spodoptera frugiperda (sf9) cell line (Wageningen University and Research)

Wageningen University and Research spodoptera frugiperda (sf9) cell line

(A) Construction of fp25k knockout bacmid. The fp25k gene in AcBacΔ cc bacmid was deleted and replaced with the zeocin resistance gene ( zeocin r ) through homologous recombination. (B) Strategy for construction of recombinant viruses, vAcΔ cc , vAcΔ cc Δ fp25k and vAcΔ cc Δ fp25k-rfp25k , the e gfp gene was inserted into the polyhedrin locus by transposition. (C) Transfection-infection assay of recombinant bacmids for viral propagation. At 48 h p.t., GFP-expressing cells were observed by fluorescence microscopy (upper panel). The fluorescent signals of GFP in cells infected by transfection supernatant were visualized at 72 h p.i. (lower panel). (D) One step growth curves of vAcΔ cc , vAcΔ cc Δ fp25k and vAcΔ cc Δ fp25k-rfp25k . <t>Sf9</t> cells were infected with each virus at an MOI of 5, the supernatants were harvested at 0, 24, 48 and 96 h p.i. and determined for the production of infectious virus by EPDA. The results were transformed logarithmically. Each point represents the average titer from three independent infections. Error bars represent standard deviations.

Spodoptera Frugiperda (Sf9) Cell Line, supplied by Wageningen University and Research, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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spodoptera frugiperda (sf9) cell line - by Bioz Stars, 2026-09

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Journal: PLoS ONE

Article Title: The FP25K Acts as a Negative Factor for the Infectivity of AcMNPV Budded Virus

doi: 10.1371/journal.pone.0128471

Figure Lengend Snippet: (A) Construction of fp25k knockout bacmid. The fp25k gene in AcBacΔ cc bacmid was deleted and replaced with the zeocin resistance gene ( zeocin r ) through homologous recombination. (B) Strategy for construction of recombinant viruses, vAcΔ cc , vAcΔ cc Δ fp25k and vAcΔ cc Δ fp25k-rfp25k , the e gfp gene was inserted into the polyhedrin locus by transposition. (C) Transfection-infection assay of recombinant bacmids for viral propagation. At 48 h p.t., GFP-expressing cells were observed by fluorescence microscopy (upper panel). The fluorescent signals of GFP in cells infected by transfection supernatant were visualized at 72 h p.i. (lower panel). (D) One step growth curves of vAcΔ cc , vAcΔ cc Δ fp25k and vAcΔ cc Δ fp25k-rfp25k . Sf9 cells were infected with each virus at an MOI of 5, the supernatants were harvested at 0, 24, 48 and 96 h p.i. and determined for the production of infectious virus by EPDA. The results were transformed logarithmically. Each point represents the average titer from three independent infections. Error bars represent standard deviations.

Article Snippet: The Spodoptera frugiperda (Sf9) cell line [ ] (as gift from Prof. Just M. Vlak, Wageningen University, The Netherlands) was cultured in Grace’s insect medium (pH 6.0; Gibco-BRL), supplemented with 10% fetal bovine serum (FBS; Gibco-BRL) at 28°C.

Techniques: Knock-Out, Homologous Recombination, Recombinant, Transfection, Infection, Expressing, Fluorescence, Microscopy, Virus, Transformation Assay

Sf9 cells were infected with vAcΔ cc or vAcΔ cc Δ fp25k at an MOI of 5. The supernatants from infected cells were collected at 0, 18, 24, 48, 72 and 96 h. p.i. Virus titers were determined by EPDA (A), genomic DNA was detected by qPCR (B), and the results were transformed logarithmically. Each point represents the average titer from three independent infections. Error bars represent standard deviations.

Journal: PLoS ONE

Article Title: The FP25K Acts as a Negative Factor for the Infectivity of AcMNPV Budded Virus

doi: 10.1371/journal.pone.0128471

Figure Lengend Snippet: Sf9 cells were infected with vAcΔ cc or vAcΔ cc Δ fp25k at an MOI of 5. The supernatants from infected cells were collected at 0, 18, 24, 48, 72 and 96 h. p.i. Virus titers were determined by EPDA (A), genomic DNA was detected by qPCR (B), and the results were transformed logarithmically. Each point represents the average titer from three independent infections. Error bars represent standard deviations.

