Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: (A and B) Images of optic nerve sections showing CTB-labeled axons (grey) in C57BL/6J mice injected with either (A) AAV-THBS1 or (B) AAV-GFP. Asterisks, lesion site. Scale bars, 100 μm.

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: Labeling, Injection

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: (A) A schematic of THBS1 mutants investigated. All constructs contain the N-terminal signal peptide and have a C-terminal HA tag. Laminin G domain (LamG), oligomerization coiled coil (CC) domain, von Willebrand complex like domain (vWC), thrombospondin type 1 repeat domain (TSR1), epidermal growth factor-like repeat domains (EGF), type 3 repeat domain (TSR3), and the thrombospondin C-terminal domain (CTD). THBS4 is shown for comparison to THBS1.

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: Construct

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: (A) Images of optic nerve section showing GFP-labeled axons (green) from HB9:GFP;Bax−/− mice and CTB (magenta) following injection with AAV-THBS1 and optic nerve crush. Asterisks, lesion site.

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: Labeling, Injection

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: shRNA, Recombinant, Multiplex Assay, Clone Assay, Software

Journal: PLoS ONE

Article Title: Optimization of a One-Step Heat-Inducible In Vivo Mini DNA Vector Production System

doi: 10.1371/journal.pone.0089345

Figure Lengend Snippet: Plasmid vectors used in this study.

Article Snippet: pGL2 , SV40P-Luc-PolyA-SV40 intron , Promega.

Techniques: Plasmid Preparation, Sequencing

Journal: bioRxiv

Article Title: Repressor sequestration activates translation of germ granule localized mRNA

doi: 10.1101/2023.10.17.562687

Figure Lengend Snippet: a , (Left) schematic of a Drosophila embryo. Germplasm (blue) is located at the posterior pole of the embryo. The dashed square represents the region imaged by confocal microscopy and presented in panel b . (Right) Schematic of a translating suntag-nanos mRNA. A repetitive array of SunTag epitopes is added to the N-terminus of the nanos coding sequence. Nascent SunTag peptides are detected by scFv-GFP binding and suntag mRNA is detected by smFISH probes (magenta dashed line). b , A representative confocal image of the posterior pole of an embryo expressing Vasa-mApple (blue), suntag-nanos (mRNA stained by suntag smFISH probes, magenta), and scFv-GFP (green). Outlined regions in germplasm and soma are magnified and presented in panel (C). Scale bar 20 µm. c , Magnified images of germplasm and soma show the different translation activities in these two parts of the embryo. Scale bar 2 µm. d , (Left) quantification of the percentage of translating mRNA in the soma and the germplasm of embryos. (Right) zoomed confocal images showing examples of a translating mRNA that co-localizes with scFv-GFP signal (arrowhead) and two non-translating mRNA which do not co-localize with scFv-GFP signal (arrows). e , Quantification of suntag-nanos mRNA translation in the soma and the posterior pole of embryos with mCherry (control) knock-down (KD) or osk KD. f , Osk-bcd 3’UTR expression induces germplasm and translation of suntag-nanos mRNA at the anterior pole. (Top) Oskar protein is immunostained with anti-Oskar antibody. (Bottom) translation of suntag-nanos mRNA in native germplasm at the posterior and induced germplasm at the anterior, which are quantified in g . Scale bar 100 µm (F top), 2 µm (F bottom).

Article Snippet: The SunTag sequence was amplified from 5’TOP-SunTag-Renilla (Addgene #119946) and inserted after the nanos start codon via In-Fusion assembly.

Techniques: Confocal Microscopy, Sequencing, Binding Assay, Expressing, Staining, Control, Knockdown

Journal: bioRxiv

Article Title: Repressor sequestration activates translation of germ granule localized mRNA

doi: 10.1101/2023.10.17.562687

Figure Lengend Snippet: a , Schematic of CRISPR knocked-in suntag-nanos allele. b , Images of germplasm in embryos expressing suntag-nanos and (top) scFv-sfGFP (super-folder GFP) or (bottom) monomeric msGFP2 (green). Suntag mRNA is stained by suntag probes (magenta). ScFv-sfGFP showed puncta GFP signals (arrowheads) which are not co-localized with mRNA signal and thus are not translating sites. ScFv-msGFP2, which is used throughout this study unless suggested otherwise, significantly reduces the aggregation. Scale bar 2 µm. c , The percentage of GFP foci co-localized with mRNA. The aggregate formation of scFv-sfGFP causes a relatively low percentage of colocalization. Using scFv-msGFP2, the majority of GFP foci (80%-90%) represent polysomes. d , Images of germplasm in embryos expressing suntag-nanos and scFv-GFP (green). Embryos were injected with 20 mM HEPES or 10 mg/ml puromycin and aged for 15min before being stained with suntag probes (magenta). Scale bar 2 µm.

