The overall goal of this procedure is to utilize high throughput RNAI screening to identify host factors involved in virus infection. This is accomplished by first depleting genes of interest by RNAI in 384 well plates. Next, the cells are challenged with the virus of interest stain for viral proteins and measured for viral infection by immunofluorescence microscopy.
Finally, automated image analysis is performed to identify candidate genes for validation and further study. Ultimately, results can be obtained that show changes in infection through quantification of the percentage of infected cells using immunofluorescence microscopy. This allows for the discovery of novel cellular factors involved in viral infection.
Though this method can provide insight into virus host interactions, it can also be applied to the study of any system that would benefit from using a visual readout of the cellular process of interest. Individuals new to this method should expect a steep learning curve because achieving reproducible infections while minimizing variability requires considerable optimization To ensure reproducibility of results, it is essential to use a single lot number for each reagent used in an entire screen. Grow S 2D RSC drosophila cells and complete Schneider's medium at 25 degrees Celsius cells should be in log phase.
For experiments in a laminar flow hood dislodge the semi adherent cells from the flask by rigorous pipetting. Count on a hemo cytometer transfer 125%of the cells needed for the experiment to a 50 milliliter conical tube pellet at 300 G for five minutes. Aspirate the super named resuspend the pellet at a final concentration of 1.7 times 10 to the six cells per milliliter in serum free Schneider's medium.
Use a sterilized and primed automated liquid handling system. Here the matrix well mate to minimize plate variability. Add 10 microliters of cell suspension to each well of a 384 well plate pre Eloqua with 250 nanograms per well of double stranded RNA Spin cells onto plates at 300 G for one minute.
Incubate the plates at 25 degrees Celsius for 45 minutes. Add 20 microliters of complete Schneider's medium to each. Well spin down the medium at 300 G for one minute to minimize the effects of evaporation.
Store plates and humidified sealed containers lined with water soaked paper towels. Incubate a 25 degrees Celsius for three days to allow for robust knockdown effects in a BSL two biosafety cabinet. Before infecting the cells, filter the crude vaccinia virus stock through a 0.8 micron syringe filter.
To remove cellular debris. Dilute the virus in Schneider's medium with 2%serum to obtain a multiplicity of infection. Abbreviated MOI of two prime.
The sterilized wellm mate with diluted virus until the tubing is free of air bubbles. Using a sterilized multichannel aspirator with vacuum manifold, gently remove medium from the plates of double stranded RNA treated drosophila cells. Dispense 30 microliters of diluted virus into each plate.
Well centrifuge the plates at 300 G for one minute. Return the plates to the humidified container and incubate at 25 degrees Celsius for 48 hours. In a BSL two biosafety cabinet aspirate media from the plate.
Then use a multichannel pipette to add 15 microliters of 4%formaldehyde fix to each plate. Well incubate room temperature for 15 minutes. Remove the liquid with a multichannel aspirator.
Use the WELLMADE to add 30 microliters of PBST to each. Well incubate at room temperature for 10 minutes. Perform a second 10 minute PBST wash in the same manner.
Removing the liquid after the incubation block and 30 microliters of PBST with 2%VSA blocking solution. Incubate at room temperature for 10 minutes. Use a multi-channel repeat pipetter to add 15 microliters of primary antibody diluted in blocking solution to each.
Well spin down plates at 300 G for one minute. Seal each plate with clear ceiling Film incubate at four degrees Celsius overnight. Use the well mate and multi-channel aspirator to wash the cells in PBST three times for 10 minutes each wash.
Use a multi-channel pipetter to add 15 microliters of fluorescently labeled secondary antibody with nuclear stain to each. Well cover with foil to protect from light incubate plates at room temperature for one hour. Wash the cells with PBST three times as before for 10 minutes each wash.
Add 30 microliters of PBST to cells and seal with clear ceiling. Film and cover with foil. Store sealed plates at four degrees Celsius for up to three weeks before imaging on an automated microscope.
Uninfected wells do not show any staining for vaccinia virus proteins while a representative infected well contains cells. Staining positive for vaccinia expressed beta GCSEs protein as measured by immunofluorescence microscopy. Automated image analysis software quantifies infection in each image using parameters set by the user.
In a representative image, the total number of cell nuclei and the number of cells expressing viral antigen are counted based on size and intensity of staining above the local background. Staining knockdown of thread serves as a positive control for robust RNAi depletion of target genes. Knockdown of this anti-apoptotic factor leads to dramatic cell death.
Knockdown of luciferase serves as a negative control for the effect of double stranded. RNA Treatment on infection Depletion of luciferase has no effect on infection relative to cells Left untreated. Knockdown of beta galacto protein serves as a positive control for a reduction in infection.
Since the assay uses beta galacto protein levels as a readout of infection, depletion of beta galacto results in a decrease in the percentage of infected cells. Knockdown of cellular factor RAB five results in a decrease in the percentage of infected cells. Since RAB five is known to participate in endocytosis, this factor likely contributes to vaccinia virus entry.
After watching this video, you should have a good understanding of how to use RNAi screening in drosophila to identify host factors that contribute to viral infection, including performing RNAi, infecting with vaccinia virus, staining imaging, and analysis of 384. Well Plates don't forget that working with DIA virus can be extremely hazardous and precautions such as wearing lab coats and protective eyeglasses should always be taken while performing this procedure. Live virus should only be opened in a BSL two biosafety cabinet, and anything in contact with live virus should be sterilized in 10%bleach before disposal.
