue trace, translation with 0.6 μM NT-dFMRP. The addition of NT-dFMRP to the IVTS inhibited luciferase mRNA translation. In contrast, the addition of BSA or MS2 coat protein to the IVTS did not inhibit the synthesis of luciferase. (C) Inhibition of translation by NT-dFMRP mutants. Time course of luciferase mRNA translation in the presence of the indicated mutant NT-dFMRP proteins. (D) Inhibition of translation by FMRP is independent of WGGA and ACUK sequences. Red trace, control mRNA; green trace, mRNA without WGGA sequence; blue trace, mRNA without ACUK sequence; orange trace, mRNA without both WGGA and ACUK sequences. In all cases, the data were normalized with respect to the control translation without NT-dFMRP. The bar graphs next to each time course show the mean ± SD from three independent experiments. (E) Inhibition of translation by FMRP in cells. Translation of control luciferase mRNA (red bars) and luciferase mRNA without WGGA and ACUK sequences (orange bars) are inhibited to a similar extent by full-length dFMRP and NT-dFMRP, as indicated. Data were normalized with respect to control cells, which were co-transfected with an empty plasmid and the appropriate luciferase plasmid. The transfection experiments were done in duplicates and the mean ± SD from three independent transfection experiments are shown. (F) Inhibition of translation by FMRP is independent of G-quadruplex and pseudoknot forming sequences in the mRNA. Red trace, control mRNA; blue trace, mRNA with ΔKC2 pseudoknot forming sequence; green trace, mRNA with SC1 G-quadruplex sequence. The bar graph shows the mean ± SD from three independent experiments. See also Figures S1 to S4.

Article Snippet: Renilla luciferase reporter mRNA constructs were capped and/or polyadenylated using the T7 mScript TM Standard mRNA Production System (CellScript).

Techniques: Luciferase, Control, RNA Binding Assay, Inhibition, Mutagenesis, Sequencing, Transfection, Plasmid Preparation

Journal: Molecular cell

Article Title: Fragile X Mental Retardation Protein Regulates Translation by Binding Directly to the Ribosome

doi: 10.1016/j.molcel.2014.03.023

Figure Lengend Snippet: FMRP (red) uses its RGG domain to bind to mRNAs having G-quadruplex (GQ) forming sequence and then docks on the 80S ribosome (blue) using the KH1 and KH2 domains. Binding of FMRP to the mRNA and the ribosome synergistically inhibit translation (OFF state). Activation of the neuron changes the post-translational modification status of FMRP, which releases FMRP from the 80S•mRNA complex and activates translation (ON state).

Article Snippet: Renilla luciferase reporter mRNA constructs were capped and/or polyadenylated using the T7 mScript TM Standard mRNA Production System (CellScript).

Techniques: Sequencing, Binding Assay, Activation Assay, Modification