gfp mrna Search Results


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  • 99
    Millipore gfp mrna
    Mesendoderm induction by Mxtx2. ( A-X ) Control <t>gfp</t> <t>mRNA</t> (50 pg) or mxtx2 mRNA (10 pg) was injected into whole zebrafish embryos (A-L), or Ctrl MO (8 ng) or Mxtx2 MO (2 ng) was injected into the yolk syncytial layer (YSL) (M-X), and embryos were fixed at 30% epiboly (4.7 hpf) and stained by whole-mount in situ hybridization (WISH) for the indicated markers. Animal pole views are shown. Fractions indicate the number of equivalent outcomes/number of embryos observed. See also Fig. S1 in the supplementary material.
    Gfp Mrna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Kaneka Corp gfp mrna
    Mesendoderm induction by Mxtx2. ( A-X ) Control <t>gfp</t> <t>mRNA</t> (50 pg) or mxtx2 mRNA (10 pg) was injected into whole zebrafish embryos (A-L), or Ctrl MO (8 ng) or Mxtx2 MO (2 ng) was injected into the yolk syncytial layer (YSL) (M-X), and embryos were fixed at 30% epiboly (4.7 hpf) and stained by whole-mount in situ hybridization (WISH) for the indicated markers. Animal pole views are shown. Fractions indicate the number of equivalent outcomes/number of embryos observed. See also Fig. S1 in the supplementary material.
    Gfp Mrna, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp mrna/product/Kaneka Corp
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    92
    Trinity Biotech gfp mrna
    Mesendoderm induction by Mxtx2. ( A-X ) Control <t>gfp</t> <t>mRNA</t> (50 pg) or mxtx2 mRNA (10 pg) was injected into whole zebrafish embryos (A-L), or Ctrl MO (8 ng) or Mxtx2 MO (2 ng) was injected into the yolk syncytial layer (YSL) (M-X), and embryos were fixed at 30% epiboly (4.7 hpf) and stained by whole-mount in situ hybridization (WISH) for the indicated markers. Animal pole views are shown. Fractions indicate the number of equivalent outcomes/number of embryos observed. See also Fig. S1 in the supplementary material.
    Gfp Mrna, supplied by Trinity Biotech, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TriLink green fluorescent protein mrna
    Characterizations of LPNs for <t>mRNA</t> delivery using a luciferase assay. ( a ) Fold changes of luciferase expression levels using different polymer incorporated LPNs as compared to TT3 LLNs; ( b ) size, ( c ) zeta potential, and ( d ) encapsulation efficiency of different <t>FLuc</t> mRNA encapsulated LPNs. Note A, B and C represents the mass ratio of polymer to mRNA equal to 9:1, 3:1 and 1:1, respectively. ( n = 3; two-tailed t test; ** p
    Green Fluorescent Protein Mrna, supplied by TriLink, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    MaxCyte gfp encoding mrna
    Characterizations of LPNs for <t>mRNA</t> delivery using a luciferase assay. ( a ) Fold changes of luciferase expression levels using different polymer incorporated LPNs as compared to TT3 LLNs; ( b ) size, ( c ) zeta potential, and ( d ) encapsulation efficiency of different <t>FLuc</t> mRNA encapsulated LPNs. Note A, B and C represents the mass ratio of polymer to mRNA equal to 9:1, 3:1 and 1:1, respectively. ( n = 3; two-tailed t test; ** p
    Gfp Encoding Mrna, supplied by MaxCyte, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Unigene clamp gfp mrna
    Absence of Gas2l2 in X. laevis Affects Cilia Rotational Polarity (A) Detection of Gas2l2 in a wild-type skin ciliated cell by immunofluorescence. (B and C) Representative immunofluorescence images visualizing basal bodies <t>(Clamp-GFP)</t> and rootlet (Centrin 4-RFP) to score basal body-rootlet alignment. (B′ and C′) Alignment of the basal body and rootlet in control - MO (n = 19, mean vector, black arrow, r = 0.98, CSD = 11.5°) and Gas2l2 -MO (n = 21, r = 0.67, CSD = 51.2). (D) The vector length (R) of each analyzed cell is represented in the graph. The average length of the vector was significantly shorter in Gas2l2 -MO (R = 0.5884 ± 0.1259) cells than in controls (R = 0.836 ± 0.0847). The introduction of human GAS2L2 <t>mRNA</t> in morpholino-treated embryos ( GAS2L2-R ) rescued the phenotype (n = 23, R = 0.7728 ± 0.1322) (ANOVA, multiple comparison, p
    Clamp Gfp Mrna, supplied by Unigene, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher gfp mrna
    Overexpression of an ectopic VSG causes silencing of the ES-resident VSG. Both (A, B) <t>mRNA</t> and (C, D) protein levels of the endogenous VSG A1.1 (green) and the ectopic VSG 121 (magenta) were monitored in growth arrested (left) and proliferating (right) clones during the course of ectopic VSG overexpression. In all graphs triangles correspond to the ES-promoter cell line <t>(GFP</t> ESpro A1.1 ES 121 tet ) and circles to the stumpy reporter cell line (GFP:PAD1 UTR A1.1 ES 121 tet ). Note that the initial VSG overexpression levels are comparable in arrested and proliferating parasites. For the quantification of (A, B) mRNA levels, total RNA samples were dot-blotted and hybridized with infrared fluorescently labeled probes, specific for VSG 121 or VSG A1 . 1 . The data were quantified and normalized to β-tubulin mRNA using the Licor Odyssey system. (C, D) VSG protein levels were quantified by dot-blotting 6x 10 5 cell equivalents. The blots were incubated with an anti-VSG 121 or an anti-VSG A1.1 antibody. A histone H3 antibody was used for normalization. The VSG expression levels are given relative to VSG 121 expression levels of MITat1.6 wild type cells and parental AnTat1.1 cells natively expressing VSG A1.1. The dashed grey line indicates wild type expression levels (100%).
    Gfp Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Horizon Discovery sirna targeting gfp mrna
    Frozen lung ( A , B ) and liver ( C ) tissue sections from transgenic mice overexpressing <t>GFP</t> 24 hours after intratracheal administration of PBS ( A1 , B1 , C1 ) or <t>GFP-siRNA</t> ( A2 , B2 , C2 ). Mice having received GFP-siRNA display reduced pulmonary but not hepatic
    Sirna Targeting Gfp Mrna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Horizon Discovery green fluorescent protein gfp mrna
    Frozen lung ( A , B ) and liver ( C ) tissue sections from transgenic mice overexpressing <t>GFP</t> 24 hours after intratracheal administration of PBS ( A1 , B1 , C1 ) or <t>GFP-siRNA</t> ( A2 , B2 , C2 ). Mice having received GFP-siRNA display reduced pulmonary but not hepatic
    Green Fluorescent Protein Gfp Mrna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BioScience Inc gfp encoding mrna
    Frozen lung ( A , B ) and liver ( C ) tissue sections from transgenic mice overexpressing <t>GFP</t> 24 hours after intratracheal administration of PBS ( A1 , B1 , C1 ) or <t>GFP-siRNA</t> ( A2 , B2 , C2 ). Mice having received GFP-siRNA display reduced pulmonary but not hepatic
    Gfp Encoding Mrna, supplied by BioScience Inc, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TriLink synthetic mrna encoding rfpamb gfp
    <t>mRNA</t> reporter assay for screening amber suppression positive stable isolate hosts. ( a) Schematic representation of the process used to generate and identify amber suppression competent cells. CHO cells were first transfected with a plasmid encoding PylRS and tRNApyl and subjected to a selection step by growth in puromycin-containing medium. Surviving isolates were transfected with mRNA encoding RFPambGFP and cells were exposed to AzK. Cells capable of efficient amber codon suppression (positive) express an <t>RFP-GFP</t> fusion and are positive for both fluorophores. Cells lacking amber suppression activity (negative) express only RFP. (b) A stable isolate identified as positive for amber suppression activity was transfected with the RFPambGFP mRNA and incubated in the presence or absence of AzK. Cells were imaged by confocal microscopy and flow cytometry. Amber suppression and incorporation of AzK was observed in presence of PylRS and tRNApyl. No GFP expression was observed in absence of AzK.
    Synthetic Mrna Encoding Rfpamb Gfp, supplied by TriLink, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Stemgent nls gfp mrna
    <t>mRNA</t> reporter assay for screening amber suppression positive stable isolate hosts. ( a) Schematic representation of the process used to generate and identify amber suppression competent cells. CHO cells were first transfected with a plasmid encoding PylRS and tRNApyl and subjected to a selection step by growth in puromycin-containing medium. Surviving isolates were transfected with mRNA encoding RFPambGFP and cells were exposed to AzK. Cells capable of efficient amber codon suppression (positive) express an <t>RFP-GFP</t> fusion and are positive for both fluorophores. Cells lacking amber suppression activity (negative) express only RFP. (b) A stable isolate identified as positive for amber suppression activity was transfected with the RFPambGFP mRNA and incubated in the presence or absence of AzK. Cells were imaged by confocal microscopy and flow cytometry. Amber suppression and incorporation of AzK was observed in presence of PylRS and tRNApyl. No GFP expression was observed in absence of AzK.
    Nls Gfp Mrna, supplied by Stemgent, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher turbo gfp mrna
    Poly-PR peptide interacts with RNA and forms aggregates. ( A ) Agarose gel electrophoresis of solutions containing <t>GFP</t> <t>mRNA</t> with HA peptide or HA-poly-(PR) 20 peptide at indicated concentrations. An arrowhead shows GFP mRNA and an arrow shows insoluble RNA
    Turbo Gfp Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TriLink gfp egfp encoding mrna
    Poly-PR peptide interacts with RNA and forms aggregates. ( A ) Agarose gel electrophoresis of solutions containing <t>GFP</t> <t>mRNA</t> with HA peptide or HA-poly-(PR) 20 peptide at indicated concentrations. An arrowhead shows GFP mRNA and an arrow shows insoluble RNA
    Gfp Egfp Encoding Mrna, supplied by TriLink, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Stemgent functional gfp mrna
    Comparative analysis of enhanced green fluorescence protein <t>(EGFP)-mRNA</t> induced antiviral responses in 10T1/2 (A) and D3 cells (B) . The cells were transfected with 3p-EGFP-mRNA (EGFP, 300ng/mL) for 12 h. The mRNA levels of genes involved in antiviral
