This protocol describes a reliable and efficient reverse genetics system for making recombinant rotaviruses. It also explains how to make recombinant rotaviruses that express fluorescent marker proteins. This method is simple requiring a minimal number of plasmids and can generate recombinant rotaviruses that express separate fluorine proteins as well as also viral proteins.
Begin by seeding BHKT7 cells onto 12-well plates. Rinse a freshly confluent monolayer of cells with PBS. Then disrupt the monolayer with trypsin-EDTA solution and resuspend the cells in five milliliters of GMEM complete medium.
Determine the concentration of viable BHKT7 cells using trypan blue and an automated cell counter. Then seed two times 10 to the fifth cells in one milliliter of GMEM into each well of a 12-well cell culture plate. Incubate the plate at 37 degrees Celsius in a 5%carbon dioxide incubator overnight.
On the next day, prepare the plasmid mixture for transfection according to manuscript directions. Then add 110 microliters of pre-warmed reduced serum medium to each plasmid mixture and gently pipette up and down to mix. Add 32 microliters of transfection reagent to the mixture.
Do a brief centrifugation following vortexing and incubate at room temperature for 20 minutes. Meanwhile, rinse the BHKT7 cells with two milliliters of GMEM incomplete medium. Add one milliliter of SMEM incomplete medium to each well and return the plate to the incubator.
After the 20-minute incubation, use a 200 microliter pipettor to add the transfection mixture drop by drop to each well. Gently rock the plate and return it to the incubator. Two days after transfection, rinse a freshly confluent monolayer of MA104 cells with PBS.
Disrupt the monolayer with trypsin-EDTA solution and resuspend the cells in five milliliters of DMEM complete medium. Count the cells and adjust the concentration to eight times 10 to the fifth cells per milliliter of DMEM incomplete medium. Add 0.25 milliliters of MA104 cells dropwise to the wells with the transfected BHKT7 cells.
Adjust the concentration of trypsin to 0.5 micrograms per milliliter by adding 0.8 microliters to one milligram per milliliter trypsin stock solution to each well. Dilute the remaining MA104 cells to a concentration of 1.5 times 10 to the fifth cells per milliliter in DMEM complete medium and place two milliliters in each well of a six-well plate. Six days after transfection, recover the recombinant virus from the transfected cells.
Subject the BHKT7 MA104 cells to three cycles of freeze/thaw under sterile conditions, moving the plates between a negative 20 degrees Celsius freezer and room temperature. Transfer lysates to 1.5 milliliter tubes. Centrifuge the tubes for 10 minutes at 500 times g and four degrees Celsius to pellet the cellular debris.
Collect the supernatant and store it at four degrees Celsius. Add trypsin to 100 microliters of clarified cell lysate to a final concentration of 10 micrograms per milliliter and incubate the mixture at 37 degrees Celsius for one hour. Then prepare a 10-fold serial dilution series ranging from 10 to the negative one to 10 to the negative seven in DMEM incomplete medium.
Rinse the MA104 monolayers twice with two milliliters of PBS and once with DMEM incomplete medium. Add 400 microliters of lysate dilutions in duplicate to the plates and incubate them at 37 degrees Celsius and 5%carbon dioxide, rocking every 10 to 15 minutes to redistribute the dilutions across the monolayer. Prepare an agarose MEM overlay solution by combining equal volumes of pre-warmed 2X EMEM with 1.5%melted and cooled agarose.
Maintain this solution at 42 degrees Celsius in a water bath and adjust the trypsin concentration to 0.5 micrograms per milliliter immediately before placing it on cells. Aspirate lysate dilutions from the six-well plate. Then rinse the cells once with two milliliters of incomplete medium.
Gently add three milliliters of the agarose MEM overlay solution onto the cell monolayer in each well. Allow the agarose to harden at room temperature. Then return the plates to the incubator.
Three days later, prepare an agarose MEM overlay solution as previously described and add neutral red to a final concentration of 50 micrograms per milliliter immediately before use. Add two milliliters of the overlay solution on top of the existing agarose in the six-well plates. Allow the agarose to harden and return the plate to the incubator, making sure to protect the plates with neutral red from light.
Over the next six hours, identify rotavirus plaques with the aid of a light box. Use disposable transfer pipettes to pick clearly defined plaques, recovering agarose plugs that extend fully to the cell layer. Expel the plug into a 1.5 milliliter tube containing 0.5 milliliters of DMEM incomplete medium and vortex the sample for 30 seconds.
Amplify the plaque isolated virus eluted into the medium by propagation on MA104 monolayers or AT25 flask with DMEM incomplete medium and 0.5 micrograms per milliliter of trypsin. Add 600 microliters of clarified infected cell lysates and 400 microliters of guanidinium thiocyanate into 1.5 milliliter microcentrifuge tubes. Vortex them for 30 seconds and incubate them at room temperature for five minutes.
Then add 200 microliters of chloroform. Vortex the solution for 30 seconds and incubate it for three minutes. After a five-minute microcentrifugation at 13, 000 times g and four degrees Celsius, transfer 550 microliters of the upper aqueous phase into a fresh tube.
Add two volumes of cold isopropyl alcohol and invert the tube four to six times. Incubate the sample at room temperature for 10 minutes. Then centrifuge it for 10 minutes at 13, 000 times g and four degrees Celsius.
Discard the supernatant leaving the RNA pellet. Wash the RNA by adding one milliliter of 75%ethanol to the tube inverting it once and centrifuging it for five minutes at 7, 500 times g and four degrees Celsius. After carefully removing the ethanol, allow RNA to air dry for five to 10 minutes.
Then dissolve the pellet in 15 microliters of nuclease free water. Add two microliters of 6X DNA loading buffer to 10 microliters of the dissolved RNA sample. Load it onto a pre-cast 10%polyacrylamide mini gel.
Resolve the RNAs by electrophoresis in tris-glycine running buffer for two hours under a constant 16 milliampere current. Once the gel run is complete, soak the gel for five to 10 minutes in water containing one microgram per milliliter ethidium bromide and detect the rotavirus genome segments with a UV transilluminator. This reverse genetics protocol was used to generate recombinant SA11 viruses that can be easily identified with a plaque assay on MA104 cells allowing for plaque isolation.
The dsRNA genomes of plaque purified rSA11 wild type and SA11-UnaG viruses were extracted with a solution containing phenol and guanidinium thiocyanate, resolved by electrophoresis on a 10%polyacrylamide gel and detected by staining with ethidium bromide. As expected, the segment seven or NSP3 dsRNA of rSA11-UnaG migrated much slower than that of the rSA11 wild type due to the presence of 2A-3xFLUnaG sequences. To check for expression of the UnaG fluorescent protein, MA104 cells were infected with wild type and recombinant virus and examined with a live cell imager.
The analysis showed that rSA11-UnaG produced green fluorescence while no fluorescence was detected in cells infected with rSA11 wild type. Immunoblot analysis was performed to investigate whether the 2A element of rSA11-NSP3-2A-xflUnaG promoted the expression of two separate proteins. The analysis showed that NSP3-2A and 3xFL-UnaG were indeed expressed as separate proteins indicating a functional 2A element.
For successful recovery of recombinant rotavirus, it is critical to use quality, well-maintained BHKT7 cells for transfection and MA104 cells for over seeding as well as accurate amounts of highly pure plasmids. Although this protocol explains how to use reverse genetics system to modify the rotavirus NSP3 gene, the same approach can be used to modify other genes such as SP1.