Techniques: Infection, Virus, Transformation Assay

(A) Electron microscopy analysis. Sf9 cells were infected with vAcΔ cc or vAcΔ cc Δ fp25k at an MOI of 5. Infected cells were fixed with glutaraldehyde at 48, 64, 72 and 96 h p.i., and nucleocapsids occlusion was observed (i-viii). Enlargements of the blocked area in (a-h). Bars represent 1 μm and 250 nm. (B) Cellular viral particle determination. Sf9 cells were infected with vAcΔ cc or vAcΔ cc Δ fp25k (MOI = 5), total cellular DNA were collected at 0, 72 and 96 h p.i. and subjected to qPCR to determine the viral genome copy number. The data are from three independent experiments. Error bars represent standard deviation. Data were analyzed by two-tailed Student's t -test.

Journal: PLoS ONE

Article Title: The FP25K Acts as a Negative Factor for the Infectivity of AcMNPV Budded Virus

doi: 10.1371/journal.pone.0128471

Figure Lengend Snippet: (A) Electron microscopy analysis. Sf9 cells were infected with vAcΔ cc or vAcΔ cc Δ fp25k at an MOI of 5. Infected cells were fixed with glutaraldehyde at 48, 64, 72 and 96 h p.i., and nucleocapsids occlusion was observed (i-viii). Enlargements of the blocked area in (a-h). Bars represent 1 μm and 250 nm. (B) Cellular viral particle determination. Sf9 cells were infected with vAcΔ cc or vAcΔ cc Δ fp25k (MOI = 5), total cellular DNA were collected at 0, 72 and 96 h p.i. and subjected to qPCR to determine the viral genome copy number. The data are from three independent experiments. Error bars represent standard deviation. Data were analyzed by two-tailed Student's t -test.

Techniques: Electron Microscopy, Infection, Standard Deviation, Two Tailed Test

(A) Sf9 cells were infected with vAcΔ cc or vAcΔ cc Δ fp25k at an MOI of 5. Infected cells were collected at 48 h p.i. and analyzed by western blotting using the corresponding antibodies, the results were repeated twice. (B) Sf9 cells infected with vAcΔ cc or vAcΔ cc Δ fp25k (MOI = 5) were collected at 48 h p.i. and analyzed by qRT-PCR. The transcriptional levels of viral genes were normalized to the internal control 28S rRNA, and the transcription difference between vAcΔ cc - and vAcΔ cc Δ fp25k -infected cells was analyzed by the 2 -ΔΔC T method. The results of corresponding genes in vAcΔ cc -infected cells were set as 100%. The data are from three independent experiments. Error bars represent standard deviation. Data were analyzed by two-tailed Student's t -test. * P <0.05.

Journal: PLoS ONE

Article Title: The FP25K Acts as a Negative Factor for the Infectivity of AcMNPV Budded Virus

doi: 10.1371/journal.pone.0128471

Figure Lengend Snippet: (A) Sf9 cells were infected with vAcΔ cc or vAcΔ cc Δ fp25k at an MOI of 5. Infected cells were collected at 48 h p.i. and analyzed by western blotting using the corresponding antibodies, the results were repeated twice. (B) Sf9 cells infected with vAcΔ cc or vAcΔ cc Δ fp25k (MOI = 5) were collected at 48 h p.i. and analyzed by qRT-PCR. The transcriptional levels of viral genes were normalized to the internal control 28S rRNA, and the transcription difference between vAcΔ cc - and vAcΔ cc Δ fp25k -infected cells was analyzed by the 2 -ΔΔC T method. The results of corresponding genes in vAcΔ cc -infected cells were set as 100%. The data are from three independent experiments. Error bars represent standard deviation. Data were analyzed by two-tailed Student's t -test. * P <0.05.

