Identification of Recombinant Vaccinia Virus-Infected Cells Using Metabolic and Fluorescent Selection

Abstract

Source: Marzook, N. B., et al., Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins. J. Vis. Exp. (2014)

This video demonstrates the generation of recombinant vaccinia viruses using plasmid-mediated homologous recombination in infected epithelial cells. Fluorescent and metabolic markers enable the selection and identification of viruses expressing tagged proteins.

Protocol

Recombinant Virus Generation

1. As per Figure 1, infect a monolayer of BS-C-1 [Buffalo Subclone C-1 (a monkey kidney epithelial cell line derived from CV-1 cells)] cells with vaccinia virus in serum-free media at a Multiplicity of Infection (MOI) > 1.
2. 1 hr post-infection, rescue cells with dulbecco's modified eagle medium (DMEM) and transfect with a mixture of recombination vector plasmid and transfection reagent in a ratio of 3:1 in serum-free media.
3. After 24 hr, scrape and recover cells in DMEM containing no fetal bovine serum (FBS) and perform three freeze-thaw cycles to break open cells and release virus particles.
4. Perform a plaque assay with a liquid overlay of 10% FBS DMEM and GPT (Guanine Phosphoribosyltransferase (from E. coli) selection reagents, mycophenolic acid (25 µg/ml), and xanthine (250 µg/ml) as follows:
   1. Seed a 6-well plate with a monolayer of BS-C-1 cells.
   2. Infect 100% confluent cell monolayers with serial dilutions of the freeze-thawed cells containing virus particles to ensure adequate separation of individual plaques.
   3. Overlay with liquid 10% FBS containing DMEM containing the GPT selection reagents - mycophenolic acid and xanthine at final concentrations of 25 µg/ml and 250 µg/ml, respectively.
5. After a 24 hr incubation, remove the liquid overlay and use a fluorescent microscope to look for plaques exhibiting diffuse red fluorescence corresponding to the incorporation of mCherry from the TDS (transient dominant selection) vector into the virus. Depending on the target gene and chosen fluorescent tag, localized fluorescence of the tagged gene may also be observed in the same red plaque.
6. Pick multiple plaques for each recombinant virus by localized scraping with a pipette tip and 100 µl of 5% FBS containing DMEM to transfer cells to an Eppendorf tube, followed by three rounds of freeze-thawing of scraped cells to release recombinant viruses.
7. Add the DMEM containing freeze-thawed virus to a monolayer of cells in a 12-well plate to amplify recombinant viruses with GPT selection reagents in the growth media. Scrape successful amplifications 24 hr post-infection.
8. Perform a plaque assay of successfully amplified plaques exhibiting red fluorescence, this time with an agarose overlay and GPT selection, as follows:
   1. Seed a 6-well plate with a monolayer of BS-C-1 cells.
   2. Perform a serial dilution of the recombinant viruses and infect wells with an increasing dilution of viruses in FBS-free DMEM.
   3. 1 hr post-infection, remove the liquid media and overlay each well with 0.5% agarose in minimal essential medium (MEM) containing 2.5% FBS, 292 µg/ml L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin, along with the GPT selection reagents.
9. 2-3 days post-infection, select plaques displaying fluorescence that is both red and the color of the chosen fluorescent tag to amplify again, this time without GPT selection.
10. Perform the next plaque assay with an agarose overlay with no selection.
11. Pick plaques that have lost their diffuse red fluorescence but retain the localized fluorescence corresponding to the tag of choice.
12. Continue plaque purifying with no selection to obtain a pure stock of recombinant viruses that have lost the mCherry and gpt selection genes but retain the localized fluorescence corresponding to the chosen tag. Check stocks are pure by plaque assay; all plaques should have a similar plaque phenotype.

Results

Figure 1. Outline of the experimental procedure to create a recombinant vaccinia virus. Events occurring at the genetic and cellular levels are depicted, along with representative plaque images outlining the steps following the creation of an A3-GFP-labeled fluorescent vaccinia virus. (A) Cells infected with vaccinia virus are transfected with the TDS recombination vector. (B) In this figure, only the result of left-hand recombination is depicted, and the example uses GFP as the fluorescent tag of choice. Right-hand recombination would result in the entire TDS plasmid being incorporated into the genome in a similar way, except the tag would be fused to the entire target gene in the intermediate step, i.e., step C. A plaque assay is performed on a cell monolayer with the recombination mix and subjected to GPT selection. (C) Plaques exhibiting both red and green fluorescence, corresponding to mCherry and GFP expression, respectively, are picked and amplified. Once selection is removed, there will be a loss of red fluorescence corresponding to the loss of the gpt and mCherry genes (D), and plaques exhibiting exclusively green fluorescence are picked and amplified (E).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Mycophenolic acid	Sigma-Aldrich	M3536-50MG	Dissolve in 0.1 N NaOH
Xanthine	Sigma-Aldrich	X0626-5G	Dissolve in 0.1 N NaoH
FuGENE HD transfection reagent	Promega	E2311
Fluorescence microscope fitted with Chroma filters 31001, 31002	Olympus, Chroma	BX51 (Olympus); 31001, 31002 (Chroma)