gene pulser mxcelltm  (Bio-Rad)

Journal: Poultry Science

Article Title: Establishment of a stable replicon cell line of Tembusu virus (TMUV) for high-throughput antiviral screening

doi: 10.1016/j.psj.2025.106109

Figure Lengend Snippet: Generation of Vero cell line expressing TMUV replicon. (A) The schematic flowchart delineates the key steps required for the establishment of a stable replicon-harboring cell line. (B) Vero cells were electroporated with viral replicon genomic RNA (10 μg) and BSD was supplemented into the medium 2 days later. Gluc activity was quantified in cell culture supernatants at indicated time points. (C) At 30 days post-electroporation, the intracellular total RNA was extracted and RT-qPCR assay was conducted to quantify viral RNA levels. Data represents the mean ± SD ( n = 3). *** p < 0.001. Statistical significance was assessed by two-tailed t -test. LOD represents limit of detection.

Article Snippet: Single electrical pulse was given with the Gene Pulser Xcell Electroporation Systems (Bio-Rad) with setting of 270 V at 950 microfarads.

Techniques: Expressing, Activity Assay, Cell Culture, Electroporation, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: ZIKV NS5-mediated STAT2 degradation as a determinant of immune evasion and pathogenicity

doi: 10.64898/2025.12.28.696729

Figure Lengend Snippet: ( A ) Schematic of recombinant ZIKV generation by reverse genetics. L162A or L162G substitutions were introduced into the NS5 coding region of the ZIKV Dakar-41525 infectious clone (GenBank: MG758785.1 ). In vitro -transcribed full-length viral genomic RNAs (10 μg) were electroporated into Vero cells (8×10 6 cells) and culture supernatants were collected 48 h post-electroporation to generate recombinant virus stocks. Immunofluorescence images show ZIKV E protein (green) and nuclei (blue) in Vero cells infected with WT ZIKV, ZIKV-NS5 L162A , ZIKV-NS5 L162G , or mock control. Scale bar, 100 μm. ( B ) A549 cells were infected with WT ZIKV or NS5 mutant viruses at an MOI of 0.1 and harvested at the indicated time points post-infection. Cell lysates were analyzed by immunoblotting using antibodies against STAT2 and NS5; β-actin served as a loading control. ( C ) HEK293T cells (1×10 6 cells) were co-transfected with plasmids expressing HA-tagged WT or mutant NS5 (L162A or L162G; 1.5μg each) together with Flag-tagged ZSWIM8 (1.5μg). At 72 h post transfection, cell lysates were subjected to anti-HA immunoprecipitation, followed by immunoblotting for co-precipitated STAT2 and Flag-ZSWIM8. Input controls are shown. ( D ) Structural mapping of residue L162 in the ZIKV NS5-STAT2 complex (PDB: 6WCZ). L162 is highlighted on the NS5 structure. All experiments were performed at least three times with similar results and representative images are shown.

Article Snippet: For virus rescue, 10 μg of in vitro -transcribed RNA was electroporated into 8×10 6 Vero cells using the Gene Pulser XcellTM Electroporation Systems (Bio-rad) at 270 V and 950 μF.

Techniques: Recombinant, In Vitro, Electroporation, Virus, Immunofluorescence, Infection, Control, Mutagenesis, Western Blot, Transfection, Expressing, Immunoprecipitation, Residue