Journal:

Article Title: Reverse Genetics for Crimean-Congo Hemorrhagic Fever Virus

doi: 10.1128/JVI.77.10.5997-6006.2003

Figure Lengend Snippet: GFP vRNA minigenome rescue by CCHF virus superinfection. 293T cells were transfected with 4 μg of plasmid pRF250 (GFP gene-containing CCHF virus minigenome construct [pol I {h} CCHF virus S CAT vRNA) and superinfected with CCHF virus 24 h later. Cell monolayers were fixed and inactivated 48 h p.i. with 4% paraformaldehyde overnight prior to removal from the containment laboratory. (a) Light microscopy of transfected and superinfected cells. (b) Immunofluorescence of transfected and superinfected cells. (c) Same cells as in panel b but at higher magnification. (d) Immunofluorescence of transfected but nonsuperinfected cells.

Article Snippet: BHK-21 (baby hamster kidney), 293T (human embryonic kidney), HeLa (human cervical carcinoma [ 21 ]), HuH-7 (human hepatoma), VeroE6 (monkey kidney), and SW13 (human adenocarcinoma) cells (American Type Culture Collection) were grown on plastic dishes in Glasgow (BHK-21), Eagle's minimal essential medium (293T, HeLa, VeroE6, and HuH-7), or Leibovitz L15 medium (SW13) supplemented with 5 to 10% fetal calf serum, 2 mM l -glutamine, 100 IU of penicillin/ml, and 100 μg of streptomycin/ml (Invitrogen/Life Technologies).

Techniques: Virus, Transfection, Plasmid Preparation, Construct, Light Microscopy, Immunofluorescence

Journal:

Article Title: Reverse Genetics for Crimean-Congo Hemorrhagic Fever Virus

doi: 10.1128/JVI.77.10.5997-6006.2003

Figure Lengend Snippet: CAT vRNA minigenome rescue by CCHF virus superinfection. Different eukaryotic cell lines were transfected with different amounts of CAT-containing CCHF virus minigenome constructs (pRF243 [pol I {m} CCHF virus S CAT {vRNA}] and pRF249 [pol I {h} CCHF virus S CAT {vRNA}]) and subsequently superinfected with CCHF virus (MOIs of 0.1 and 0.001 PFU/cell). Cells were harvested 72 h posttransfection (48 h p.i.) and analyzed for CAT activity. Lane 1, reporter gene background activity in BHK-21, HeLa, 293T, and SW13 cells upon CCHF virus infection; lane 2, reporter gene background activity after transfection of pol I vector plasmids (pRF207 and pRF240); lanes 3, 7, 11, and 15, reporter gene background activity after transfection of different CAT vRNA minigenomes (nonsuperinfected); lanes 4 to 6, CAT activity in BHK-21 cells transfected with plasmid pRF243 (Fig. ​(Fig.1C)1C) and superinfected with CCHF virus; lanes 8 to 10, HeLa cells transfected with plasmid pRF249 (Fig. ​(Fig.1C)1C) and superinfected with CCHF virus; lanes 12 to 14, 293T cells transfected with plasmid pRF249 and superinfected with CCHF virus; lanes 16 to 18, SW13 cells transfected with plasmid pRF249 and superinfected with CCHF virus.

Article Snippet: BHK-21 (baby hamster kidney), 293T (human embryonic kidney), HeLa (human cervical carcinoma [ 21 ]), HuH-7 (human hepatoma), VeroE6 (monkey kidney), and SW13 (human adenocarcinoma) cells (American Type Culture Collection) were grown on plastic dishes in Glasgow (BHK-21), Eagle's minimal essential medium (293T, HeLa, VeroE6, and HuH-7), or Leibovitz L15 medium (SW13) supplemented with 5 to 10% fetal calf serum, 2 mM l -glutamine, 100 IU of penicillin/ml, and 100 μg of streptomycin/ml (Invitrogen/Life Technologies).

