HCT116, BHK21 and Vero E6 cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 and cultured in Dulbecco’s Modified Eagle medium (DMEM; Invitrogen) supplemented with 10% FBS. Viral stocks were prepared from infectious clones (kindly provided by Dr Carla Saleh, Institut Pasteur, Paris, France) carrying either the wild-type or a GFP containing version of the SINV genomic sequence as previously described [29 (link)]. Briefly, the plasmids were linearized by XhoI digestion and used as a substrate for in vitro transcription using the mMESSAGE mMACHINE capped RNA transcription kit (Ambion, Thermo Fisher Scientific Inc.). In vitro synthesized viral RNA was transfected in BHK21 cells, viral infectious particles were collected 48 hpt and used to infect BHK21 for the full viral production. Titers were measured by standard plaque assay in Vero E6 cells. Cells were infected at a MOI of 1, 10−1 or 10−2 as indicated in the figure legends.
Mmessage mmachine capped rna transcription kit
Manufactured by Thermo Fisher Scientific
The MMESSAGE mMACHINE capped RNA transcription kit is a laboratory tool designed for the in vitro synthesis of capped RNA transcripts. The kit includes reagents and protocols for the efficient production of capped RNA from DNA templates.
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3 protocols using mmessage mmachine capped rna transcription kit
1
SINV Viral Stock Preparation
2
Cell Culture and Virus Production Protocol
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Cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO2. HCT116, BHK-21 and Vero cells were cultured in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% FBS. SK-N-BE(2) cells (95011815, Sigma-Aldrich) were maintained in 1:1 medium composed of Ham's F12 medium (Gibco, Thermo Fisher Scientific ) supplemented with 15% FBS and Eagle's minimal essential medium (ENEM) supplemented with 1% NEAA (Gibco, Thermo Fisher Scientific). SK-N-BE(2) cell differentiation was induced by 10 µM retinoic acid (RA) treatment (R2625, Sigma-Aldrich) for 5 d. Plasmids carrying a green fluorescent protein (GFP)-SINV genomic sequence or wild-type SINV genomic sequence (kindly provided by Dr. Carla Saleh, Institut Pasteur) were linearized and used as a substrate for in vitro transcription using mMESSAGE mMACHINE capped RNA transcription kit (Ambion, Thermo Fisher Scientific) as in López et al. (2020) (link). Viral stocks were prepared in BHK-21 baby hamster kidney cells, and titers were measured by plaque assay in Vero cells. Cells were infected at a MOI of 10−2 or 10−1 as indicated in the figure legends and samples were harvested at 24 h post-infection (hpi) unless specified otherwise.
3
Recombinant SINV-GFP Virus Production
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Plasmid carrying a green fluorescent protein (GFP)‐SINV genomic sequence (a kind gift of C. Saleh) was linearized with XhoI as in Girardi et al (2013 ). It was used as a substrate for in vitro transcription using mMESSAGE mMACHINE capped RNA transcription kit (Ambion, Thermo Fisher Scientific Inc.) following the manufacturer's instructions. GFP expression is driven by duplication of the subgenomic promoter. SINV‐GFP viral stocks were prepared in BHK21 cells, and titers were measured by plaque assay. Cells were infected with SINV‐GFP at a MOI of 1 or 10−1, and samples were harvested at 48 h post‐infection (hpi).
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