    Functional Gfp Mrna, supplied by Stemgent, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher chimeric gfp dele mrna
    X. laevis PGC can migrate in vitro in the under-agarose migration assay. (A) Ventral explants were dissected from the embryos at developmental stage 17–19 or 28–30 injected at 2-cell stage vegetally with <t>GFP_DELE</t> <t>mRNA</t> to label PGCs. Explants were treated with accutase and the dissociated cells were transferred between 0.5% agarose gel and a 5% BSA-coated Petri dish in 0.8× MBSH buffer. (B) Time-lapse images of an under-agarose migration assay with dissociated endodermal cells from stage 28–30 embryos. PGCs can be identified as GFP-positive (green) in contrast to the GFP-negative somatic cells. Red arrows indicate migrating PGC. Relative time from the start of the time-lapse imaging is shown in the upper left corner of each image panel (min:sec). Scale bars: 20 µm. (C) Time-lapse microscopy was used to monitor the behavior of PGCs. Relative amount of migratory and non-migratory cells in the total amount of analyzed PGCs was calculated for each experiment. N – number of experiments. Error bars represent standard deviation. *** corresponds to P
    Chimeric Gfp Dele Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Horizon Discovery gfp mrnas
    X. laevis PGC can migrate in vitro in the under-agarose migration assay. (A) Ventral explants were dissected from the embryos at developmental stage 17–19 or 28–30 injected at 2-cell stage vegetally with <t>GFP_DELE</t> <t>mRNA</t> to label PGCs. Explants were treated with accutase and the dissociated cells were transferred between 0.5% agarose gel and a 5% BSA-coated Petri dish in 0.8× MBSH buffer. (B) Time-lapse images of an under-agarose migration assay with dissociated endodermal cells from stage 28–30 embryos. PGCs can be identified as GFP-positive (green) in contrast to the GFP-negative somatic cells. Red arrows indicate migrating PGC. Relative time from the start of the time-lapse imaging is shown in the upper left corner of each image panel (min:sec). Scale bars: 20 µm. (C) Time-lapse microscopy was used to monitor the behavior of PGCs. Relative amount of migratory and non-migratory cells in the total amount of analyzed PGCs was calculated for each experiment. N – number of experiments. Error bars represent standard deviation. *** corresponds to P
    Gfp Mrnas, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher sirna against gfp mrna
    Overexpression or down-regulation of SCCAs significantly affected UV-induced apoptosis. (A) FACS analyses of apoptotic cells using SCCA1- or SCCA2-transfected 3T3/J2 cells, 48 h after UV irradiation (30 mJ/cm 2 ). Cells were stained with FITC-conjugated Annexin V and propidium iodide. (B) Analyses of five experiments are summarized. (C) 12 clones whose expression levels of SCCA1 <t>mRNA</t> distributed from 1 to 2,772-fold were established. Using these clones, the effects of UV irradiation were examined. Cells were harvested 48 h after UV irradiation (50 mJ/cm 2 ) and FACS analyses were performed. The antiapoptotic activity correlated with SCCA1 expression. r = 0.734. (D) Using pSilencer vector, an <t>siRNA</t> construct targeted to a homologous sequence of SCCAs was stably transfected into HaCaT keratinocytes. Typical FACS analyses of nonirradiated (UV−) and UV-irradiated (UV+) siSCCA/HaCaT cells were shown. Apoptotic cells were analyzed 48 h after UV irradiation (75 mJ/cm 2 ). (E) Statistical analyses of five experiments. Error bars represent the mean of five wells ± SD.
    Sirna Against Gfp Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher gfp 5 ggcuacguccaggagcgca 3 mrnas
    Overexpression or down-regulation of SCCAs significantly affected UV-induced apoptosis. (A) FACS analyses of apoptotic cells using SCCA1- or SCCA2-transfected 3T3/J2 cells, 48 h after UV irradiation (30 mJ/cm 2 ). Cells were stained with FITC-conjugated Annexin V and propidium iodide. (B) Analyses of five experiments are summarized. (C) 12 clones whose expression levels of SCCA1 <t>mRNA</t> distributed from 1 to 2,772-fold were established. Using these clones, the effects of UV irradiation were examined. Cells were harvested 48 h after UV irradiation (50 mJ/cm 2 ) and FACS analyses were performed. The antiapoptotic activity correlated with SCCA1 expression. r = 0.734. (D) Using pSilencer vector, an <t>siRNA</t> construct targeted to a homologous sequence of SCCAs was stably transfected into HaCaT keratinocytes. Typical FACS analyses of nonirradiated (UV−) and UV-irradiated (UV+) siSCCA/HaCaT cells were shown. Apoptotic cells were analyzed 48 h after UV irradiation (75 mJ/cm 2 ). (E) Statistical analyses of five experiments. Error bars represent the mean of five wells ± SD.
    Gfp 5 Ggcuacguccaggagcgca 3 Mrnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher gfp mrna expression trizol
    Overexpression or down-regulation of SCCAs significantly affected UV-induced apoptosis. (A) FACS analyses of apoptotic cells using SCCA1- or SCCA2-transfected 3T3/J2 cells, 48 h after UV irradiation (30 mJ/cm 2 ). Cells were stained with FITC-conjugated Annexin V and propidium iodide. (B) Analyses of five experiments are summarized. (C) 12 clones whose expression levels of SCCA1 <t>mRNA</t> distributed from 1 to 2,772-fold were established. Using these clones, the effects of UV irradiation were examined. Cells were harvested 48 h after UV irradiation (50 mJ/cm 2 ) and FACS analyses were performed. The antiapoptotic activity correlated with SCCA1 expression. r = 0.734. (D) Using pSilencer vector, an <t>siRNA</t> construct targeted to a homologous sequence of SCCAs was stably transfected into HaCaT keratinocytes. Typical FACS analyses of nonirradiated (UV−) and UV-irradiated (UV+) siSCCA/HaCaT cells were shown. Apoptotic cells were analyzed 48 h after UV irradiation (75 mJ/cm 2 ). (E) Statistical analyses of five experiments. Error bars represent the mean of five wells ± SD.
    Gfp Mrna Expression Trizol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Miltenyi Biotec gfp expressing mrna plasmid
    Overexpression or down-regulation of SCCAs significantly affected UV-induced apoptosis. (A) FACS analyses of apoptotic cells using SCCA1- or SCCA2-transfected 3T3/J2 cells, 48 h after UV irradiation (30 mJ/cm 2 ). Cells were stained with FITC-conjugated Annexin V and propidium iodide. (B) Analyses of five experiments are summarized. (C) 12 clones whose expression levels of SCCA1 <t>mRNA</t> distributed from 1 to 2,772-fold were established. Using these clones, the effects of UV irradiation were examined. Cells were harvested 48 h after UV irradiation (50 mJ/cm 2 ) and FACS analyses were performed. The antiapoptotic activity correlated with SCCA1 expression. r = 0.734. (D) Using pSilencer vector, an <t>siRNA</t> construct targeted to a homologous sequence of SCCAs was stably transfected into HaCaT keratinocytes. Typical FACS analyses of nonirradiated (UV−) and UV-irradiated (UV+) siSCCA/HaCaT cells were shown. Apoptotic cells were analyzed 48 h after UV irradiation (75 mJ/cm 2 ). (E) Statistical analyses of five experiments. Error bars represent the mean of five wells ± SD.
    Gfp Expressing Mrna Plasmid, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TriLink pdna enhanced gfp egfp mrna
    Overexpression or down-regulation of SCCAs significantly affected UV-induced apoptosis. (A) FACS analyses of apoptotic cells using SCCA1- or SCCA2-transfected 3T3/J2 cells, 48 h after UV irradiation (30 mJ/cm 2 ). Cells were stained with FITC-conjugated Annexin V and propidium iodide. (B) Analyses of five experiments are summarized. (C) 12 clones whose expression levels of SCCA1 <t>mRNA</t> distributed from 1 to 2,772-fold were established. Using these clones, the effects of UV irradiation were examined. Cells were harvested 48 h after UV irradiation (50 mJ/cm 2 ) and FACS analyses were performed. The antiapoptotic activity correlated with SCCA1 expression. r = 0.734. (D) Using pSilencer vector, an <t>siRNA</t> construct targeted to a homologous sequence of SCCAs was stably transfected into HaCaT keratinocytes. Typical FACS analyses of nonirradiated (UV−) and UV-irradiated (UV+) siSCCA/HaCaT cells were shown. Apoptotic cells were analyzed 48 h after UV irradiation (75 mJ/cm 2 ). (E) Statistical analyses of five experiments. Error bars represent the mean of five wells ± SD.
    Pdna Enhanced Gfp Egfp Mrna, supplied by TriLink, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    VANGL2 LTD synthetic gfp vangl2 mrna
    <t>Vangl2</t> membrane localization in embryos with defective PCP signaling. (A-B′) Live embryos mosaically expressing <t>GFP-VANGL2</t> and mCherry. Arrows indicate individual cells showing anteriorly biased Vangl2 membrane localization. (C) Quantification of mGFP (green) and mCherry (red) posterior/anterior membrane FI ratios obtained by analyzing individual cells in the notochord of 5-6 s stage (11.7-12 hpf) embryos in specified mutants or Xdd1 RNA-injected embryos. The data plot shows the quantification of either GFP-Vangl2 from Tg(vangl2:GFP-Vangl2) cells in MZ vangl2 vu67/vu67 and Xdd1 -injected embryos, or of GFP-VANGL2 in MZ fz7a e3/e3 ; MZ fz7b hu3465/hu3465 and kny fr6/fr6 embryos. GFP-Vangl2 and GFP-VANGL2 quantification data are combined for the WT embryos. Blue bar indicates the average posterior/anterior FI ratio for each condition. ** P
    Synthetic Gfp Vangl2 Mrna, supplied by VANGL2 LTD, used in various techniques. Bioz Stars score: 88/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mesendoderm induction by Mxtx2. ( A-X ) Control gfp mRNA (50 pg) or mxtx2 mRNA (10 pg) was injected into whole zebrafish embryos (A-L), or Ctrl MO (8 ng) or Mxtx2 MO (2 ng) was injected into the yolk syncytial layer (YSL) (M-X), and embryos were fixed at 30% epiboly (4.7 hpf) and stained by whole-mount in situ hybridization (WISH) for the indicated markers. Animal pole views are shown. Fractions indicate the number of equivalent outcomes/number of embryos observed. See also Fig. S1 in the supplementary material.

