This protocol can be used to screen arrayed lentiviral or retroviral assay libraries to identify novel regulators of transcription factors in cancer cells. The main advantages of this technique are that it is rapid, medium throughput, and inexpensive, and that it uses equipment and reagents commonly accessible to most investigators. To expand and purify each lentiviral vector in the library, first add 1.3 milliliters of Luria broth supplemented with 100 micrograms per milliliter of ampicillin to each well of a 96-well deep well plate.
Inoculate each well with two microliters of glycerol stock for an overnight incubation at 37 degrees Celsius and 225 revolutions per minute. The next day, transfer each bacteria culture into individual 1.5 milliliter microcentrifuge tubes for centrifugation. Purify each vector with an appropriate bacterial miniprep kit according to the manufacturer's instructions.
For each vector in the prepared library, seed one well of a 24-well plate with one times 10 to the fifth 293FT cells and incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours. To prepare a transfection mixture which will be set up for each vector in the library, first elute each lentiviral vector to a final concentration of 50 nanograms per microliter with nuclease-free water. Transfer five microliters of each dilution into individual wells of a 96-well PCR plate.
Make a transfection super mix by mixing 1.25 microliters times x of transfection reagent one with 23.75 microliters times x of pre-warmed transfection buffer. Incubate the transfection super mix at room temperature for five minutes. Then add 125 nanograms times x of psPAX2 and 125 nanograms times x of VSVG to the super mix with gentle pipetting.
Immediately aliquot the super mix into each tube of a PCR strip and use a multichannel pipette to transfer 25 microliters of the mixture into each well of diluted viral vector. After a 20 minute incubation at room temperature, use a multichannel pipette to transfer 30 microliters of each transfection mixture from each well into a corresponding well of 293FT cells in the previously prepared 24-well plate. Incubate the cells for 24 hours before replacing the medium in each well with 500 microliters of fresh complete growth medium for another 24-hour incubation in the cell culture incubator.
The next day, use a multichannel pipette to collect the viral supernatant from each well and aliquot 220 microliters of each supernatant into two wells in a new 96-well plate to generate an arrayed viral supernatant plate. To prepare A375 cells for infection, seed one times 10 to the fifth cells in a 24-well plate in 0.5 milliliters of complete growth medium per well for each viral vector in the library. Include an extra well that will not be infected to serve as a control for drug selection.
After a 24-hour incubation in the cell culture incubator, thaw the frozen arrayed lentiviral supernatant set to room temperature before replacing the growth medium from each well of the prepared cell culture plate with 200 microliters of growth medium supplemented with 20 micrograms per milliliter of polybrene. Using a multichannel pipette, transfer 200 microliters of viral supernatant from each 96 well to each well of the 24-well plate. Return the cells to the cell culture incubator for 24 to 48 hours.
At the end of the incubation, replace the medium in each well with 500 microliters of growth medium supplemented with puromycin and return the cells to the cell culture incubator for an additional 48 hours. Following the 48-hour puromycin selection, sort the cells into three or four groups such that all of the wells in a single group have a similar cell density and add 200 microliters of trypsin-EDTA to one representative well from each group for a five-minute incubation at 37 degrees Celsius. Once the cells have detached, neutralize the trypsin with 400 microliters of growth medium supplemented with puromycin and count the number of cells from each trypsinized well.
Dilute each cell suspension to a two times 10 to the fifth cells per milliliter concentration with growth medium supplemented with puromycin and seed 500 microliters of cells from each well into the same well position in a new 24-well plate. After using the counts from each representative well to estimate the cell number in each remaining well in each group, add the appropriate volume of trypsin-EDTA to each well to achieve a one times 10 to the sixth cells per milliliter concentration. During the trypsinization, use a multichannel pipette to add 400 microliters of growth medium supplemented with puromycin to the appropriate corresponding wells in the new 24-well plate.
Once cells have detached, gently mix the contents of each well and transfer 100 microliters of each cell suspension to the appropriate corresponding well in the new 24-well plate. Then return the cells to the cell culture incubator for 24 hours. To transfect the cells with the dual-luciferase reporter constructs, prepare the transfection dilution mixture as indicated in the table.
To prepare the reporter dilution mixture, mix the volumes of each reagent multiplied by the total number of transfections plus several extra volumes. Next, mix the transfection dilution mixture with the reporter dilution mixture and incubate the resulting solution at room temperature for 15 minutes to produce the transfection mixture. During the incubation, rinse each well of the cell culture plate with 250 microliters of PBS and add 447 microliters of fresh complete growth medium to each well.
Then use a multichannel pipette to distribute 53 microliters of transfection mix to each well of the cell culture plate. Place the plate at 37 degrees Celsius and 5%carbon dioxide for 24 hours. To measure the luciferase activity, dilute 5X passive lysis buffer from the luciferase activity kit at a one-to-five dilution with deionized water and thaw the necessary volumes of reagent A and reagent B buffer from the kit.
When the reagents are ready, replace the supernatant in each well with 75 microliters of passive lysis buffer and incubate the plate for 30 minutes at room temperature with occasional shaking. During the incubation, dilute the 50X reagent B substrate from the kit to a one-to-50 concentration with thawed reagent B buffer. At the end of the incubation, add 30 microliters of passive lysis buffer to four blank control wells and transfer 30 microliters of lysate from each well into duplicate wells of a 96-well flat bottom white assay plate.
When all of the lysate has been plated, use a multichannel pipette to add 50 microliters of reagent A to each well and read the firefly luciferase signal with a plate reader. Then use a multichannel pipette to add 50 microliters of reagent B to each well and read the Renilla luciferase signal with a plate reader. As expected, YAP2SA increases firefly luciferase activity but not Renilla luciferase activity as compared to the control vector.
In contrast, YAP2SA S94A does not increase firefly luciferase activity. Importantly, YAP2SA does not alter the activity of a minimal promoter construct that lacks the MCAT TEAD binding elements. In cells transfected with the YAP-TAZ-TEAD reporter construct, the tandem YAP and TAZ short hairpins significantly reduced firefly luciferase levels, but the control short hairpin non-targeting control does not.
In contrast, in cells transfected with the minimal reporter construct, the YAP-TAZ short hairpin RNA does not significantly alter firefly luciferase levels. The Renilla signal in each well is also not significantly altered by the short hairpin non-targeting control or the YAP-TAZ short hairpin RNA. As expected, short hairpin RNAs targeting YAP and TAZ, SARC or PI3 kinase significantly reduces YAP-TAZ-TEAD activity.
Short hairpin RNAs targeting ATM, CDH1, CSK, ERBB2, and gelsolin each increased normalized firefly luciferase levels consistent with published studies showing that these proteins were YAP and/or TAZ. Despite established roles for ATR, CCNE2, and ERBB4 in other cell types, short hairpin RNAs targeting these genes do not significantly change normalized firefly luciferase levels in A375 cells. Following this screen, identifying regulators should be validated using other readouts for their transcription factor activity.
Effective knockdown of the identified regulator by the assay should also be confirmed. Remember to use caution and to follow safety guidelines when working with infectious lentiviruses, particularly if the viruses are human infectious.