Techniques: Virus, Transfection, Construct, Activity Assay, Infection, Plasmid Preparation

Journal:

Article Title: Reverse Genetics for Crimean-Congo Hemorrhagic Fever Virus

doi: 10.1128/JVI.77.10.5997-6006.2003

Figure Lengend Snippet: Optimization of the reverse genetics system. (A) Time course of reporter gene expression. 293T cells were transfected with the pol I expression cassette plasmid pRF249 (pol I [h] CCHF virus S CAT [vRNA]; Fig. ​Fig.1C)1C) 24 h prior to superinfection with CCHF virus (see scheme for the experimental procedure). Cells were harvested at different time points postsuperinfection and analyzed for CAT activity. The diagram summarizes the results from two independent experiments. (B) Effect of additional nucleoprotein on reporter gene expression. BHK-21 cells were transfected with 4 (lanes 2 and 4) or 8 μg (lanes 1, 3, and 5) of plasmid pRF243 (pol I [m] CCHF virus S CAT [vRNA]; Fig. ​Fig.1C)1C) and cotransfected with 1 μg of plasmid pCMV CCHF N (lanes 1, 4, and 5) or 1 μg of plasmid pcDNA3(+) (lanes 2 and 3) to maintain similar transfection conditions. Subsequently, cells were superinfected with CCHF virus 24 h posttransfection, harvested 72 h posttransfection (48 h p.i.), and analyzed for CAT activity. A transfected (8 μg of RF243) but noninfected culture was used as a control (lane 1).

Article Snippet: BHK-21 (baby hamster kidney), 293T (human embryonic kidney), HeLa (human cervical carcinoma [ 21 ]), HuH-7 (human hepatoma), VeroE6 (monkey kidney), and SW13 (human adenocarcinoma) cells (American Type Culture Collection) were grown on plastic dishes in Glasgow (BHK-21), Eagle's minimal essential medium (293T, HeLa, VeroE6, and HuH-7), or Leibovitz L15 medium (SW13) supplemented with 5 to 10% fetal calf serum, 2 mM l -glutamine, 100 IU of penicillin/ml, and 100 μg of streptomycin/ml (Invitrogen/Life Technologies).

Techniques: Gene Expression, Transfection, Expressing, Plasmid Preparation, Virus, Activity Assay, Control

Journal:

Article Title: Reverse Genetics for Crimean-Congo Hemorrhagic Fever Virus

doi: 10.1128/JVI.77.10.5997-6006.2003

Figure Lengend Snippet: CCHF virus S CAT cRNA minigenome rescue. 293T and BHK-21 cell lines were transfected with different amounts of the human (pRF283) and murine (pRF284) pol I-driven, CAT-containing CCHF virus cRNA minigenome plasmids (Fig. ​(Fig.1C)1C) as indicated. Twenty-four hours later, cells were superinfected with CCHF virus (MOI as indicated), harvested 72 h posttransfection (48 h p.i.), and analyzed for CAT activity. Lanes 1 and 4, transfected but noninfected controls; lanes 2 and 3, 293T cells transfected with pRF283 and superinfected with CCHF virus; lanes 5 and 6, BHK-21 cells transfected with pRF284 and superinfected with CCHF virus.

Article Snippet: BHK-21 (baby hamster kidney), 293T (human embryonic kidney), HeLa (human cervical carcinoma [ 21 ]), HuH-7 (human hepatoma), VeroE6 (monkey kidney), and SW13 (human adenocarcinoma) cells (American Type Culture Collection) were grown on plastic dishes in Glasgow (BHK-21), Eagle's minimal essential medium (293T, HeLa, VeroE6, and HuH-7), or Leibovitz L15 medium (SW13) supplemented with 5 to 10% fetal calf serum, 2 mM l -glutamine, 100 IU of penicillin/ml, and 100 μg of streptomycin/ml (Invitrogen/Life Technologies).

