The overall goal of this protocol is to identify host targets that can be manipulated to enhance oncolytic virus therapy. This method can help answer key questions in the field of oncolytic virus therapy, such as what specific host factors and cellular pathways modulate oncolytic virus replication. The main advantage of this technique is that it allows one to identify host targets that can be manipulated to enhance oncolytic virus therapy in a comprehensive and unbiased manner.
This protocol can also be utilized to identify host factors that modulate virus replication in general. After developing a high-throughput screening assay according to the text protocol, remove a batch of microtiter plates from the freezer and allow them to defrost for 20 to 30 minutes. In the meantime, fill two bottles with 70%ethanol, then fill two 50mL conical tubes with PBS and one with distilled water.
Immerse liquid dispenser tubing into a bottle of 70%ethanol then place a piece of masking tape around the tubing to mark the point below which the tubing can be considered sterile. Use approximately 25mL of 70%ethanol to prime the tubing. Pour additional ethanol into the bottle to replace the lost volume, then let the tubing sit in ethanol for a minimum of five minutes.
After preparing the cell culture hood, centrifuge the sealed plates and sets at 1026 times G in room temperature for two minutes. Next, remove the seals from the plates. Then pipette the required amount of transfection reagent into the conical tubes containing the pre-warmed transfection reagent diluent.
Begin trypsinizing the cells by aspirating the medium from each plate. Use nine mL of PBS to rinse the cells, then pipette three mL of trypsin per plate. Incubate the cells at 37 degrees celsius for approximately five minutes.
Then, use 22mL of medium to resuspend the cells pipetting up and down 10 times. Combine the cell suspension from each tissue culture plate into a sterile bottle and mix the cell suspension by inverting the bottle. Then pipette one mL aliquats in the cell suspension into two separate microfuge tubes.
Remove the sample from each microfuge tube and count the cells. Then calculate the volume of cell suspension required for the density required, and prepare enough diluted cell suspension to dispense across one batch of plates. While the cells are being prepared, empty the liquid dispenser tubing of ethanol.
Use approximately 25mL of PBS to prime the tubing, and then empty the tubing. Ensure that the portion of the tubing that will be immersed in transfection reagent, and later in the cell suspension, is kept sterile. Set apart plates that can safely be done with one conical tube of transfection reagent solution.
Pipette the transfection reagent solutions up and down 10 times. Then use the solution to prime the tubing before dispensing 5 microliters per well. Centrifuge the plates at 1026 times G in room temperature for one minute.
Then incubate the plates at room temperature for the time prescribed for the transfection reagent being used. During the incubation, empty the tubing of the transfection reagent solution and place it into a full 50mL conical tube containing PBS. Then use approximately 25mL of PBS to rinse the tubing by priming and then emptying it.
Mix the bottle containing the cell suspension, then place the tubing in the suspension and prime the tubing enough to see that each tip is dispensing properly. Dispense 30 microliters per well of the cell suspension across all plates. Empty the cell suspension from the tubing, then rinse the tubing with approximately 25mL of PBS followed by approximately 50mL of distilled water.
Allow the plates to sit at room temperature for 45 to 60 minutes from the time of cell seeding. Then incubate the plates in an undisturbed incubator for the desired length of gene silencing. To save time, the day prior to infecting the cells, pipette into sterile bottles the precise amount of medium required to infect each batch of plates, including medium for uninfected wells.
Warm the medium at 37 degrees celsius, then fill two bottles with 70%ethanol, two 50mL conical tubes with PBS, and one with distilled water. Sterilize the tubing with 70%ethanol as demonstrated earlier. While the tubing is sitting in the ethanol, first vortex the virus and then pipette the precise volume of virus required into the bottle of infection medium.
Then rinse the tubing by using 25mL of PBS to prime it, and then empty it. After removing the plates from the incubator, place them into the tissue culture hood. Use medium to prime the tubing, then dispense 40 microliters of medium into the uninfected control wells.
Empty the tubing of medium, and use 25mL of PBS to rinse it. After mixing the virus, place the tubing into the bottle containing the virus, dispense 40 microliters of virus across the plates. Centrifuge the plates at 400 times G in room temperature for 30 minutes.
Incubate the plates for the previously determined length of time. The day prior to fixing, prepare a bottle of fixing and standing solution containing four percent formaldehyde and PBS for each batch of plates, but omit the Hoechst stain. Store the solution in the flammable cabinet away from light.
The following day, use 70%ethanol and PBS to rinse the tubing as demonstrated earlier. Dispense 30 microliters of the solution across all the plates, then place the plates in the incubator for 30 minutes. Following incubation, apply the seals to the plates and store them at four degrees celsius protected from light.
Carry out imaging and analysis according to the text protocol. Shown here are 786 O-cells stained with Hoechst 33342 following a 72 hour incubation with transfection reagent diluent alone. Transfection reagent and diluent, non-targeting siRNA, and with siRNA targeting PLK1.
The survival plot seen here indicates that 22%of cells transfected with PLK1 siRNA survived, and 94%of cells transfected with non-targeting siRNA survived. This graph depicts the results of the live time course of 786 O-cells infected with oncolytic vaccinia virus expressing enhanced GFP at various MOIs. Representative images of cells infected at an MOI of 0.05 over a live time course are shown here.
An incubation period of 21 hours would allow for the detection of increases and decreases in spread as the time point is within a linear range. Following genome-wide RNAi screens across three tumor cell lines, 1008 hits common to at least two out of three cancer cell lines were identified using the MAD method. 10 hits within the UPR and ERAD pathways were selected for secondary validation via siRNA with different targeting sequences.
When dispensing reagents, it is critical that you have the correct batch of plates, have selected the correct volume for dispensing, and that the tubing has been filled with the correct reagent and is always fully submerged in the reagent. After watching this video you should have a good understanding of how to conduct a high-throughput RNAi screen to identify host targets that can be manipulated to enhance oncolytic virus therapy, or to identify host factors that modulate virus replication in general.
