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    Thermo Fisher drosophila s2 cells
    Infectivity of Cricket paralysis virus (CrPV) in Diaphorina citri . (A) Insects were intrathoracically microinjected with either purified CrPV virions or buffer solution (as control). Microinjected insects were maintained in a single leaf system for six days and were monitored daily for mortality and symptoms; n, number of insects used. (B) Survival curve of adult insects injected with CrPV. Survival rates were monitored at three time points post injection. Three biological replicates of n = 20 insects each per condition were analyzed. Error bars indicate standard error of the mean. (C) Cluster of flightless insects (a known paralysis symptom) observed in CrPV-injected D. citri under light microscope. Viral RNA accumulation was checked over a time-course by RT-qPCR (D) and confirmed by northern blot analysis on the total RNA from the injected insects (E). RNA from CrPV- or mock-infected <t>S2</t> cells was used as a control. In (D), viral RNA accumulation is expressed as log10 genome copy number per μL (10 ng of RNA), and each time point represents data from three replicates of three pooled insects, and error bars indicate standard error of the mean. (−) Mock, buffer-injected insects; (+) CrPV-injected insects; dpi, days post injection; MB, methylene blue staining.
    Drosophila S2 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/drosophila s2 cells/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    drosophila s2 cells - by Bioz Stars, 2022-05
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    99
    Thermo Fisher puromycin
    Infectivity of Cricket paralysis virus (CrPV) in Diaphorina citri . (A) Insects were intrathoracically microinjected with either purified CrPV virions or buffer solution (as control). Microinjected insects were maintained in a single leaf system for six days and were monitored daily for mortality and symptoms; n, number of insects used. (B) Survival curve of adult insects injected with CrPV. Survival rates were monitored at three time points post injection. Three biological replicates of n = 20 insects each per condition were analyzed. Error bars indicate standard error of the mean. (C) Cluster of flightless insects (a known paralysis symptom) observed in CrPV-injected D. citri under light microscope. Viral RNA accumulation was checked over a time-course by RT-qPCR (D) and confirmed by northern blot analysis on the total RNA from the injected insects (E). RNA from CrPV- or mock-infected <t>S2</t> cells was used as a control. In (D), viral RNA accumulation is expressed as log10 genome copy number per μL (10 ng of RNA), and each time point represents data from three replicates of three pooled insects, and error bars indicate standard error of the mean. (−) Mock, buffer-injected insects; (+) CrPV-injected insects; dpi, days post injection; MB, methylene blue staining.
    Puromycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/puromycin/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    puromycin - by Bioz Stars, 2022-05
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    99
    Thermo Fisher geneticin
    Infectivity of Cricket paralysis virus (CrPV) in Diaphorina citri . (A) Insects were intrathoracically microinjected with either purified CrPV virions or buffer solution (as control). Microinjected insects were maintained in a single leaf system for six days and were monitored daily for mortality and symptoms; n, number of insects used. (B) Survival curve of adult insects injected with CrPV. Survival rates were monitored at three time points post injection. Three biological replicates of n = 20 insects each per condition were analyzed. Error bars indicate standard error of the mean. (C) Cluster of flightless insects (a known paralysis symptom) observed in CrPV-injected D. citri under light microscope. Viral RNA accumulation was checked over a time-course by RT-qPCR (D) and confirmed by northern blot analysis on the total RNA from the injected insects (E). RNA from CrPV- or mock-infected <t>S2</t> cells was used as a control. In (D), viral RNA accumulation is expressed as log10 genome copy number per μL (10 ng of RNA), and each time point represents data from three replicates of three pooled insects, and error bars indicate standard error of the mean. (−) Mock, buffer-injected insects; (+) CrPV-injected insects; dpi, days post injection; MB, methylene blue staining.
    Geneticin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/geneticin/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    geneticin - by Bioz Stars, 2022-05
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    Infectivity of Cricket paralysis virus (CrPV) in Diaphorina citri . (A) Insects were intrathoracically microinjected with either purified CrPV virions or buffer solution (as control). Microinjected insects were maintained in a single leaf system for six days and were monitored daily for mortality and symptoms; n, number of insects used. (B) Survival curve of adult insects injected with CrPV. Survival rates were monitored at three time points post injection. Three biological replicates of n = 20 insects each per condition were analyzed. Error bars indicate standard error of the mean. (C) Cluster of flightless insects (a known paralysis symptom) observed in CrPV-injected D. citri under light microscope. Viral RNA accumulation was checked over a time-course by RT-qPCR (D) and confirmed by northern blot analysis on the total RNA from the injected insects (E). RNA from CrPV- or mock-infected S2 cells was used as a control. In (D), viral RNA accumulation is expressed as log10 genome copy number per μL (10 ng of RNA), and each time point represents data from three replicates of three pooled insects, and error bars indicate standard error of the mean. (−) Mock, buffer-injected insects; (+) CrPV-injected insects; dpi, days post injection; MB, methylene blue staining.