    Journal: Development (Cambridge, England)

    Article Title: Embryonic mesoderm and endoderm induction requires the actions of non-embryonic Nodal-related ligands and Mxtx2

    doi: 10.1242/dev.058974

    Figure Lengend Snippet: Mesendoderm induction by Mxtx2. ( A-X ) Control gfp mRNA (50 pg) or mxtx2 mRNA (10 pg) was injected into whole zebrafish embryos (A-L), or Ctrl MO (8 ng) or Mxtx2 MO (2 ng) was injected into the yolk syncytial layer (YSL) (M-X), and embryos were fixed at 30% epiboly (4.7 hpf) and stained by whole-mount in situ hybridization (WISH) for the indicated markers. Animal pole views are shown. Fractions indicate the number of equivalent outcomes/number of embryos observed. See also Fig. S1 in the supplementary material.

    Article Snippet: Briefly, eng-mxtx2, vp16-mxtx2 mRNA or gfp mRNA was injected as above, but a portion of the embryos were then treated with 50 μg/ml cycloheximide (Sigma) beginning at the 64- to 128-cell stage, and all embryos were fixed when untreated embryos reached the 30% epiboly stage and stained for ndr2 expression.

    Techniques: Injection, Staining, In Situ Hybridization

    AUF1 p37 and p42 bind to the IL-6 3′UTR in NIH 3T3 cells only in the presence of the critical ARE at site L. (A) Each of the four myc-tagged AUF1 isoforms was expressed in mouse NIH 3T3 cells in the presence of the GFP-IL-6_wt construct. The proteins of whole-cell lysates were immunoprecipitated with anti-myc tag antibody. The amounts of GFP-IL-6 mRNA bound to antibody-coupled beads and GFP-IL-6 mRNA left in the supernatant were quantified by real-time PCR. Their sum was considered the total mRNA recovered (100%). The ratio of coprecipitated to total mRNA was calculated for each isoform and is shown on the graph. mARP0 mRNA served as a control for nonspecific coprecipitation. The values reported are averages from at least three independent experiments ± SD. (B) The binding of myc-tagged AUF1 p37 to various GFP-IL-6 constructs was assessed by the same method. The values reported are averages from three experiments ± SD.

    Journal: Molecular and Cellular Biology

    Article Title: Destabilization of Interleukin-6 mRNA Requires a Putative RNA Stem-Loop Structure, an AU-Rich Element, and the RNA-Binding Protein AUF1 ▿

    doi: 10.1128/MCB.01155-06

    Figure Lengend Snippet: AUF1 p37 and p42 bind to the IL-6 3′UTR in NIH 3T3 cells only in the presence of the critical ARE at site L. (A) Each of the four myc-tagged AUF1 isoforms was expressed in mouse NIH 3T3 cells in the presence of the GFP-IL-6_wt construct. The proteins of whole-cell lysates were immunoprecipitated with anti-myc tag antibody. The amounts of GFP-IL-6 mRNA bound to antibody-coupled beads and GFP-IL-6 mRNA left in the supernatant were quantified by real-time PCR. Their sum was considered the total mRNA recovered (100%). The ratio of coprecipitated to total mRNA was calculated for each isoform and is shown on the graph. mARP0 mRNA served as a control for nonspecific coprecipitation. The values reported are averages from at least three independent experiments ± SD. (B) The binding of myc-tagged AUF1 p37 to various GFP-IL-6 constructs was assessed by the same method. The values reported are averages from three experiments ± SD.

    Article Snippet: To perform IP of mRNA with AUF1, 0.5 × 106 to 1 × 106 NIH 3T3 cells stably expressing myc-tagged AUF1 isoforms and GFP mRNA constructs were lysed in 400 μl CelLytic-M cell lysis reagent (C-2978; Sigma).

    Techniques: Construct, Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    Effect of the proteasome inhibitor MG132 on IL-6 mRNA and AUF1 stability. (A) The decay of GFP-IL-6_wt mRNA in mouse NIH 3T3 cells was analyzed using the Tet-Off system and real-time PCR after a treatment for 4 h with 40 μM MG132. Average results ± SD of four experiments are shown, and half-lives were calculated by linear regression of semilogarithmic plots. (B) Exogenous myc-tagged AUF1 p37 was induced with mifepristone in NIH 3T3 cells and its level analyzed on a Western blot before and after treatment for 4 h with 40 μM MG132. Expression of γ-tubulin was unchanged by MG132 and served as a loading control.

    Journal: Molecular and Cellular Biology

    Article Title: Destabilization of Interleukin-6 mRNA Requires a Putative RNA Stem-Loop Structure, an AU-Rich Element, and the RNA-Binding Protein AUF1 ▿

    doi: 10.1128/MCB.01155-06

    Figure Lengend Snippet: Effect of the proteasome inhibitor MG132 on IL-6 mRNA and AUF1 stability. (A) The decay of GFP-IL-6_wt mRNA in mouse NIH 3T3 cells was analyzed using the Tet-Off system and real-time PCR after a treatment for 4 h with 40 μM MG132. Average results ± SD of four experiments are shown, and half-lives were calculated by linear regression of semilogarithmic plots. (B) Exogenous myc-tagged AUF1 p37 was induced with mifepristone in NIH 3T3 cells and its level analyzed on a Western blot before and after treatment for 4 h with 40 μM MG132. Expression of γ-tubulin was unchanged by MG132 and served as a loading control.