Techniques: Infection, Western Blot, Quantitative RT-PCR, Control, Standard Deviation, Two Tailed Test

(A) Western blot analysis of structural proteins incorporated into recombinant BVs. Equal copy numbers of vAcΔ cc and vAcΔ cc Δ fp25k virions were purified from the supernatants of infected Sf9 cells at 48 h p.i. and subjected to SDS-PAGE, then blotted with antibodies against GP64, Ac23, and VP39. (B) Transformation assay. Genomic DNA isolated from vAcΔ cc or vAcΔ cc Δ fp25k BVs harvested at 36 h and 48 h p.i. was quantified by qPCR. Equal copy numbers (5×10 8 ) of genomic DNA was used to transform competent E . coli DH10B cells, and the number of CFUs was calculated. (C) Transfection assay. Equal copy numbers of genomic DNA isolated from vAcΔ cc and vAcΔ cc Δ fp25k BVs were transfected into Sf9 cell. Transfection efficiency was calculated as the number of cells expressing EGFP. The mean number of cells transfected by BV genomic DNA of control virus was set as 100%. Data are representative of three independent experiments. Error bars represent standard deviation. Data were analyzed by two-tailed Student’s t -test. * P <0.05.

Journal: PLoS ONE

Article Title: The FP25K Acts as a Negative Factor for the Infectivity of AcMNPV Budded Virus

doi: 10.1371/journal.pone.0128471

Figure Lengend Snippet: (A) Western blot analysis of structural proteins incorporated into recombinant BVs. Equal copy numbers of vAcΔ cc and vAcΔ cc Δ fp25k virions were purified from the supernatants of infected Sf9 cells at 48 h p.i. and subjected to SDS-PAGE, then blotted with antibodies against GP64, Ac23, and VP39. (B) Transformation assay. Genomic DNA isolated from vAcΔ cc or vAcΔ cc Δ fp25k BVs harvested at 36 h and 48 h p.i. was quantified by qPCR. Equal copy numbers (5×10 8 ) of genomic DNA was used to transform competent E . coli DH10B cells, and the number of CFUs was calculated. (C) Transfection assay. Equal copy numbers of genomic DNA isolated from vAcΔ cc and vAcΔ cc Δ fp25k BVs were transfected into Sf9 cell. Transfection efficiency was calculated as the number of cells expressing EGFP. The mean number of cells transfected by BV genomic DNA of control virus was set as 100%. Data are representative of three independent experiments. Error bars represent standard deviation. Data were analyzed by two-tailed Student’s t -test. * P <0.05.

Techniques: Western Blot, Recombinant, Purification, Infection, SDS Page, Transformation Assay, Isolation, Transfection, Expressing, Control, Virus, Standard Deviation, Two Tailed Test

(A) Genomic organization of AcBacΔ cc - gp64 . (B) Western blot analysis of cells infected with vAcΔ cc or vAcΔ cc - gp64 and virions of vAcΔ cc or vAcΔ cc - gp64 . For expression of GP64 in infected cells, Sf9 cells were infected with vAcΔ cc or vAcΔ cc - gp64 at an MOI of 5. Infected cells were collected at 48 h p.i. and analyzed by western blotting. For GP64 incorporation into virions, equal copy numbers of vAcΔ cc and vAcΔ cc - gp64 virions were purified from the supernatants of infected Sf9 cells at 48 h p.i. and subjected to SDS-PAGE, then blotted with antibody against GP64, experiments were repeated twice. (C) One-step growth curve of vAcΔ cc and vAcΔ cc - gp64 , virus titers were determined by EPDA. Data are representative of three independent experiments. Error bars represent standard deviation.

Journal: PLoS ONE

Article Title: The FP25K Acts as a Negative Factor for the Infectivity of AcMNPV Budded Virus

doi: 10.1371/journal.pone.0128471

Figure Lengend Snippet: (A) Genomic organization of AcBacΔ cc - gp64 . (B) Western blot analysis of cells infected with vAcΔ cc or vAcΔ cc - gp64 and virions of vAcΔ cc or vAcΔ cc - gp64 . For expression of GP64 in infected cells, Sf9 cells were infected with vAcΔ cc or vAcΔ cc - gp64 at an MOI of 5. Infected cells were collected at 48 h p.i. and analyzed by western blotting. For GP64 incorporation into virions, equal copy numbers of vAcΔ cc and vAcΔ cc - gp64 virions were purified from the supernatants of infected Sf9 cells at 48 h p.i. and subjected to SDS-PAGE, then blotted with antibody against GP64, experiments were repeated twice. (C) One-step growth curve of vAcΔ cc and vAcΔ cc - gp64 , virus titers were determined by EPDA. Data are representative of three independent experiments. Error bars represent standard deviation.

Techniques: Western Blot, Infection, Expressing, Purification, SDS Page, Virus, Standard Deviation

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