This experiment generates fluorescently labeled recombinant EO viruses through a modular and rapid method based on transient dominant selection. First, create a recombinant vector containing a fluorescent tag surrounded by synthesized minimal regions of homology, as well as markers for metabolic and fluorescent selection. Transfect this recombination vector into infected cells and amplify for recombinant viruses using metabolic selection and fluorescence.
Next, remove the metabolic selection to excise the marker cassette and isolate the cells containing recombinant viruses by fluorescent marker.Selection. Results of viral plaques on cell monolayers allow visualization of individual virus particles that are characteristic of the fluorescently tagged gene. We developed this method as we wanted to generate recombinant viruses with multiple genetic modifications using a selection method that could be used repeatedly and universally regardless of the tag.
Whilst the principle of transient dominant selection has been described previously, we wanted to apply it on a larger scale and in a modular manner to give us more flexibility in making genomic modifications. A visual demonstration of this method is critical as recombinant viral plaque screening and isolation can be tricky. Their frequency can be low, and therefore some unique methods are required for efficient recovery of the virus.
So labeled a viral gene of interest at the N or C terminus of the protein. Accordingly, identify 150 base pair long flanking regions of homology. Then design an oligonucleotide sequence comprising the 150 base pair left and right arms, separated by a pair of restriction sites of choice.
Also flank this region by a second pair of restriction sites that are different from the first pair to allow incorporation into the TDS vector. Once synthesized, now verify that the oligonucleotide with the restriction sites is designed to be in frame with the desired insertion site. For the oligonucleotide synthesis, ensure the restriction sites incorporated in between the left and right arms.
Match those flanking the open reading frame of the fluorescence tag of choice. Incorporate these same restriction sites on either side of the fluorescence tag by PCR. Clone the synthesized fragment into the TDS vector.
Also clone the fluorescence tag into the resulting recombination vector using the restriction sites in between the left and right arm. Regions of homology. Infect a monolayer of BSC one cells with vaccinia virus and serum free media.
One hour post infection. Rescue the cells with ECCOs modified eagle medium transfect with a mixture of recombination, vector plasmid, and transfection reagents in a ratio of three to one in serum free media. After 24 hours, scrape and recover cells in DMEM containing no fetal bovine serum.
Subject the cells to three free thaw cycles to break open cells and release the virus particles next to obtain adequate separation of individual plaques. Infect 100%confluent cell monolayers of BSC one cells with serial dilutions of the preparation of virus particles. Overlay with liquid 10%FBS and DMEM plus the GPT selection, reagents and incubate for 24 hours.
Aspirate the liquid overlay and use a fluorescent microscope to look for plaques. Exhibiting diffuse red fluorescence corresponding to the incorporation of M cherry from the TDS vector into the virus. Depending on the target gene and chosen fluorescence tag, localized fluorescence of the tag gene may also be observed in the same red plaque.
Now to pick multiple plaques for each recombinant virus, scrape with the pipette tip and with 100 microliters of 5%FBS containing DMEM transferred the cells to an einor tube. Perform three rounds of freeze thawing of the scraped cells. To release the recombinant viruses.
Add the DMEM containing freeze thawed virus to a monolayer of cells. In a 12 well plate to amplify the recombinant viruses with GPT selection reagents in the growth media. Then scrape the successful amplifications at 24 hours post-infection for a plaque assay of successfully amplified plaques, exhibiting red fluorescence C two six, well played with a monolayer of BSC one cells.
Perform a serial dilution of the recombinant viruses and infect the wells with an increasing dilution of viruses in FBS free DME M1 hour post infection. Remove the liquid media and overlay each well with 0.5%agros in complete minimal essential medium three days post infection. Pick the plaques displaying fluorescence that is both red and the color of the chosen fluorescence tag.
Amplify these again without GPT selection. Pick plaques that have lost their diffuse red fluorescence, but retain the localized fluorescence corresponding to the tag of choice. Continue plaque purifying with no selection to obtain a pure stock of recombinant viruses that have lost the M cherry and GPT selection genes, but retain the localized fluorescence corresponding to the chosen tag.
Evaluate purity of the stocks with a plaque assay aiming for all plaques of a similar plaque phenotype. Amplify the test viral stocks in a monolayer of BSC one cells. After 24 hours post infection, scrape the infected cells into 250 microliters of DMEM centrifuge the infected cells.
Remove the supernat and resuspend the cell pellet in 500 microliters of 0.1%SDS in TE buffer vortex to lyce the cells. Then add 500 microliters of phenol, chloroform, isoamyl alcohol, and invert to mix centrifuge. Harvest the upper phase and repeat the extraction.
Then to precipitate the DNA from the aqueous layer, add one milliliter of chilled, 100%ethanol and 50 microliters of sodium acetate. Mix by inversion and place the sample at negative 20 degrees Celsius overnight. After pelleting the DNA by centrifugation, remove all the liquid and air dry the DNA pellet and resus.
Suspend it in 50 microliters of TE buffer. Use this genomic DNA template to PCR screen for viral insertion. Generate recombinant viruses with multiple tags as detailed in the text protocol.
These recombinant vaccinia viruses express proteins tagged with fluorescent markers targeted at viral structural proteins, A three and F 13, which are part of the inner virus core and outer envelope respectively. Genes corresponding to virion localized proteins were selected to visualize virus particles. A three is a core protein of the vaccinia virion, and F 13 is a viral envelope protein.
A double tagged virus with both fluorescently labeled A three and F 13 is also shown realtime fluorescence microscopy can be performed with a dual labeled virus. In this case, the inner core protein of vaccinia virus. A three is labeled in red and the outer envelope protein F 13 is labeled in green.
Unwrapped virus particles from the virus factory are visualized in red and wrapped virion are represented by colocalization of red and green channels. Quantitative analysis was used to determine recombination efficiencies between recombinant vectors and the vaccinia virus genome. The efficiency of homologous recombination of vectors with the vaccinia virus genome can be detected with as low as 70 base pair regions of homology.
This method has been used to create viruses with multiple fluorescent tags and or multiple gene deletions. These have been employed in the study of the virus infectious life cycle, the role of viral proteins in subverting host defense systems, as well as interactions with host signaling processes. After watching this video, you should have a good understanding of how to generate recombinant vaccinia viruses using a method that is modular in the tags used, meticulous in resolving desired recombinants and applicable in a variety of research contexts.