    Article Snippet: To perform IP of mRNA with AUF1, 0.5 × 106 to 1 × 106 NIH 3T3 cells stably expressing myc-tagged AUF1 isoforms and GFP mRNA constructs were lysed in 400 μl CelLytic-M cell lysis reagent (C-2978; Sigma).

    Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing

    RNAi against endogenous AUF1 stabilizes GFP-IL-6 mRNA. (A) Western blot analyzed with polyclonal anti-AUF1 and anti-γ-tubulin antibodies after expression of three different interfering RNAs in mouse NIH 3T3 cells. Oligo, oligonucleotide. (B) Half-lives of GFP-IL-6 mRNA measured by the Tet-Off system before and after infection with a retroviral RNAi vector carrying the oligonucleotide 301 sequence, which targets AUF1 exon 5.

    Journal: Molecular and Cellular Biology

    Article Title: Destabilization of Interleukin-6 mRNA Requires a Putative RNA Stem-Loop Structure, an AU-Rich Element, and the RNA-Binding Protein AUF1 ▿

    doi: 10.1128/MCB.01155-06

    Figure Lengend Snippet: RNAi against endogenous AUF1 stabilizes GFP-IL-6 mRNA. (A) Western blot analyzed with polyclonal anti-AUF1 and anti-γ-tubulin antibodies after expression of three different interfering RNAs in mouse NIH 3T3 cells. Oligo, oligonucleotide. (B) Half-lives of GFP-IL-6 mRNA measured by the Tet-Off system before and after infection with a retroviral RNAi vector carrying the oligonucleotide 301 sequence, which targets AUF1 exon 5.

    Article Snippet: To perform IP of mRNA with AUF1, 0.5 × 106 to 1 × 106 NIH 3T3 cells stably expressing myc-tagged AUF1 isoforms and GFP mRNA constructs were lysed in 400 μl CelLytic-M cell lysis reagent (C-2978; Sigma).

    Techniques: Western Blot, Expressing, Infection, Plasmid Preparation, Sequencing

    The IL-6 3′UTR confers mRNA instability on a stable GFP mRNA. GFP mRNA with or without the 3′UTR of human IL-6 mRNA was stably expressed in COS-7 cells. Transcription was blocked by actinomycin D (Act D). (A) GFP mRNA was measured at different time points on Northern blots by hybridization with a GFP-specific probe and normalized to endogenous GAPDH mRNA. (B) The mRNA decay was plotted on a semilogarithmic scale and the half-life calculated by linear regression. Error bars indicate standard deviations (SD) from at least three independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Destabilization of Interleukin-6 mRNA Requires a Putative RNA Stem-Loop Structure, an AU-Rich Element, and the RNA-Binding Protein AUF1 ▿

    doi: 10.1128/MCB.01155-06

    Figure Lengend Snippet: The IL-6 3′UTR confers mRNA instability on a stable GFP mRNA. GFP mRNA with or without the 3′UTR of human IL-6 mRNA was stably expressed in COS-7 cells. Transcription was blocked by actinomycin D (Act D). (A) GFP mRNA was measured at different time points on Northern blots by hybridization with a GFP-specific probe and normalized to endogenous GAPDH mRNA. (B) The mRNA decay was plotted on a semilogarithmic scale and the half-life calculated by linear regression. Error bars indicate standard deviations (SD) from at least three independent experiments.

    Article Snippet: To perform IP of mRNA with AUF1, 0.5 × 106 to 1 × 106 NIH 3T3 cells stably expressing myc-tagged AUF1 isoforms and GFP mRNA constructs were lysed in 400 μl CelLytic-M cell lysis reagent (C-2978; Sigma).

    Techniques: Stable Transfection, Activated Clotting Time Assay, Northern Blot, Hybridization

    Characterizations of LPNs for mRNA delivery using a luciferase assay. ( a ) Fold changes of luciferase expression levels using different polymer incorporated LPNs as compared to TT3 LLNs; ( b ) size, ( c ) zeta potential, and ( d ) encapsulation efficiency of different FLuc mRNA encapsulated LPNs. Note A, B and C represents the mass ratio of polymer to mRNA equal to 9:1, 3:1 and 1:1, respectively. ( n = 3; two-tailed t test; ** p

    Journal: Cellular and Molecular Bioengineering

    Article Title: Lipid Polymer Hybrid Nanomaterials for mRNA Delivery

    doi: 10.1007/s12195-018-0536-9

    Figure Lengend Snippet: Characterizations of LPNs for mRNA delivery using a luciferase assay. ( a ) Fold changes of luciferase expression levels using different polymer incorporated LPNs as compared to TT3 LLNs; ( b ) size, ( c ) zeta potential, and ( d ) encapsulation efficiency of different FLuc mRNA encapsulated LPNs. Note A, B and C represents the mass ratio of polymer to mRNA equal to 9:1, 3:1 and 1:1, respectively. ( n = 3; two-tailed t test; ** p

    Article Snippet: Firefly luciferase mRNAs (FLuc mRNAs) and enhanced green fluorescent protein mRNA (eGFP mRNAs) were purchased from TriLink Biotechnologies, Inc. (San Diego, CA).

    Techniques: Luciferase, Expressing, Two Tailed Test

    Evaluation of LPNs to deliver eGFP mRNA. ( a ) Fold changes of eGFP expression levels using different polymer incorporated LPNs as compared to TT3 LLNs; ( b ) size, ( c ) zeta potential, and ( d ) encapsulation efficiency of different eGFP encapsulated LPNs. ( n = 3; two-tailed t -test; n.s . not significant; *** p

    Journal: Cellular and Molecular Bioengineering

    Article Title: Lipid Polymer Hybrid Nanomaterials for mRNA Delivery

    doi: 10.1007/s12195-018-0536-9

    Figure Lengend Snippet: Evaluation of LPNs to deliver eGFP mRNA. ( a ) Fold changes of eGFP expression levels using different polymer incorporated LPNs as compared to TT3 LLNs; ( b ) size, ( c ) zeta potential, and ( d ) encapsulation efficiency of different eGFP encapsulated LPNs. ( n = 3; two-tailed t -test; n.s . not significant; *** p

    Article Snippet: Firefly luciferase mRNAs (FLuc mRNAs) and enhanced green fluorescent protein mRNA (eGFP mRNAs) were purchased from TriLink Biotechnologies, Inc. (San Diego, CA).

    Techniques: Expressing, Two Tailed Test

    Absence of Gas2l2 in X. laevis Affects Cilia Rotational Polarity (A) Detection of Gas2l2 in a wild-type skin ciliated cell by immunofluorescence. (B and C) Representative immunofluorescence images visualizing basal bodies (Clamp-GFP) and rootlet (Centrin 4-RFP) to score basal body-rootlet alignment. (B′ and C′) Alignment of the basal body and rootlet in control - MO (n = 19, mean vector, black arrow, r = 0.98, CSD = 11.5°) and Gas2l2 -MO (n = 21, r = 0.67, CSD = 51.2). (D) The vector length (R) of each analyzed cell is represented in the graph. The average length of the vector was significantly shorter in Gas2l2 -MO (R = 0.5884 ± 0.1259) cells than in controls (R = 0.836 ± 0.0847). The introduction of human GAS2L2 mRNA in morpholino-treated embryos ( GAS2L2-R ) rescued the phenotype (n = 23, R = 0.7728 ± 0.1322) (ANOVA, multiple comparison, p

    Journal: American Journal of Human Genetics

    Article Title: Lack of GAS2L2 Causes PCD by Impairing Cilia Orientation and Mucociliary Clearance

    doi: 10.1016/j.ajhg.2018.12.009

    Figure Lengend Snippet: Absence of Gas2l2 in X. laevis Affects Cilia Rotational Polarity (A) Detection of Gas2l2 in a wild-type skin ciliated cell by immunofluorescence. (B and C) Representative immunofluorescence images visualizing basal bodies (Clamp-GFP) and rootlet (Centrin 4-RFP) to score basal body-rootlet alignment. (B′ and C′) Alignment of the basal body and rootlet in control - MO (n = 19, mean vector, black arrow, r = 0.98, CSD = 11.5°) and Gas2l2 -MO (n = 21, r = 0.67, CSD = 51.2). (D) The vector length (R) of each analyzed cell is represented in the graph. The average length of the vector was significantly shorter in Gas2l2 -MO (R = 0.5884 ± 0.1259) cells than in controls (R = 0.836 ± 0.0847). The introduction of human GAS2L2 mRNA in morpholino-treated embryos ( GAS2L2-R ) rescued the phenotype (n = 23, R = 0.7728 ± 0.1322) (ANOVA, multiple comparison, p

    Article Snippet: Coinjection of Centrin 4-RFP mRNA (Unigene ID: Xl.50473) and Clamp-GFP mRNA (Unigene ID: Xl.26316) labeled basal bodies and ciliary rootlets, respectively.