Article Snippet: The SunTag sequence was amplified from 5’TOP-SunTag-Renilla (Addgene #119946) and inserted after the nanos start codon via In-Fusion assembly.

Techniques: CRISPR, Expressing, Staining, Injection

Journal: bioRxiv

Article Title: Repressor sequestration activates translation of germ granule localized mRNA

doi: 10.1101/2023.10.17.562687

Figure Lengend Snippet: a , Schematics of stage-1, stage-3, and stage-5 embryos. Germplasm or pole cells are labeled in blue. Outlined regions are imaged and presented in panel (B). b , Example images of stage-1, stage-3, and stage-5 embryos expressing suntag-nanos . SunTag is stained by anti-GCN4 (green); suntag mRNA is stained by smFISH (magenta); germplasm or pole cells are marked by Vasa-mApple (blue). Scale bar 20 µm. c , Images of a stage-1 embryo expressing suntag-nanos stained by anti-GCN4 (top) and a stage-1 embryo from a w 1118 fly stained by anti-Nanos (bottom). Scale bar 100 µm. d , Images of germplasm in stage-1 embryos (top) and pole cells in stage-5 embryos expressing suntag-nanos . SunTag (green) is stained by anti-GCN4 (left) or by endogenous scFv-GFP (right); suntag mRNA is stained by smFISH (magenta). Scale bar 2 µm. e , Quantification of the percentage of mRNA foci co-localized with SunTag staining signal in stage-1 germplasm and stage-5 pole cells when SunTag is stained by scFv-GFP or anti-GCN4.

Article Snippet: The SunTag sequence was amplified from 5’TOP-SunTag-Renilla (Addgene #119946) and inserted after the nanos start codon via In-Fusion assembly.

Techniques: Labeling, Expressing, Staining

Journal: bioRxiv

Article Title: Repressor sequestration activates translation of germ granule localized mRNA

doi: 10.1101/2023.10.17.562687

Figure Lengend Snippet: a , Images of embryos expressing suntag-nanos with mcherry knockdown (top) or osk knockdown (bottom). SunTag is stained by anti-GCN4 (green) and germplasm is marked by Vasa-mApple (magenta). Scale bar 100 µm. b , Images of embryos expressing Vasa-mApple and osk-bcd3’UTR , forming germplasm and localizing nanos mRNA at the anterior pole. Germplasm is marked by Vasa-mApple (magenta). Endogenous nanos mRNA is stained by smFISH probes against nanos (green). Scale bar 100 µm.

Article Snippet: The SunTag sequence was amplified from 5’TOP-SunTag-Renilla (Addgene #119946) and inserted after the nanos start codon via In-Fusion assembly.

Techniques: Expressing, Knockdown, Staining

Journal: bioRxiv

Article Title: Repressor sequestration activates translation of germ granule localized mRNA

doi: 10.1101/2023.10.17.562687

Figure Lengend Snippet: a , Schematic of Drosophila oogenesis stages. b , Representative images of germplasm (top) and soma (middle) in stage 14 oocyte and cytoplasm of stage 7 nurse cells (bottom) expressing suntag-nanos and scFv-GFP. Blue, Vasa; magenta, suntag smFISH; green, scFv-GFP. Scale bar 1 µm. c , Translating fraction of suntag-nanos mRNA in stage 4-10 nurse cells, soma, and germplasm of stage 10-12 (developing) oocytes, stage 14 (mature) oocytes, and stage 1-2 embryos. d , Protocol of in vitro activation of oocytes and live imaging. Mature oocytes are dissected from Vasa-mApple/+; suntag-nanos , scFv-GFP/+ flies and activated with 30% Robb’s buffer (see method for details). Activated eggs are mounted onto a coverslip and imaged by confocal microscopy. e , Representative time-lapse images of the germplasm of an activated egg with an increasing number of polysome (green foci). Germplasm is marked by Vasa-mApple (magenta) and SunTag is detected by endogenous scFv-GFP (green). The top shows the merged image, and the bottom shows scFv-GFP channel only. Scale bar 20 µm. Schematics in a and d were generated with BioRender ( https://www.biorender.com/ )

