Recombinant Merkel Cell Polyomavirus (MCPyV) Virion Preparation: A Technique to Produce High-titer Recombinant MCPyV Virion
Trascrizione
– Merkel cell polyomavirus, or MCPyV, is a double-stranded DNA oncovirus with a circular genome that causes merkel cell carcinoma, a type of skin cancer. To prepare MCPyV virions, first, treat an adherent culture of packaging cells with a cocktail containing a transfection agent, the viral genome, and two expression plasmids encoding proteins required for promoting viral replication.
During incubation, the transfection reagent forms lypoplexes with the plasmids, facilitating their delivery into the packaging cells. Once inside the nucleus, the cell machinery allows the expression of the viral and plasmid genomes. The expression plasmids encode large-tumor antigen and small-tumor antigen proteins. These proteins translocate to the nucleus and drive viral DNA replication. Subsequently, the viral genome encodes structural capsid proteins. These proteins move into the nucleus and help in the assembly of recombinant virions.
Next, harvest the cultured cells. Treat them with a solution containing detergent and DNase. The detergent lyses the cells to release the viruses. DNase degrades any free plasmid DNA from the suspension. Centrifuge to separate cellular debris in the pellet. Load the virus-containing supernatant into a tube carrying a suitable density gradient medium. Centrifuge to obtain concentrated virion-rich fractions within the gradient medium.
In the following protocol, we will show the preparation of high-titer recombinant MCPyV virions using 293TT cells.
– In the late afternoon or evening, seed 6 million 293TT-28 cells into a 10 centimeter dish containing supplemented DMEM medium. Incubate at 37 degrees Celsius with 5% carbon dioxide. The next morning, ensure that the cells are about 50% confluent. Then, transfect the cells with 66 microliters of transfection reagent, 12 micrograms of re-ligated MCPyV isolate R17b DNA, 8.4 micrograms of ST expression plasmid pMtB, and 9.6 micrograms of LT expression plasmid pADL. Incubate the cells overnight at 37 degrees Celsius with 5% carbon dioxide.
The following day, when the transfected cells are nearly confluent, trypsinize the cells and transfer them to a 15 centimeter dish for continued expansion. When that dish becomes nearly confluent, transfer the cells into three new 15 centimeter dishes. When those new dishes become nearly confluent, harvest the cells as outlined in the text protocol. Centrifuge at 180 x g at room temperature for 5 minutes. Then, remove the supernatant and add 1 cell volume of DPBS-Mg.
Add 25 micromolar ammonium sulfate, followed by 0.5% Triton X-100, 0.1% Benzonase, and 0.1% of an ATP-dependent DNase. Mix well and incubate at 37 degrees Celsius overnight. The next day, cool the mixture on ice for 15 minutes. Next, add 0.17 volume of sodium chloride. Mix and incubate on ice for another 15 minutes. Centrifuge at 12000 x g at 4 degrees Celsius for 10 minutes. If the supernatant is not clear, gently invert the tube and repeat the centrifugation.
After this, transfer the supernatant to a new tube. Resuspend the pellet in one volume of DBPS supplemented with 0.8 moles of sodium chloride. Centrifuge at 12000 x g at 4 degrees Celsius for 10 minutes. If the supernatant is not clear, gently invert the tube and repeat the centrifugation. Combine the two supernatants and centrifuge again at 12000 x g at 4 degrees Celsius for 10 minutes.
Then, use a pipette to deposit gradients of iodixonal into thin-walled 5 milliliter polyallomer tubes as outlined in the text protocol. Load 3 milliliters of the clarified, virus-containing supernatant on top of the prepared iodixonal gradient. Centrifuge with an SW55ti rotor at 234,000 x g and at 16 degrees Celsius for 3.5 hours, making sure to set the acceleration and deceleration to slow. After the ultracentrifugation is complete, collect 12 fractions in siliconized tubes.