    Techniques: Immunofluorescence, Plasmid Preparation

    Overexpression of an ectopic VSG causes silencing of the ES-resident VSG. Both (A, B) mRNA and (C, D) protein levels of the endogenous VSG A1.1 (green) and the ectopic VSG 121 (magenta) were monitored in growth arrested (left) and proliferating (right) clones during the course of ectopic VSG overexpression. In all graphs triangles correspond to the ES-promoter cell line (GFP ESpro A1.1 ES 121 tet ) and circles to the stumpy reporter cell line (GFP:PAD1 UTR A1.1 ES 121 tet ). Note that the initial VSG overexpression levels are comparable in arrested and proliferating parasites. For the quantification of (A, B) mRNA levels, total RNA samples were dot-blotted and hybridized with infrared fluorescently labeled probes, specific for VSG 121 or VSG A1 . 1 . The data were quantified and normalized to β-tubulin mRNA using the Licor Odyssey system. (C, D) VSG protein levels were quantified by dot-blotting 6x 10 5 cell equivalents. The blots were incubated with an anti-VSG 121 or an anti-VSG A1.1 antibody. A histone H3 antibody was used for normalization. The VSG expression levels are given relative to VSG 121 expression levels of MITat1.6 wild type cells and parental AnTat1.1 cells natively expressing VSG A1.1. The dashed grey line indicates wild type expression levels (100%).

    Journal: PLoS Pathogens

    Article Title: A quorum sensing-independent path to stumpy development in Trypanosoma brucei

    doi: 10.1371/journal.ppat.1006324

    Figure Lengend Snippet: Overexpression of an ectopic VSG causes silencing of the ES-resident VSG. Both (A, B) mRNA and (C, D) protein levels of the endogenous VSG A1.1 (green) and the ectopic VSG 121 (magenta) were monitored in growth arrested (left) and proliferating (right) clones during the course of ectopic VSG overexpression. In all graphs triangles correspond to the ES-promoter cell line (GFP ESpro A1.1 ES 121 tet ) and circles to the stumpy reporter cell line (GFP:PAD1 UTR A1.1 ES 121 tet ). Note that the initial VSG overexpression levels are comparable in arrested and proliferating parasites. For the quantification of (A, B) mRNA levels, total RNA samples were dot-blotted and hybridized with infrared fluorescently labeled probes, specific for VSG 121 or VSG A1 . 1 . The data were quantified and normalized to β-tubulin mRNA using the Licor Odyssey system. (C, D) VSG protein levels were quantified by dot-blotting 6x 10 5 cell equivalents. The blots were incubated with an anti-VSG 121 or an anti-VSG A1.1 antibody. A histone H3 antibody was used for normalization. The VSG expression levels are given relative to VSG 121 expression levels of MITat1.6 wild type cells and parental AnTat1.1 cells natively expressing VSG A1.1. The dashed grey line indicates wild type expression levels (100%).

    Article Snippet: GFP mRNA was detected with a 32 P-labeled probe (complete eGFP ORF, Thermo Scientific DecaLabel DNA Labeling Kit) and quantified using a Phosphorimager.

    Techniques: Over Expression, Labeling, Incubation, Expressing

    Silencing of the ES-resident VSG is independent of ES-attenuation. The transcriptional status of the active ES of growth arrested (left) and proliferating (right) ectopic VSG 121 overexpressors was monitored. (A, B) The GFP ESpro reporter was used to detect transcripts from the ES promotor region ( GFP ) and (C, D) ESAG6 was measured as an example for a native ES transcript. All data points reflect measurements on the single cell level using mRNA FISH (Affymetrix). The signal intensity in deconvolved, summed slice projections (100 images, z-step 100 nm) was measured with Image J and is represented as relative fluorescent unit (RFU). Two different induction times (24 and 48 h) were analyzed, and non-induced long slender (0 h) or density-induced short stumpy cells (st) served as controls. Only parasites in G1-phase of the cell cycle were analyzed. The magenta bars are means ± SD (n > 100). Statistical analysis was conducted using an unpaired t-test (not significant (n.s.) p-value > 0.05; ** p-value

    Journal: PLoS Pathogens

    Article Title: A quorum sensing-independent path to stumpy development in Trypanosoma brucei

    doi: 10.1371/journal.ppat.1006324

    Figure Lengend Snippet: Silencing of the ES-resident VSG is independent of ES-attenuation. The transcriptional status of the active ES of growth arrested (left) and proliferating (right) ectopic VSG 121 overexpressors was monitored. (A, B) The GFP ESpro reporter was used to detect transcripts from the ES promotor region ( GFP ) and (C, D) ESAG6 was measured as an example for a native ES transcript. All data points reflect measurements on the single cell level using mRNA FISH (Affymetrix). The signal intensity in deconvolved, summed slice projections (100 images, z-step 100 nm) was measured with Image J and is represented as relative fluorescent unit (RFU). Two different induction times (24 and 48 h) were analyzed, and non-induced long slender (0 h) or density-induced short stumpy cells (st) served as controls. Only parasites in G1-phase of the cell cycle were analyzed. The magenta bars are means ± SD (n > 100). Statistical analysis was conducted using an unpaired t-test (not significant (n.s.) p-value > 0.05; ** p-value

    Article Snippet: GFP mRNA was detected with a 32 P-labeled probe (complete eGFP ORF, Thermo Scientific DecaLabel DNA Labeling Kit) and quantified using a Phosphorimager.

    Techniques: Fluorescence In Situ Hybridization

    Frozen lung ( A , B ) and liver ( C ) tissue sections from transgenic mice overexpressing GFP 24 hours after intratracheal administration of PBS ( A1 , B1 , C1 ) or GFP-siRNA ( A2 , B2 , C2 ). Mice having received GFP-siRNA display reduced pulmonary but not hepatic

    Journal:

    Article Title: Silencing of Fas, but Not Caspase-8, in Lung Epithelial Cells Ameliorates Pulmonary Apoptosis, Inflammation, and Neutrophil Influx after Hemorrhagic Shock and Sepsis

    doi:

    Figure Lengend Snippet: Frozen lung ( A , B ) and liver ( C ) tissue sections from transgenic mice overexpressing GFP 24 hours after intratracheal administration of PBS ( A1 , B1 , C1 ) or GFP-siRNA ( A2 , B2 , C2 ). Mice having received GFP-siRNA display reduced pulmonary but not hepatic

    Article Snippet: C57BL/6-TgN(ACTbEGFP)1Osb mice were subjected to intratracheal delivery of siRNA targeting GFP mRNA (Dharmacon, Lafayette, CO), C3H/HeN mice were challenged with hemorrhagic shock, intratracheal delivery of siRNA (see below), and cecal ligation and puncture.

    Techniques: Transgenic Assay, Mouse Assay

    Lung tissue MPO staining 24 hours after polymicrobial sepsis and 52 hours after hemorrhagic shock. Mice underwent sham procedures and administration of GFP-siRNA ( A ) or hemorrhage and administration of GFP-siRNA and sepsis ( B ) or hemorrhage and administration

    Journal:

    Article Title: Silencing of Fas, but Not Caspase-8, in Lung Epithelial Cells Ameliorates Pulmonary Apoptosis, Inflammation, and Neutrophil Influx after Hemorrhagic Shock and Sepsis

    doi:

    Figure Lengend Snippet: Lung tissue MPO staining 24 hours after polymicrobial sepsis and 52 hours after hemorrhagic shock. Mice underwent sham procedures and administration of GFP-siRNA ( A ) or hemorrhage and administration of GFP-siRNA and sepsis ( B ) or hemorrhage and administration

    Article Snippet: C57BL/6-TgN(ACTbEGFP)1Osb mice were subjected to intratracheal delivery of siRNA targeting GFP mRNA (Dharmacon, Lafayette, CO), C3H/HeN mice were challenged with hemorrhagic shock, intratracheal delivery of siRNA (see below), and cecal ligation and puncture.