Article Snippet: The SunTag sequence was amplified from 5’TOP-SunTag-Renilla (Addgene #119946) and inserted after the nanos start codon via In-Fusion assembly.

Techniques: Expressing, In Vitro, Activation Assay, Imaging, Confocal Microscopy, Generated

Journal: bioRxiv

Article Title: Repressor sequestration activates translation of germ granule localized mRNA

doi: 10.1101/2023.10.17.562687

Figure Lengend Snippet: a , Images of germ granules are segmented with the Ilastik program. The top shows the original grayscale images of Vasa-mApple at the posterior pole of an embryo (left) and zoomed image of the outline region in the germplasm (right). The bottom shows the black-and-white binary images of segmented germ granules (granules in white). Scale bar 20 µm (left), and 2 µm (right). b , Schematic of the distance measurement program. The granule surface is defined after segmentation by Ilastik. The coordinates of mRNA smFISH or scFv-GFP/anti-GCN4 spots are determined by FISH-Quant and used to measure to distance to the closest granule surface. The schematic is drawn in 2D but the actual data and measurement are in 3D (see methods). c - f , Control and validation experiments of distance measurement. (Left) representative images of germplasm with germ granules marked by Vasa-mApple (blue). Magenta: c suntag mRNA smFISH; d is the same image as c with mRNA channel rotated by 180° to shuffle the mRNA distribution; e has the same Vasa channel image as c with simulated points randomly distributed within the image; f smFISH of osk mRNA. Scale bar 2 µm. The distributions of mRNA foci or simulated points are plotted in the relative frequency histograms on the right. The x-axis refers to the distance of foci centroids to the border of the closest granule; the zero marks granule border; a negative value denotes being inside a granule and positive denotes outside. The two bins around 0 have abnormally low counts in all experiments, likely an artifact caused by the design of the distance measuring program. g The distributions of mRNA foci or simulated points in ( c - f ) are plotted together as a kernel density estimate (KDE) plot. The distributions of shuffled mRNA, random points, and osk mRNA show a shift away from the surface of germ granules when compared to suntag-nanos mRNA.

Article Snippet: The SunTag sequence was amplified from 5’TOP-SunTag-Renilla (Addgene #119946) and inserted after the nanos start codon via In-Fusion assembly.

Techniques: Control, Biomarker Discovery

Journal: bioRxiv

Article Title: Repressor sequestration activates translation of germ granule localized mRNA

doi: 10.1101/2023.10.17.562687

Figure Lengend Snippet: a-d , Orientation of translating mRNA. Translating suntag-nanos mRNAs in embryos from Vasa-mApple/+; suntag-nanos , scFv-GFP/ Df(nanos) flies (see methods) are detected with smFISH against suntag ( a and b ) and nanos 3’UTR ( c and d ). Example germplasm images are shown in a and c . Scale bar 1 µm. The orthogonal views of outline regions are shown on the right. Scale bar 0.3 µm. Blue, Vasa; magenta, mRNA smFISH; green, scFv-GFP. The distributions of scFv-GFP and smFISH foci were mapped and plotted in relative frequency histograms overlaid with kernel density estimate (KDE) in b and d . The x-axis refers to the distance of foci centroids to the border of the closest granule; the zero marks granule border; a negative value denotes being inside a granule and positive denotes outside. In total, 12684 smFISH foci and 12733 scFv-GFP foci from 7 images were mapped in b . 5663 smFISH foci and 5649 scFv-GFP foci from 3 images were mapped in d . e - g , Polysomes distribute preferentially on the surface of germ granules. e , Example image showing the distribution of translating (arrows) and non-translating (arrowheads) mRNA stained by suntag smFISH probes. Blue, Vasa; magenta, suntag mRNA; green, scFv-GFP. Scale bar 1 µm. f , Relative frequency histogram with KDE of translating and non-translating mRNA distribution in germplasm. 12684 translating and 19712 non-translating foci from 7 images were plotted. g , The average translating fraction in each bin of the x-axis from 7 images was calculated and plotted. The average translating fraction in entire germplasm is indicated as the dashed line. The translating fractions on the granule surface (0 ≤ x ≤ 200 nm) were compared with the ones within granules (x < 0) or ones not localized to granules (x > 400 nm) using Welch’s t-test. h , Distribution of total suntag-nanos mRNA stained by suntag probes in stage 1 embryos and stage 14 oocytes. 6468 foci from 4 oocyte images and 32473 foci from 7 embryo images were mapped. i , A model of the predicted orientation and distribution of translating and non-translating mRNAs in germ granules.

