Plasmid-Mediated Generation of Recombinant Rotavirus in Cultured Cells

Begin with a tube containing a recombinant plasmid mixture encoding the full set of rotavirus genes in a reduced-serum medium.
Add a transfection reagent to facilitate plasmid uptake.
Introduce the transfection mixture dropwise onto a monolayer of fibroblast cells in a multiwell plate and gently rock the plate.
Incubate to allow plasmid entry, enabling transcription of viral RNAs and protein synthesis.
This results in the assembly of immature virions that remain intracellular or loosely attached to the cell surface.
Next, overlay with epithelial cells, the natural targets of rotavirus, to support viral replication.
Add trypsin to cleave the outer capsid protein on surface-associated virions.
Cleavage activates the virions, allowing entry into epithelial cells and productive infection.
Perform freeze-thaw cycles to lyse the cells and release recombinant virions.
Transfer the lysate to a tube and centrifuge to pellet debris.
Collect the supernatant containing recombinant rotavirus for downstream applications.
Seed two times ten to the fifth cells in 1 milliliter of GMEM into each well of a 12-well cell culture plate. Incubate the plate at 37 degrees Celsius in a 5% carbon dioxide incubator over