    Techniques: Staining, Mouse Assay

    Lung tissue IFN-α ( A ), IL-6 ( B ), and TNF-α ( C ) concentrations at 18 hours after intratracheal instillation of polyinosinic-polycytidylic acid [poly(I:C)], GFP-siRNA, PBS, or caspase-8-siRNA (C-8) show that administration

    Journal:

    Article Title: Silencing of Fas, but Not Caspase-8, in Lung Epithelial Cells Ameliorates Pulmonary Apoptosis, Inflammation, and Neutrophil Influx after Hemorrhagic Shock and Sepsis

    doi:

    Figure Lengend Snippet: Lung tissue IFN-α ( A ), IL-6 ( B ), and TNF-α ( C ) concentrations at 18 hours after intratracheal instillation of polyinosinic-polycytidylic acid [poly(I:C)], GFP-siRNA, PBS, or caspase-8-siRNA (C-8) show that administration

    Article Snippet: C57BL/6-TgN(ACTbEGFP)1Osb mice were subjected to intratracheal delivery of siRNA targeting GFP mRNA (Dharmacon, Lafayette, CO), C3H/HeN mice were challenged with hemorrhagic shock, intratracheal delivery of siRNA (see below), and cecal ligation and puncture.

    Techniques:

    Representative H E preparation of lung tissue slides from animals 24 hours after polymicrobial sepsis and 52 hours after hemorrhagic shock. Mice underwent sham procedures and GFP-siRNA administration ( A ) or hemorrhage and GFP-siRNA administration

    Journal:

    Article Title: Silencing of Fas, but Not Caspase-8, in Lung Epithelial Cells Ameliorates Pulmonary Apoptosis, Inflammation, and Neutrophil Influx after Hemorrhagic Shock and Sepsis

    doi:

    Figure Lengend Snippet: Representative H E preparation of lung tissue slides from animals 24 hours after polymicrobial sepsis and 52 hours after hemorrhagic shock. Mice underwent sham procedures and GFP-siRNA administration ( A ) or hemorrhage and GFP-siRNA administration

    Article Snippet: C57BL/6-TgN(ACTbEGFP)1Osb mice were subjected to intratracheal delivery of siRNA targeting GFP mRNA (Dharmacon, Lafayette, CO), C3H/HeN mice were challenged with hemorrhagic shock, intratracheal delivery of siRNA (see below), and cecal ligation and puncture.

    Techniques: Mouse Assay

    mRNA reporter assay for screening amber suppression positive stable isolate hosts. ( a) Schematic representation of the process used to generate and identify amber suppression competent cells. CHO cells were first transfected with a plasmid encoding PylRS and tRNApyl and subjected to a selection step by growth in puromycin-containing medium. Surviving isolates were transfected with mRNA encoding RFPambGFP and cells were exposed to AzK. Cells capable of efficient amber codon suppression (positive) express an RFP-GFP fusion and are positive for both fluorophores. Cells lacking amber suppression activity (negative) express only RFP. (b) A stable isolate identified as positive for amber suppression activity was transfected with the RFPambGFP mRNA and incubated in the presence or absence of AzK. Cells were imaged by confocal microscopy and flow cytometry. Amber suppression and incorporation of AzK was observed in presence of PylRS and tRNApyl. No GFP expression was observed in absence of AzK.

    Journal: mAbs

    Article Title: Development of a high yielding expression platform for the introduction of non-natural amino acids in protein sequences

    doi: 10.1080/19420862.2019.1684749

    Figure Lengend Snippet: mRNA reporter assay for screening amber suppression positive stable isolate hosts. ( a) Schematic representation of the process used to generate and identify amber suppression competent cells. CHO cells were first transfected with a plasmid encoding PylRS and tRNApyl and subjected to a selection step by growth in puromycin-containing medium. Surviving isolates were transfected with mRNA encoding RFPambGFP and cells were exposed to AzK. Cells capable of efficient amber codon suppression (positive) express an RFP-GFP fusion and are positive for both fluorophores. Cells lacking amber suppression activity (negative) express only RFP. (b) A stable isolate identified as positive for amber suppression activity was transfected with the RFPambGFP mRNA and incubated in the presence or absence of AzK. Cells were imaged by confocal microscopy and flow cytometry. Amber suppression and incorporation of AzK was observed in presence of PylRS and tRNApyl. No GFP expression was observed in absence of AzK.

    Article Snippet: Synthetic mRNA encoding RFPamb-GFP (TriLink Biotechnologies) transfections were optimized using 1 × 106 cells and 1.7–3.4 pmol mRNA construct using program EH118 in a 96-well shuttle (Lonza).

    Techniques: Reporter Assay, Transfection, Plasmid Preparation, Selection, Activity Assay, Incubation, Confocal Microscopy, Flow Cytometry, Cytometry, Expressing

    Poly-PR peptide interacts with RNA and forms aggregates. ( A ) Agarose gel electrophoresis of solutions containing GFP mRNA with HA peptide or HA-poly-(PR) 20 peptide at indicated concentrations. An arrowhead shows GFP mRNA and an arrow shows insoluble RNA

    Journal: Human Molecular Genetics

    Article Title: Poly-dipeptides encoded by the C9ORF72 repeats block global protein translation

    doi: 10.1093/hmg/ddw052

    Figure Lengend Snippet: Poly-PR peptide interacts with RNA and forms aggregates. ( A ) Agarose gel electrophoresis of solutions containing GFP mRNA with HA peptide or HA-poly-(PR) 20 peptide at indicated concentrations. An arrowhead shows GFP mRNA and an arrow shows insoluble RNA

    Article Snippet: Anti-HA antibody-conjugated agarose beads, 1-step human in vitro protein expression kits, anti-turbo GFP antibody and turbo GFP mRNA were purchased from Pierce (Rockford, IL, USA).

    Techniques: Agarose Gel Electrophoresis

    Comparative analysis of enhanced green fluorescence protein (EGFP)-mRNA induced antiviral responses in 10T1/2 (A) and D3 cells (B) . The cells were transfected with 3p-EGFP-mRNA (EGFP, 300ng/mL) for 12 h. The mRNA levels of genes involved in antiviral

    Journal: Stem Cells and Development

    Article Title: Mouse Embryonic Stem Cells Have Underdeveloped Antiviral Mechanisms That Can Be Exploited for the Development of mRNA-Mediated Gene Expression Strategy

    doi: 10.1089/scd.2013.0417

    Figure Lengend Snippet: Comparative analysis of enhanced green fluorescence protein (EGFP)-mRNA induced antiviral responses in 10T1/2 (A) and D3 cells (B) . The cells were transfected with 3p-EGFP-mRNA (EGFP, 300ng/mL) for 12 h. The mRNA levels of genes involved in antiviral

    Article Snippet: To test how mESCs respond to mRNA modifications, we used a fully functional GFP mRNA with base modifications (purchased from Stemgent).

    Techniques: Fluorescence, Transfection

    X. laevis PGC can migrate in vitro in the under-agarose migration assay. (A) Ventral explants were dissected from the embryos at developmental stage 17–19 or 28–30 injected at 2-cell stage vegetally with GFP_DELE mRNA to label PGCs. Explants were treated with accutase and the dissociated cells were transferred between 0.5% agarose gel and a 5% BSA-coated Petri dish in 0.8× MBSH buffer. (B) Time-lapse images of an under-agarose migration assay with dissociated endodermal cells from stage 28–30 embryos. PGCs can be identified as GFP-positive (green) in contrast to the GFP-negative somatic cells. Red arrows indicate migrating PGC. Relative time from the start of the time-lapse imaging is shown in the upper left corner of each image panel (min:sec). Scale bars: 20 µm. (C) Time-lapse microscopy was used to monitor the behavior of PGCs. Relative amount of migratory and non-migratory cells in the total amount of analyzed PGCs was calculated for each experiment. N – number of experiments. Error bars represent standard deviation. *** corresponds to P

    Journal: Biology Open

    Article Title: Migratory and adhesive properties of Xenopus laevis primordial germ cells in vitro

    doi: 10.1242/bio.20135140

    Figure Lengend Snippet: X. laevis PGC can migrate in vitro in the under-agarose migration assay. (A) Ventral explants were dissected from the embryos at developmental stage 17–19 or 28–30 injected at 2-cell stage vegetally with GFP_DELE mRNA to label PGCs. Explants were treated with accutase and the dissociated cells were transferred between 0.5% agarose gel and a 5% BSA-coated Petri dish in 0.8× MBSH buffer. (B) Time-lapse images of an under-agarose migration assay with dissociated endodermal cells from stage 28–30 embryos. PGCs can be identified as GFP-positive (green) in contrast to the GFP-negative somatic cells. Red arrows indicate migrating PGC. Relative time from the start of the time-lapse imaging is shown in the upper left corner of each image panel (min:sec). Scale bars: 20 µm. (C) Time-lapse microscopy was used to monitor the behavior of PGCs. Relative amount of migratory and non-migratory cells in the total amount of analyzed PGCs was calculated for each experiment. N – number of experiments. Error bars represent standard deviation. *** corresponds to P

    Article Snippet: Chimeric GFP_DELE mRNA was in vitro transcribed from NotI (FastDigest, Fermentas, Vilnius, Lithuania) linearized pCS2+gfpDELE plasmid (pCS2+ vector containing EGFP ORF followed by Xenopus Dead end (dnd1 ) localization element (DELE)) using mMESSAGE mMACHINE SP6 Kit (Ambion, Austin, Texas, USA).