Techniques: Virus, Transfection, Activity Assay

Journal:

Article Title: Reverse Genetics for Crimean-Congo Hemorrhagic Fever Virus

doi: 10.1128/JVI.77.10.5997-6006.2003

Figure Lengend Snippet: Packaging of pol I-driven minigenome segments into CCHF particles. Murine (pRF243 and pRF284) and human (pRF249 and pRF283) pol I-driven CCHF virus vRNA and cRNA minigenomes (Fig. ​(Fig.1C)1C) were transfected into BHK-21 or 293T cells 24 h prior to superinfection with CCHF virus (MOI, 0.001 PFU/cell). Cell lysates were prepared 48 h later and assayed for CAT activity, while the cell culture media were collected and 2-ml undiluted samples were transferred to fresh HuH-7 or VeroE6 cells (P1). This was repeated twice (P2 and P3), and CAT activity was assayed after each passage. (A) vRNA minigenomes. Two different cell lines (HuH-7 and VeroE6) were used for passaging recombinant CCHF viruses derived from either the murine (lanes 3 to 8) or human (lanes 11 to 16) pol I system. Lane 1, pRF243-transfected but nonsuperinfected BHK-21 cells; lane 2, pRF243-transfected and superinfected BHK-21 cells; lanes 3 to 5, P1 to P3 in HuH-7 cells; lanes 6 to 8, P1 to P3 in VeroE6 cells; lane 9, pRF249-transfected but nonsuperinfected 293T cells; lane 10, pRF249-transfected and superinfected 293T cells; lanes 11 to 13, P1 to P3 in HuH-7 cells; lanes 14 to 16, P1 to P3 in VeroE6 cells. (B) cRNA minigenomes. VeroE6 cells were used for passaging recombinant CCHF viruses derived from either the human (lanes 2 to 5) or murine (lane 7 to 10) pol I system. Lane 1, pRF283-transfected but nonsuperinfected 293T cells; lane 2, pRF283-transfected and superinfected 293T cells; lanes 3 to 5, P1 to P3 in VeroE6 cells; lane 6, pRF284-transfected but nonsuperinfected BHK-21 cells; lane 7, pRF284-transfected and superinfected BHK-21 cells; lanes 8 to 10, P1 to P3 in VeroE6 cells. The results of two independent experiments are summarized.

Article Snippet: BHK-21 (baby hamster kidney), 293T (human embryonic kidney), HeLa (human cervical carcinoma [ 21 ]), HuH-7 (human hepatoma), VeroE6 (monkey kidney), and SW13 (human adenocarcinoma) cells (American Type Culture Collection) were grown on plastic dishes in Glasgow (BHK-21), Eagle's minimal essential medium (293T, HeLa, VeroE6, and HuH-7), or Leibovitz L15 medium (SW13) supplemented with 5 to 10% fetal calf serum, 2 mM l -glutamine, 100 IU of penicillin/ml, and 100 μg of streptomycin/ml (Invitrogen/Life Technologies).

Techniques: Virus, Transfection, Activity Assay, Cell Culture, Passaging, Recombinant, Derivative Assay

Journal: STAR Protocols

Article Title: Protocol to use de-epithelialized porcine urinary bladder as a tissue scaffold for propagation of pancreatic cells

doi: 10.1016/j.xpro.2022.101869

Figure Lengend Snippet:

Article Snippet: HA-R-Spondin1-Fc 293T Cells (recommended: <5 passages after selection) , Trevigen/Bio-Techne , Cat.: 3710-001-1.

Techniques: Transduction, Recombinant, Marker, Purification, DNA Purification, Selection, Software, Imaging, Cell Culture

Journal: STAR Protocols

Article Title: Protocol to use de-epithelialized porcine urinary bladder as a tissue scaffold for propagation of pancreatic cells

doi: 10.1016/j.xpro.2022.101869

Figure Lengend Snippet: Medium for culturing patient-derived organoids (see also Beutel et al. ⁴ )

Article Snippet: HA-R-Spondin1-Fc 293T Cells (recommended: <5 passages after selection) , Trevigen/Bio-Techne , Cat.: 3710-001-1.

Techniques: Concentration Assay