    Journal: bioRxiv

    Article Title: Development of a cricket paralysis virus-based system for inducing RNA interference-mediated gene silencing in Diaphorina citri

    doi: 10.1101/2020.11.15.383588

    Figure Lengend Snippet: Infectivity of Cricket paralysis virus (CrPV) in Diaphorina citri . (A) Insects were intrathoracically microinjected with either purified CrPV virions or buffer solution (as control). Microinjected insects were maintained in a single leaf system for six days and were monitored daily for mortality and symptoms; n, number of insects used. (B) Survival curve of adult insects injected with CrPV. Survival rates were monitored at three time points post injection. Three biological replicates of n = 20 insects each per condition were analyzed. Error bars indicate standard error of the mean. (C) Cluster of flightless insects (a known paralysis symptom) observed in CrPV-injected D. citri under light microscope. Viral RNA accumulation was checked over a time-course by RT-qPCR (D) and confirmed by northern blot analysis on the total RNA from the injected insects (E). RNA from CrPV- or mock-infected S2 cells was used as a control. In (D), viral RNA accumulation is expressed as log10 genome copy number per μL (10 ng of RNA), and each time point represents data from three replicates of three pooled insects, and error bars indicate standard error of the mean. (−) Mock, buffer-injected insects; (+) CrPV-injected insects; dpi, days post injection; MB, methylene blue staining.

    Article Snippet: The stability of the new recombinant CrPV vectors carrying different insert lengths was checked by recovering the recombinant virions in S2 cells, as already described.

    Techniques: Infection, Purification, Injection, Light Microscopy, Quantitative RT-PCR, Northern Blot, Staining

    Recombinant CrPV (rCrPV) virions carrying the target gene-derived sequences were propagated in Drosophila S2 cells and checked by SDS-PAGE (A) and by RT-PCR of the insertion site region to confirm the retention of the target-derived inserts (B). M, Page ruler prestained protein ladder; L, 1 Kb plus DNA ladder.

    Journal: bioRxiv

    Article Title: Development of a cricket paralysis virus-based system for inducing RNA interference-mediated gene silencing in Diaphorina citri

    doi: 10.1101/2020.11.15.383588

    Figure Lengend Snippet: Recombinant CrPV (rCrPV) virions carrying the target gene-derived sequences were propagated in Drosophila S2 cells and checked by SDS-PAGE (A) and by RT-PCR of the insertion site region to confirm the retention of the target-derived inserts (B). M, Page ruler prestained protein ladder; L, 1 Kb plus DNA ladder.

    Article Snippet: The stability of the new recombinant CrPV vectors carrying different insert lengths was checked by recovering the recombinant virions in S2 cells, as already described.

    Techniques: Recombinant, Derivative Assay, SDS Page, Reverse Transcription Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis

    Construction and stability analysis of the recombinant clones of Cricket paralysis virus (CrPV). (A) Schematic representation of the genome structures of CrPV-DN* mutant (from this work, top) and its derivative recombinant CrPV (rCrPV, bottom) showing the insertion site for cloning the desired inserts. (B) List of the green fluorescent protein (GFP) gene-derived sequences (shown as DNA sequences) individually inserted into the CrPV insertion site to generate the rCrPVs; the respective nucleotide (nt) lengths of each sequence are shown. Recombinant CrPV virions were recovered from Drosophila S2 cells transfected with the rCrPV-derived transcripts. Stability of the rCrPVs was evaluated by checking insert retention in the virion RNA, by RT-PCR (C); and by transmission electron microscopy of the rCrPV virions (D). Scale bars in (D) are 60 nm.

