Journal: Insects

Article Title: Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells

doi: 10.3390/insects15060375

Figure Lengend Snippet: Primers used for qPCR.

Article Snippet: The apoptosis rate of insect Sf9 cells at different time points after virus infection was analyzed using the Annexin V-Elab Fluor ® 647/PI apoptosis detection kit (Elabscience, Wuhan, China) and flow cytometry.

Techniques: Sequencing

Journal: Insects

Article Title: Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells

doi: 10.3390/insects15060375

Figure Lengend Snippet: Insect Sf9 cells exhibit normal biological functions even when heterologous VSR is overexpressed. ( A ) The schematic diagram illustrates the construction of transient expression plasmids: (a) Plasmid pie1-p0, (b) Plasmid pie1-p19, and (c) Plasmid pie1-core. ( B ) Transfection of recombinant plasmids into Insect Sf9 cells for 48 h does not induce any morphological changes in their structure. ( C ) Western blot analysis using His-tag antibody confirms successful overexpression of VSR protein in Sf9 cells (48 h post-transfection), with α-tubulin serving as a control. ( D ) qPCR results reveal no significant alteration in the expression levels of apoptotic genes in normal insect Sf9 cells after 24–72 h of VSR protein overexpression. ( E ) Flow cytometry analysis demonstrates that the apoptosis rate of Sf9 cells remains unchanged across different groups following VSR protein overexpression (48 h post-transfection; Annexin V-647 and Propidium Iodide used as fluorescent dyes).

Article Snippet: The apoptosis rate of insect Sf9 cells at different time points after virus infection was analyzed using the Annexin V-Elab Fluor ® 647/PI apoptosis detection kit (Elabscience, Wuhan, China) and flow cytometry.

Techniques: Expressing, Plasmid Preparation, Transfection, Recombinant, Western Blot, Over Expression, Control, Flow Cytometry

Journal: Insects

Article Title: Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells

doi: 10.3390/insects15060375

Figure Lengend Snippet: Schematic representation illustrating the generation of a recombinant baculovirus harboring the vsr gene, luciferase gene, and fluorescent protein gene. ( A ) Schematic diagram depicting the construction of recombinant plasmids pFBDM-vsr-polh-egfp driven by various promoters (vsr: p64-p0, p64-p19, p64-core, p10-p0, p10-p19, p10-core). ( B ) Schematic diagram outlining the assembly of recombinant plasmid pUCDM-p64-Rluc-polh-Fluc-polh-mCherry containing luciferase genes. ( C ) Illustration presenting E. coli Sw106 strain hosting MultiBacmid. ( D ) Diagram demonstrating the integration of the vsr gene, luciferase gene, and fluorescent protein gene into recombinant E. coli Sw106 MultiBacmid using Tn7 and Cre-loxP transposition technology. ( E ) Representation showcasing normal insect Sf9 cells. ( F ) Successful invasion of Sf9 cells by recombinant E. coli Sw106 facilitated by Invasin, resulting in rBacmid release. ( G ) Schematic diagram displaying the construction of high-expression recombinant baculoviruses carrying different vsr genes, luciferase genes, and fluorescent protein genes.

Article Snippet: The apoptosis rate of insect Sf9 cells at different time points after virus infection was analyzed using the Annexin V-Elab Fluor ® 647/PI apoptosis detection kit (Elabscience, Wuhan, China) and flow cytometry.

Techniques: Recombinant, Luciferase, Plasmid Preparation, Expressing

Journal: Insects

Article Title: Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells

doi: 10.3390/insects15060375

Figure Lengend Snippet: The recombinant baculovirus carrying the target gene successfully infected insect Sf9 cells, and the expression of VSR protein in the cells was identified using Western blot. ( A ) After incubation of the recombinant baculovirus with normal insect Sf9 cells for 48 h (MOI = 1), fluorescence microscopy revealed the presence of both green and red fluorescence signals within the insect Sf9 cells, confirming successful infection by the recombinant virus and expression of the target protein (Bar = 50 µm). ( B ) After infecting insect Sf9 cells with the recombinant baculovirus, cell pellets were collected at various time points during infection. Subsequently, intracellular proteins were identified using Western blot analysis with His-tag antibody and α-tubulin antibody. The results demonstrated a significant upregulation of VSR in Sf9 cells between 48- and 96 h post-infection, with the highest expression level observed at 72 h post-infection. All the Western blot experiments were conducted as parallel experiments, and gel blots were processed simultaneously .

