JoVE Encyclopedia of Experiments
Microbiology
0 views • 2:53 min • September 30th, 2026
Begin with epithelial cells infected with vaccinia virus and transfected with a recombination plasmid vector.
The vector carries a green fluorescently tagged target gene and a red fluorescently tagged metabolic selection gene under a viral promoter.
During viral DNA replication, the plasmid cassette is inserted into the viral genome.
Collect the cells in serum-free media.
Use freeze-thaw cycles to lyse the cells and release the viruses.
Prepare serial dilutions of the lysate.
Infect fresh epithelial cells with the diluted lysate to get separate individual plaques.
Add media containing serum for cell survival, and a metabolic selection reagent, then Incubate.
The selection reagent inhibits enzymes involved in nucleotide synthesis. Only infected cells carrying the metabolic selection gene express an alternative enzyme that allows nucleotide synthesis, enabling survival.
Remove the media and observe under a fluorescence microscope.
The presence of both red and green fluorescence in a plaque shows successful cassette integration and expression of the target gene.
Infect a monolayer of BS-C-one cells with vaccinia virus in serum free media.
One hour post infection, rescue the cells with Dulbecco's
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