    Journal: bioRxiv

    Article Title: Development of a cricket paralysis virus-based system for inducing RNA interference-mediated gene silencing in Diaphorina citri

    doi: 10.1101/2020.11.15.383588

    Figure Lengend Snippet: Construction and stability analysis of the recombinant clones of Cricket paralysis virus (CrPV). (A) Schematic representation of the genome structures of CrPV-DN* mutant (from this work, top) and its derivative recombinant CrPV (rCrPV, bottom) showing the insertion site for cloning the desired inserts. (B) List of the green fluorescent protein (GFP) gene-derived sequences (shown as DNA sequences) individually inserted into the CrPV insertion site to generate the rCrPVs; the respective nucleotide (nt) lengths of each sequence are shown. Recombinant CrPV virions were recovered from Drosophila S2 cells transfected with the rCrPV-derived transcripts. Stability of the rCrPVs was evaluated by checking insert retention in the virion RNA, by RT-PCR (C); and by transmission electron microscopy of the rCrPV virions (D). Scale bars in (D) are 60 nm.

    Article Snippet: The stability of the new recombinant CrPV vectors carrying different insert lengths was checked by recovering the recombinant virions in S2 cells, as already described.

    Techniques: Recombinant, Clone Assay, Mutagenesis, Derivative Assay, Sequencing, Transfection, Reverse Transcription Polymerase Chain Reaction, Transmission Assay, Electron Microscopy

    Construction of an attenuated variant of Cricket paralysis virus (CrPV) by mutational analysis on the CrPV DvExNPGP motif: a signal motif for cleavage of the 1A silencing suppressor protein. (A) Schematic representation of the CrPV genome organization showing the location of the DvExNPGP motif as well as the cleavage site (red arrow). (B) List of single and double amino acid (aa) mutations introduced into CrPV DvExNPGP motif; mutated aa’s are highlighted in red. CrPV mutant-derived transcripts were transfected into S2 cells and viral RNA accumulation in the supernatant was evaluated 96 hours post transfection by both RT-qPCR (C) and northern blot analysis (D). In (C), viral RNA accumulation is expressed as log10 genome copy number per μL (10 ng of RNA), and error bars represent standard error of the mean of four biological replicates. The infectivity of the CrPV-DN* mutant (B), selected as an attenuated variant of CrPV, was evaluated in Diaphorina citri by microinjecting CrPV-DN*-derived virions into adult insects. Microinjected insects were monitored daily for mortality (E). Three biological replicates of n = 20 insects each per condition were analyzed. Error bars indicate standard error of the mean. Viral replication rates were checked over a time-course by RT-qPCR (F). Each time point represents data from three replicates of three pooled insects, and error bars indicate standard error of the mean. For comparison, this experiment was simultaneously performed with wild-type (WT) CrPV. MB, methylene blue staining; n, number of insects used.

    Journal: bioRxiv

    Article Title: Development of a cricket paralysis virus-based system for inducing RNA interference-mediated gene silencing in Diaphorina citri

    doi: 10.1101/2020.11.15.383588

    Figure Lengend Snippet: Construction of an attenuated variant of Cricket paralysis virus (CrPV) by mutational analysis on the CrPV DvExNPGP motif: a signal motif for cleavage of the 1A silencing suppressor protein. (A) Schematic representation of the CrPV genome organization showing the location of the DvExNPGP motif as well as the cleavage site (red arrow). (B) List of single and double amino acid (aa) mutations introduced into CrPV DvExNPGP motif; mutated aa’s are highlighted in red. CrPV mutant-derived transcripts were transfected into S2 cells and viral RNA accumulation in the supernatant was evaluated 96 hours post transfection by both RT-qPCR (C) and northern blot analysis (D). In (C), viral RNA accumulation is expressed as log10 genome copy number per μL (10 ng of RNA), and error bars represent standard error of the mean of four biological replicates. The infectivity of the CrPV-DN* mutant (B), selected as an attenuated variant of CrPV, was evaluated in Diaphorina citri by microinjecting CrPV-DN*-derived virions into adult insects. Microinjected insects were monitored daily for mortality (E). Three biological replicates of n = 20 insects each per condition were analyzed. Error bars indicate standard error of the mean. Viral replication rates were checked over a time-course by RT-qPCR (F). Each time point represents data from three replicates of three pooled insects, and error bars indicate standard error of the mean. For comparison, this experiment was simultaneously performed with wild-type (WT) CrPV. MB, methylene blue staining; n, number of insects used.

    Article Snippet: The stability of the new recombinant CrPV vectors carrying different insert lengths was checked by recovering the recombinant virions in S2 cells, as already described.

    Techniques: Variant Assay, Mutagenesis, Derivative Assay, Transfection, Quantitative RT-PCR, Northern Blot, Infection, Staining