Article Snippet: The apoptosis rate of insect Sf9 cells at different time points after virus infection was analyzed using the Annexin V-Elab Fluor ® 647/PI apoptosis detection kit (Elabscience, Wuhan, China) and flow cytometry.

Techniques: Recombinant, Infection, Expressing, Western Blot, Incubation, Fluorescence, Microscopy, Virus

Journal: Insects

Article Title: Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells

doi: 10.3390/insects15060375

Figure Lengend Snippet: Impact of early or later expression of heterologous VSR on the rate of virus-induced apoptosis in Sf9 insect cells at distinct time intervals. (*: p < 0.05; **: p < 0.01; Compared with the control group).

Article Snippet: The apoptosis rate of insect Sf9 cells at different time points after virus infection was analyzed using the Annexin V-Elab Fluor ® 647/PI apoptosis detection kit (Elabscience, Wuhan, China) and flow cytometry.

Techniques: Expressing, Virus, Control

Journal: Insects

Article Title: Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells

doi: 10.3390/insects15060375

Figure Lengend Snippet: Repercussions of heterologous VSR on the regulation of apoptotic gene expression in virus-infected host Sf9 cells. ( A ) Heat map illustrating the impact of heterologous VSR on the regulation of apoptotic gene expression in Sf9 cells during viral infection, depicting early and late expression patterns. ( B ) Significance analysis of differential expression levels of host apoptotic genes at different time points during viral infection, with values above the green dotted line or below the yellow dotted line indicating statistical significance ( p < 0.05).

Article Snippet: The apoptosis rate of insect Sf9 cells at different time points after virus infection was analyzed using the Annexin V-Elab Fluor ® 647/PI apoptosis detection kit (Elabscience, Wuhan, China) and flow cytometry.

Techniques: Gene Expression, Virus, Infection, Expressing, Quantitative Proteomics

Journal: Insects

Article Title: Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells

doi: 10.3390/insects15060375

Figure Lengend Snippet: Impact of heterologous VSR on protein expression efficiency in insect cell bioreactors. ( A ) The expression of early protein Renilla-luciferase in insect Sf9 cells was significantly enhanced by heterologous VSR. ( B ) The expression of late protein Firefly-luciferase in insect Sf9 cells was significantly increased upon the introduction of heterologous VSR.

Article Snippet: The apoptosis rate of insect Sf9 cells at different time points after virus infection was analyzed using the Annexin V-Elab Fluor ® 647/PI apoptosis detection kit (Elabscience, Wuhan, China) and flow cytometry.

Techniques: Expressing, Luciferase

Journal: Viruses

Article Title: A Novel Hemocyte-Specific Small Protein Participates in White Spot Syndrome Virus Infection via Binding to Viral Envelope Protein

doi: 10.3390/v15010227

Figure Lengend Snippet: Encoding capability of start codon in the predicted ORF in LvHSSP transcript. ( A ) Diagram of the GFP fusion constructs used for transfection. The start codon ATGGTG of the GFP (GFPwt) gene is mutated to ATTGTT (GFPmut). The start codon ATG of the ORF in LvHSSP transcript is mutated to ATT. ( B ) GFP fluorescence signals in different constructs of transfected Sf9 cells.

Article Snippet: Sf9 cells were purchased from the China Center for Type Culture Collection (Wuhan, China), cultured in Sf-900TM II SFM (Gibco, Grand Island, NE, USA) at 27 °C, and sub-cultured every 3–4 days.

Techniques: Construct, Transfection, Fluorescence