Article Snippet: The SunTag sequence was amplified from 5’TOP-SunTag-Renilla (Addgene #119946) and inserted after the nanos start codon via In-Fusion assembly.

Techniques: Staining

Journal: bioRxiv

Article Title: Repressor sequestration activates translation of germ granule localized mRNA

doi: 10.1101/2023.10.17.562687

Figure Lengend Snippet: a , Distribution of suntag-nanos mRNA in germplasm after injecting 20 mM HEPES buffer (control, top) or 10mg/ml puromycin (bottom) and 30 min aging. Blue, Vasa; magenta, suntag smFISH; green, scFv-GFP. Scale bar 1 µm. b , Distributions of total mRNA detected by suntag smFISH in germplasm of HEPES-injected embryos and puromycin-injected embryos were plotted in relative frequency histograms with KDE curves. Spots from three embryos of each condition were mapped and plotted.

Article Snippet: The SunTag sequence was amplified from 5’TOP-SunTag-Renilla (Addgene #119946) and inserted after the nanos start codon via In-Fusion assembly.

Techniques: Control, Injection

Journal: bioRxiv

Article Title: Repressor sequestration activates translation of germ granule localized mRNA

doi: 10.1101/2023.10.17.562687

Figure Lengend Snippet: a , Example image of the posterior of an embryo expressing Vasa-mApple (blue), nanos-suntag -SREmut (mRNA stained by suntag probes, magenta). SunTag is stained with anti-GCN4 (green). An image of the anti-GCN4 channel is shown on the right with germplasm and soma outlined, showing that translation is prevalent in both soma and germplasm. Scale bar 20 µm. b , Quantification of the translating fractions in embryos from flies expressing transgenic suntag-nanos-WT , suntag-nanos-SREmut , or suntag-nanos-tub3’UTR . Stage-2 embryos were used for quantification. Pairwise statistical comparisons were conducted using Welch’s t-test. c , The intensities of polysomes (anti-GCN4 staining) in soma and germplasm of embryos from flies expressing UAS-suntag-nanos-SREmut . Quantification results from four embryos were plotted in a super-plot. Individual dots represent the intensities of individual polysomes, each color-coded by embryos. Each colored circle represents the mean intensity of each embryo. The black lines and error bars indicate the mean and standard deviation (SD) of the four embryos. Statistical comparison was performed on the mean intensities of individual embryos using Welch’s t-test. d , (Left) Schematic of the live imaging setup. The posterior pole of the live embryo is stuck onto a coverslip and imaged by an upright microscope. (Right) a representative image of germplasm during live imaging. Blue, Vasa; green, scFv-GFP. Scale bar 10 µm. e , The intensities of polysomes over time during live imaging suntag-nanos mRNA translation with (red curve, average of 10 curves) or without (blue curve, average of 23 curves) photo-bleaching when Time= 30s. The elongation rate calculated from the plot is indicated. f , Polysome intensities of suntag-nanos-SREmut mRNA in germplasm (blue curve, average of 35 curves) and soma (red curve, average of 19 curves) over time with photo-bleaching when Time= 30s. The elongation rates in germplasm and soma calculated from the plot are indicated. g , Representative time-lapse image of fluorescence recovery after photobleaching (FRAP) of two translation sites (arrowheads). Blue, Vasa; green, scFv-GFP. Scale bar 500 nm.

