Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection

Our method uses host range selection and fluorescent markers for the efficient and seamless generation of recombinant vaccina virus in a variety of cell types. This technique combines the speed of homologous recombination with the scarless nature of transient dominant selection. In addition, we host range selection to eliminate the possibility of chemically induced changes.

This method can also be used to modify vaccinia virus or other poxviruses to express foreign genes, for example, to generate vaccines. If you're performing this protocol with fluorescence selection only, you must ensure that you're screening enough viruses to detect a relatively rare recombinants without PKR selective pressure. This method relies on the gain and loss of fluorescent markers to successfully select viruses that have acquired the recombination cassettes and to detect viruses that have scarless recombined.

To generate a recombination vector, first add 17 microliters of DNase free water, 1.2 microliters of each primer, five microliters of 5x PCR reaction buffer, template DNA, and 0.5 microliters of DNA polymerase to one PCR tube per amplicon. Add additional DNase free water to bring the final volume in each tube to 50 microliters. And place the tubes into a thermocycler.

Melt the DNA at 98 degree Celsius for 30 seconds before using 25 rounds of a three-step PCR protocol as indicated. To visualize the amplification products on a 1%agarose gel, add 10 microliters of each DNA product and 2 microliters of loading buffer to each well. Run the gel for one hour at eight volts per centimeter.

At the end of the run, use a DNA gel extraction kit according to the manufacturer's protocol to gel purify each amplicon. Elute the amplicons from the column with 50 microliters of DNase free water and immediate centrifugation. To linearize a cloning vector, first add one microgram of the vector, 17 microliters of DNase free water, 2 microliters of reaction buffer, and 1 microliter of EcoRI to a tube for a one hour incubation at 37 degree Celsius.

At the end of the incubation, add 0.2 picomoles of the linearized vector 10 microliters of DNase free water, and 10 microliters of DNA assembly master mix to each isolated amplicon. Incubate samples at 50 degree Celsius for one hour. At the end of the incubation, transform chemically competent E.coli with two microliters of the assembled product according to standard protocols.

Plate 100 microliters of transformed cells onto LB agarose plates supplemented with 100 micrograms per milliliter of ampicillin. After overnight incubation at 37 degree Celsius, select well-isolated colonies for inoculation in Luria broth supplemented with 100 micrograms per milliliter of ampicillin overnight at 37 degree Celsius and 225 revolutions per minute. The next morning, use a plasmid miniprep kit to isolate the plasmids.

And check the concentration and purity of the DNA on a spectrophotometer. For recombinant virus generation, infect a confluent monolayer of suitable cells with the virus to be recombined at a multiplicity of infection of one in a six-well plate. And incubate the infected cells at 37 degree Celsius and 5%carbon dioxide for one hour.

At the end of the incubation, replace the supernatant in each well with fresh DMEM and using a commercially available transfection reagent according to the manufacturer's protocol. Add a three micrograms of the recombination vector of interest to each well. Place the plate in the incubator for additional 48 hours.

Before pooling the cells from each transfection condition into individual 1.5 milliliters tubes. Then freeze-thaw the pooled cells three times before sonicating the resulting lysates for 15 seconds at a 50%amplitude. Next, serially 10-fold dilute the sonicated lysate, harvested from a 10 to the first to 10 to the sixth concentration in fresh DMEM and add one milliliter of each dilution to individual confluent wells of a protein kinase R competent cell line.

Place the cells in the cell culture incubator for one hour before replacing the supernatants with fresh medium and returning the cells to the cell culture incubator. After 24 to 48 hours, identify the recombinant viruses by fluorescence microscopy. Plaques from recombinant viruses express red fluorescence due to integration of the mCherry-E3L fusion gene.

Plaque purify the recombinant viruses three times on wild type RK13 cells. After three rounds of plaque purification, infect RK13+E3L+K3L cells with serial dilutions of the plaque lysate. To identify the collapsed viruses, by fluorescence microscopy using an appropriate imaging system equipped with GFP and RFP filter cubes.

Then plaque purify green-only VC-R4, or colorless E3L plaques three times on RK13+E3L+K3L cells. Ensuring that no plaques fluoresce red. Infect the cells with at least 100 plaque forming units to increase your chances of identifying a colorless plaque.

In rare cases, multiple rounds of infection might be required. Red fluorescent plaques in protein kinase R competent RK13 cells are indicative of viral expression of mCherry-E3L. And colorless plaques or plaques expressing only eGFP in RK13+E3L+K3L cells confirm the collapse of the mCherry-E3L selection marker.

The recombinant virus VC-R4, which lacks both protein kinase R antagonists cannot replicate in protein kinase R competent RK13 cells while the apparent virus VP872, which expresses E3L is replication competent. To confirm that this inability to replicate in RK13 cells was only due to the loss of E3L, enhanced GFP was replaced with E3L in the VC-R4 virus. It generated a relevant virus using the same selection protocol.

Interestingly, while making this virus, colorless plaques consistent with collapse of the mCherry-E3L selection marker were identified prior to selection in RK13+E3L+K3L cells that are generally required to select scarless recombinants. Likely because of the extended sequence identity between the mCherry-E3L recombination cassette and the E3L gene being inserted into VC-R4. On average 12.6%of progeny virions underwent recombination with the transfected plasmid, similar to previously reported frequencies.

Here, the frequency of colorless plaques relative to the total plaques RK13+E3L+K3L cells is shown, demonstrating that the rate of collapse and the loss of the mCherry-E3L selection marker occurred at an approximately 1.8%frequency. In the first stage using PKR competent cells ensures that all of the plaques with contained recombinant virus. In the second stage using PKR deficient cells enables the detection of recombinant virus with a collapse mCherry-E3L locus.

This approach allows us to rapidly generate viruses containing exogenous genes, for example, for vaccine production offer the expression of different viral PKR antagonists. to facilitate the analysis of species-specific interactions with PKR from various hosts.