    Techniques: Pyrolysis Gas Chromatography, In Vitro, Migration, Injection, Agarose Gel Electrophoresis, Imaging, Size-exclusion Chromatography, Time-lapse Microscopy, Standard Deviation

    PGCs reduce overall cell-cell adhesion after transition to the active migration state. (A) Cells were isolated from GFP_DELE mRNA injected embryos and transferred to the Petri dish half coated with fibronectin and half coated with bovine serum albumin (BSA). Weakly adhering cells from BSA-coated region were attached to an atomic force microscope cantilever (1). Subsequently, the attached cell is brought into contact with a cell spread on fibronectin-coated part of the Petri dish (2). (B) Fluorescence image of a labeled primordial germ cell spread on a fibronectin-coated Petri dish. (C) Bright-field image of a migratory PGC attached to a poly-D-lysin coated cantilever. Scale bars: 50 µm. (D) Example of a force-distance curve of two somatic cells from the same developmental stage. The approach curve (grey), as well as the retraction curve (black) are shown. (E) Maximum adhesion force either between PGCs and somatic endodermal cells (PGC-Som) or between two somatic endodermal cells (Som-Som) isolated from embryos at stages 17–19 (pre-migratory PGCs) or stages 28–30 (migratory PGCs). Box-whisker plots: lines reflect the median of the distribution, boxes comprise the 25th and 75th percentile, whisker tops and bottoms are drawn to the 10th and 90th percentiles, respectively. N corresponds to the number of curves that have been analyzed per category. *** corresponds to P -values

    Journal: Biology Open

    Article Title: Migratory and adhesive properties of Xenopus laevis primordial germ cells in vitro

    doi: 10.1242/bio.20135140

    Figure Lengend Snippet: PGCs reduce overall cell-cell adhesion after transition to the active migration state. (A) Cells were isolated from GFP_DELE mRNA injected embryos and transferred to the Petri dish half coated with fibronectin and half coated with bovine serum albumin (BSA). Weakly adhering cells from BSA-coated region were attached to an atomic force microscope cantilever (1). Subsequently, the attached cell is brought into contact with a cell spread on fibronectin-coated part of the Petri dish (2). (B) Fluorescence image of a labeled primordial germ cell spread on a fibronectin-coated Petri dish. (C) Bright-field image of a migratory PGC attached to a poly-D-lysin coated cantilever. Scale bars: 50 µm. (D) Example of a force-distance curve of two somatic cells from the same developmental stage. The approach curve (grey), as well as the retraction curve (black) are shown. (E) Maximum adhesion force either between PGCs and somatic endodermal cells (PGC-Som) or between two somatic endodermal cells (Som-Som) isolated from embryos at stages 17–19 (pre-migratory PGCs) or stages 28–30 (migratory PGCs). Box-whisker plots: lines reflect the median of the distribution, boxes comprise the 25th and 75th percentile, whisker tops and bottoms are drawn to the 10th and 90th percentiles, respectively. N corresponds to the number of curves that have been analyzed per category. *** corresponds to P -values

    Article Snippet: Chimeric GFP_DELE mRNA was in vitro transcribed from NotI (FastDigest, Fermentas, Vilnius, Lithuania) linearized pCS2+gfpDELE plasmid (pCS2+ vector containing EGFP ORF followed by Xenopus Dead end (dnd1 ) localization element (DELE)) using mMESSAGE mMACHINE SP6 Kit (Ambion, Austin, Texas, USA).

    Techniques: Migration, Isolation, Injection, Microscopy, Fluorescence, Labeling, Pyrolysis Gas Chromatography, Whisker Assay

    Pre-migratory PGCs show high affinity to fibronectin. (A) Schematic drawing of a Petri dish being divided into three sectors coated either with fibronectin, collagen I or bovine serum albumin (BSA). Either PGCs or somatic cells from both stages were brought into contact with the different substrates using single-cell force spectroscopy. (B) Maximum adhesion forces of either PGCs or somatic cells (Som) isolated from GFP_DELE mRNA injected embryos at developmental stage 17–19 (pre-migratory PGCs) or stage 28–30 (migratory PGCs) during interaction with BSA (B), collagen I (C) or fibronectin (F) coated surfaces. The most relevant change in interaction, the decline in binding strength of PGCs to fibronectin during development, is shown in blue bars. At least 37 force curves per category have been recorded and analyzed. Values are obtained from AFM force-distance curves. Negative forces correspond to positive adhesion forces. Error bars show standard deviations. All p-values obtained from either Wilcoxon rank sum tests or two-tailed t-tests are given in supplementary material Table S2 . (C,D) Typical AFM force-distance curves monitoring the interaction of a pre-migratory primordial germ cell with a (C) BSA-coated and a (D) fibronectin-coated part of the Petri dish.

    Journal: Biology Open

    Article Title: Migratory and adhesive properties of Xenopus laevis primordial germ cells in vitro

    doi: 10.1242/bio.20135140

    Figure Lengend Snippet: Pre-migratory PGCs show high affinity to fibronectin. (A) Schematic drawing of a Petri dish being divided into three sectors coated either with fibronectin, collagen I or bovine serum albumin (BSA). Either PGCs or somatic cells from both stages were brought into contact with the different substrates using single-cell force spectroscopy. (B) Maximum adhesion forces of either PGCs or somatic cells (Som) isolated from GFP_DELE mRNA injected embryos at developmental stage 17–19 (pre-migratory PGCs) or stage 28–30 (migratory PGCs) during interaction with BSA (B), collagen I (C) or fibronectin (F) coated surfaces. The most relevant change in interaction, the decline in binding strength of PGCs to fibronectin during development, is shown in blue bars. At least 37 force curves per category have been recorded and analyzed. Values are obtained from AFM force-distance curves. Negative forces correspond to positive adhesion forces. Error bars show standard deviations. All p-values obtained from either Wilcoxon rank sum tests or two-tailed t-tests are given in supplementary material Table S2 . (C,D) Typical AFM force-distance curves monitoring the interaction of a pre-migratory primordial germ cell with a (C) BSA-coated and a (D) fibronectin-coated part of the Petri dish.

    Article Snippet: Chimeric GFP_DELE mRNA was in vitro transcribed from NotI (FastDigest, Fermentas, Vilnius, Lithuania) linearized pCS2+gfpDELE plasmid (pCS2+ vector containing EGFP ORF followed by Xenopus Dead end (dnd1 ) localization element (DELE)) using mMESSAGE mMACHINE SP6 Kit (Ambion, Austin, Texas, USA).

    Techniques: Spectroscopy, Isolation, Injection, Binding Assay, Two Tailed Test

    Overexpression or down-regulation of SCCAs significantly affected UV-induced apoptosis. (A) FACS analyses of apoptotic cells using SCCA1- or SCCA2-transfected 3T3/J2 cells, 48 h after UV irradiation (30 mJ/cm 2 ). Cells were stained with FITC-conjugated Annexin V and propidium iodide. (B) Analyses of five experiments are summarized. (C) 12 clones whose expression levels of SCCA1 mRNA distributed from 1 to 2,772-fold were established. Using these clones, the effects of UV irradiation were examined. Cells were harvested 48 h after UV irradiation (50 mJ/cm 2 ) and FACS analyses were performed. The antiapoptotic activity correlated with SCCA1 expression. r = 0.734. (D) Using pSilencer vector, an siRNA construct targeted to a homologous sequence of SCCAs was stably transfected into HaCaT keratinocytes. Typical FACS analyses of nonirradiated (UV−) and UV-irradiated (UV+) siSCCA/HaCaT cells were shown. Apoptotic cells were analyzed 48 h after UV irradiation (75 mJ/cm 2 ). (E) Statistical analyses of five experiments. Error bars represent the mean of five wells ± SD.

    Journal: The Journal of Cell Biology

    Article Title: Serpin squamous cell carcinoma antigen inhibits UV-induced apoptosis via suppression of c-JUN NH2-terminal kinase

    doi: 10.1083/jcb.200508064

    Figure Lengend Snippet: Overexpression or down-regulation of SCCAs significantly affected UV-induced apoptosis. (A) FACS analyses of apoptotic cells using SCCA1- or SCCA2-transfected 3T3/J2 cells, 48 h after UV irradiation (30 mJ/cm 2 ). Cells were stained with FITC-conjugated Annexin V and propidium iodide. (B) Analyses of five experiments are summarized. (C) 12 clones whose expression levels of SCCA1 mRNA distributed from 1 to 2,772-fold were established. Using these clones, the effects of UV irradiation were examined. Cells were harvested 48 h after UV irradiation (50 mJ/cm 2 ) and FACS analyses were performed. The antiapoptotic activity correlated with SCCA1 expression. r = 0.734. (D) Using pSilencer vector, an siRNA construct targeted to a homologous sequence of SCCAs was stably transfected into HaCaT keratinocytes. Typical FACS analyses of nonirradiated (UV−) and UV-irradiated (UV+) siSCCA/HaCaT cells were shown. Apoptotic cells were analyzed 48 h after UV irradiation (75 mJ/cm 2 ). (E) Statistical analyses of five experiments. Error bars represent the mean of five wells ± SD.