Article Snippet: The SunTag sequence was amplified from 5’TOP-SunTag-Renilla (Addgene #119946) and inserted after the nanos start codon via In-Fusion assembly.

Techniques: Expressing, Staining, Transgenic Assay, Standard Deviation, Comparison, Imaging, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Repressor sequestration activates translation of germ granule localized mRNA

doi: 10.1101/2023.10.17.562687

Figure Lengend Snippet: a , Schematics of transgenic constructs of UAS-suntag-nanos , UAS-suntag-nanos-SREmut , and UAS-suntag-nanos-tubulin3’UTR . The red asterisks in nanos 3’UTR represent the two SREs mutated in the construct. b , Representative images of embryos expressing suntag-nanos (top), suntag-nanos-SREmut (middle), and suntag-nanos-tubulin3’UTR (bottom). Note that the translation activities in the soma of embryos expressing suntag-nanos-SREmut and suntag-nanos-tubulin3’UTR are higher than suntag-nanos . Blue, Vasa; magenta, suntag smFISH; green, anti-GCN4. Scale bar 20 µm.

Article Snippet: The SunTag sequence was amplified from 5’TOP-SunTag-Renilla (Addgene #119946) and inserted after the nanos start codon via In-Fusion assembly.

Techniques: Transgenic Assay, Construct, Expressing

Journal: bioRxiv

Article Title: Repressor sequestration activates translation of germ granule localized mRNA

doi: 10.1101/2023.10.17.562687

Figure Lengend Snippet: a , Germplasm of an embryo expressing suntag-nanos flies with SunTag detected by anti-GCN4. The polysomes (arrows) have stronger fluorescence intensities and are co-localized with the mRNA signal (not shown). Individual synthesized SunTag-Nanos proteins (examples pointed out by arrowheads) have lower intensities and are not co-localized with mRNA. Scale bar 2 µm. b , Fluorescence intensities of polysomes and single SunTag-Nanos protein, extracted from FISH-Quant analysis (see methods). Data from five embryos, represented by different colors, are plotted as a super-plot. c , Calculated ribosome occupancy on suntag-nanos mRNA using data from b . d , Theoretical process of fluorescence recovery after photo-bleaching (FRAP). Before photo-bleaching, suntag-nanos mRNA is translated at a steady state with SunTag bound by fluorescent scFv-GFP (phase 1). Photo-bleaching diminishes the fluorescence of bound scFv-GFP (phase 2). Newly synthesized SunTag epitopes after photo-bleaching bind fluorescent scFv-GFP, causing fluorescence recovery of the polysome. Assuming a constant elongation rate, the initial phase of recovery is linear (phase 3). When the peptide that contains the first SunTag synthesized post-bleaching leaves polysome, which counteracts the increase of newly synthesized SunTag, the increase of signal starts to slow down (phase 4). When the first ribosome loaded after photo-bleaching finishes the translation, the signal reaches a plateau (phase 5) with the same intensity as before photo-bleaching because all the SunTags are bound by fluorescent scFv-GFP again. e , A hypothetical FRAP curve (top) based on the theoretical FRAP process, and the FRAP experimental data (bottom, same as ), which shows a similar curve as the theoretical curve.

Article Snippet: The SunTag sequence was amplified from 5’TOP-SunTag-Renilla (Addgene #119946) and inserted after the nanos start codon via In-Fusion assembly.

Techniques: Expressing, Fluorescence, Synthesized

Journal: bioRxiv

Article Title: Repressor sequestration activates translation of germ granule localized mRNA

doi: 10.1101/2023.10.17.562687

Figure Lengend Snippet: a , AlphaFold structure model of short Oskar protein, with LOTUS domain in red, SGNH-like domain in blue, and linker region in green. b , Percentage of glutamine (Q) and asparagine (N) in three regions of short Oskar proteins from eleven Drosophila species. Each dot represents an Oskar of a particular Drosophila species. c , Oskar-NQmut-bcd3’UTR induces anterior germplasm similarly to Oskar-WT-bcd3’UTR . Oskar-WT/NQmut proteins are immunostained with anti-Oskar antibody. Scale bar 100 µm. d , Distribution of Smaug in germplasm. Images of induced germplasm by Oskar-WT or Oskar-NQmut at the anterior pole. Germ granules are labeled by Vasa-mApple (magenta). Smaug is visualized with Smaug-GFP (green). Scale bar 5 µm. e , Intensity profiles of Vasa-mApple (magenta) and Smaug-GFP (green) along the lines across the germ granules induced by Oskar-WT or Oskar-NQmut. Intensity profiles of 20 germ granules were combined for each genotype, where the curves represent the mean value and the color-filled areas cover the standard deviation. f , Representative images showing the translation of suntag-nanos mRNA in germplasm induced by Oskar-WT or Oskar-NQmut. Blue, Vasa; magenta, suntag smFISH; green, anti-GCN4. Scale bar 5 µm. g , The fraction of suntag-nanos-WT or suntag-nanos-SREmut mRNA being translated in anterior germplasm induced by Oskar-WT or Oskar-NQmut. Each dot represents the normalized measurement of an embryo where the translating fraction in the anterior germplasm is divided by the translating fraction in the native germplasm at the posterior. Statistical comparisons between Oskar-WT and NQmut were performed by t-test, and p-values are indicated. h , The cuticle phenotypes generated by Oskar-WT/NQmut-bcd3’UTR . The cuticle images show a range of cuticle phenotypes corresponding to different levels of anteriorly-expressed Nanos protein. The bar graph shows the frequency of each cuticle phenotype caused by Oskar-WT/NQmut-bcd3’UTR expression. Statistical comparison was performed using Chi-square test. i , Oskar mediates Smaug localization and translational de-repression of nanos mRNA. With WT Oskar, Smaug, but not its co-factors in translational repression (Cup/CCR4-NOT), is localized to germ granules. Localized Smaug is dysfunctional in translational repression, allowing the translation of nanos mRNA. With Oskar-NQmut, Smaug loses the localization but remains functional inside germ granules, thus repressing the translation of nanos mRNA.

Article Snippet: The SunTag sequence was amplified from 5’TOP-SunTag-Renilla (Addgene #119946) and inserted after the nanos start codon via In-Fusion assembly.

Techniques: Labeling, Standard Deviation, Generated, Expressing, Comparison, Functional Assay

Journal: PLoS Pathogens

Article Title: A Homolog Pentameric Complex Dictates Viral Epithelial Tropism, Pathogenicity and Congenital Infection Rate in Guinea Pig Cytomegalovirus

doi: 10.1371/journal.ppat.1005755

Figure Lengend Snippet: (i) Structure of parental BAC virus (NRD13) and modified mutant GP129FRT GPCMV. Modified mutant encodes an ectopic GP129 (myc tagged) cDNA under SV40 promoter control in the GP25/GP26 intergenic locus as described in materials and methods . Arrows (right) indicate virus tropism to epithelial cells: red, no tropism; green, tropism. (ii) Western blot analysis of sucrose gradient purified GP129FRT. Purified virus was evaluated for the presence of structural proteins present in the viral membrane by western blot analysis: gB (detected by mouse anti-gB), lanes 1–3; gH (rabbit anti-gH), lanes 4–6; GP131 (mouse anti-GP131), lanes 7–9; GP129 (mouse anti-myc), lanes 10–12. Additionally, a control GFP protein (mouse anti-GFP) expressed by the virus was also evaluated (lanes 13–15). Secondary antibodies were either anti-mouse IgG/HRP or anti-rabbit IgG/HRP. Lanes: 1, 4, 7, 10 and 13 total cell lysate (GP129FRT); 2, 5, 8, 11 and 14 total cell lysate (uninfected); 3, 6, 9, 12, and 15 (purified virus particle). Equivalent protein loading was determined by Bradford assay.

Article Snippet: Next, the SV40 promoter/ SV40 polyA sequence from pSI (Promega) was isolated as a Bgl II/ Bam H I fragment and cloned into the unique Bam H I site (GPCMV 22122 strain, position 38,538 bases) in the intergenic locus between GP25 (sense strand) and GP26 (complementary strand) genes in linearized pUCGP25/GP26 to generate pSIGP25/GP26.

Techniques: Virus, Modification, Mutagenesis, Control, Western Blot, Purification, Membrane, Bradford Assay