    Article Snippet: Vector-based siRNA A double-stranded oligonucleotide was designed corresponding to a common sequence of human SCCAs (5′-AAGCCAACACCAAGTTCATGT-3′) to allow formation of the hairpin structure in the expressed oligo-RNA, cloned into the pSilencer vector (Ambion), and transfected into human keratinocyte cell line HaCaT cells. siRNA against GFP mRNA (Ambion) was used as a control.

    Techniques: Over Expression, FACS, Transfection, Irradiation, Staining, Clone Assay, Expressing, Activity Assay, Plasmid Preparation, Construct, Sequencing, Stable Transfection

    Vangl2 membrane localization in embryos with defective PCP signaling. (A-B′) Live embryos mosaically expressing GFP-VANGL2 and mCherry. Arrows indicate individual cells showing anteriorly biased Vangl2 membrane localization. (C) Quantification of mGFP (green) and mCherry (red) posterior/anterior membrane FI ratios obtained by analyzing individual cells in the notochord of 5-6 s stage (11.7-12 hpf) embryos in specified mutants or Xdd1 RNA-injected embryos. The data plot shows the quantification of either GFP-Vangl2 from Tg(vangl2:GFP-Vangl2) cells in MZ vangl2 vu67/vu67 and Xdd1 -injected embryos, or of GFP-VANGL2 in MZ fz7a e3/e3 ; MZ fz7b hu3465/hu3465 and kny fr6/fr6 embryos. GFP-Vangl2 and GFP-VANGL2 quantification data are combined for the WT embryos. Blue bar indicates the average posterior/anterior FI ratio for each condition. ** P

    Journal: Development (Cambridge, England)

    Article Title: A dynamic intracellular distribution of Vangl2 accompanies cell polarization during zebrafish gastrulation

    doi: 10.1242/dev.119032

    Figure Lengend Snippet: Vangl2 membrane localization in embryos with defective PCP signaling. (A-B′) Live embryos mosaically expressing GFP-VANGL2 and mCherry. Arrows indicate individual cells showing anteriorly biased Vangl2 membrane localization. (C) Quantification of mGFP (green) and mCherry (red) posterior/anterior membrane FI ratios obtained by analyzing individual cells in the notochord of 5-6 s stage (11.7-12 hpf) embryos in specified mutants or Xdd1 RNA-injected embryos. The data plot shows the quantification of either GFP-Vangl2 from Tg(vangl2:GFP-Vangl2) cells in MZ vangl2 vu67/vu67 and Xdd1 -injected embryos, or of GFP-VANGL2 in MZ fz7a e3/e3 ; MZ fz7b hu3465/hu3465 and kny fr6/fr6 embryos. GFP-Vangl2 and GFP-VANGL2 quantification data are combined for the WT embryos. Blue bar indicates the average posterior/anterior FI ratio for each condition. ** P

    Article Snippet: To express GFP-VANGL2 in a mosaic fashion, we injected synthetic GFP-VANGL2 mRNA into one blastomere of WT embryos at the 64-cell stage ( F), after first titrating the mRNA to establish the concentration that would enable us to visualize fluorescence without causing gastrulation defects due to excess VANGL2 expression ( ). mCherry mRNA was co-injected as a control.

    Techniques: Expressing, Injection

    Membrane localization of Vangl2 in embryos with patterning defects. Confocal images of whole-mount immunostaining with antibodies against zebrafish Vangl2 C-terminus (A-C) and β-catenin (A′-C′) in MZ oep tz257/tz257 (A,A′) and MZ ichabod (B,B′) embryos, and WT embryos injected with 100 pg noggin synthetic mRNA (C,C′). Scale bars: 20 µm.

    Journal: Development (Cambridge, England)

    Article Title: A dynamic intracellular distribution of Vangl2 accompanies cell polarization during zebrafish gastrulation

    doi: 10.1242/dev.119032

    Figure Lengend Snippet: Membrane localization of Vangl2 in embryos with patterning defects. Confocal images of whole-mount immunostaining with antibodies against zebrafish Vangl2 C-terminus (A-C) and β-catenin (A′-C′) in MZ oep tz257/tz257 (A,A′) and MZ ichabod (B,B′) embryos, and WT embryos injected with 100 pg noggin synthetic mRNA (C,C′). Scale bars: 20 µm.

    Article Snippet: To express GFP-VANGL2 in a mosaic fashion, we injected synthetic GFP-VANGL2 mRNA into one blastomere of WT embryos at the 64-cell stage ( F), after first titrating the mRNA to establish the concentration that would enable us to visualize fluorescence without causing gastrulation defects due to excess VANGL2 expression ( ). mCherry mRNA was co-injected as a control.

    Techniques: Immunostaining, Injection

    Enrichment of Vangl2 at anterior membranes of mediolaterally elongated cells. (A-C′) Live embryos at 90% epiboly stage (9 hpf; A,A′) or 5 s stage (11.7 hpf; B-C′). mCherry- and GFP-Vangl2-expressing cells were transplanted from Tg(vangl2:GFP-Vangl2) embryos into the WT unlabeled host. Yellow arrowheads indicate the anterior cell membranes where Vangl2 is enriched. (D,E) GFP-Vangl2 and mCherry posterior/anterior membrane FI ratios of individual cells in the notochord at 90% epiboly stage (9 hpf) and at 5-6 s stage (11.7-12 hpf) (D) or in the neuroectoderm at 5-6 s stage (11.7-12 hpf) (E). The blue bars represent the average posterior/anterior FI ratios for each condition. (F-I) Vangl2 asymmetry analysis using GFP-VANGL2 mosaic expression. (F) Modified image of 64-cell stage embryo showing the mosaic synthetic mRNA injection performed. (G) Live embryos mosaically expressing GFP-VANGL2 (green)/H2B-RFP (red) in notochord cells. (H) Plot Profile (Fiji) quantification of FI of two cells (white rectangle in G) at four different z -planes (confocal optical slices). (I) Comparison of GFP-VANGL2, GFP-Vangl2 and corresponding mCherry membrane FI ratios of individual cells in the notochord at 5-6 s (11.7-12 hpf) stage. * P

    Journal: Development (Cambridge, England)

    Article Title: A dynamic intracellular distribution of Vangl2 accompanies cell polarization during zebrafish gastrulation

    doi: 10.1242/dev.119032

    Figure Lengend Snippet: Enrichment of Vangl2 at anterior membranes of mediolaterally elongated cells. (A-C′) Live embryos at 90% epiboly stage (9 hpf; A,A′) or 5 s stage (11.7 hpf; B-C′). mCherry- and GFP-Vangl2-expressing cells were transplanted from Tg(vangl2:GFP-Vangl2) embryos into the WT unlabeled host. Yellow arrowheads indicate the anterior cell membranes where Vangl2 is enriched. (D,E) GFP-Vangl2 and mCherry posterior/anterior membrane FI ratios of individual cells in the notochord at 90% epiboly stage (9 hpf) and at 5-6 s stage (11.7-12 hpf) (D) or in the neuroectoderm at 5-6 s stage (11.7-12 hpf) (E). The blue bars represent the average posterior/anterior FI ratios for each condition. (F-I) Vangl2 asymmetry analysis using GFP-VANGL2 mosaic expression. (F) Modified image of 64-cell stage embryo showing the mosaic synthetic mRNA injection performed. (G) Live embryos mosaically expressing GFP-VANGL2 (green)/H2B-RFP (red) in notochord cells. (H) Plot Profile (Fiji) quantification of FI of two cells (white rectangle in G) at four different z -planes (confocal optical slices). (I) Comparison of GFP-VANGL2, GFP-Vangl2 and corresponding mCherry membrane FI ratios of individual cells in the notochord at 5-6 s (11.7-12 hpf) stage. * P

    Article Snippet: To express GFP-VANGL2 in a mosaic fashion, we injected synthetic GFP-VANGL2 mRNA into one blastomere of WT embryos at the 64-cell stage ( F), after first titrating the mRNA to establish the concentration that would enable us to visualize fluorescence without causing gastrulation defects due to excess VANGL2 expression ( ). mCherry mRNA was co-injected as a control.

    Techniques: Expressing, Modification, Injection