The overall goal of this procedure is to identify transcriptional regulators of any gene of interest using a simple high throughput transfection protocol and a homemade dual glow luciferase assay. This is accomplished by first designing and cloning a unique set of dual luciferase reporter plasmids. These plasmids are cot transfected into 2 9 3 T cells in microplate with plasmids from a DNA expression library, such that each well is transfected with a single library plasmid.
After 48 hours, Firefly luciferase activity is assayed, followed by an assay of vanilla luciferase activity. The final step is to calculate firefly to vanilla luciferase activity ratios. Ultimately, the relative induction of the gene of interest in response to each library.
Gene is inferred by the ratio of firefly to vanilla luciferase activity in each well after plate based normalization. The main advantage of this technique over existing reporter assay protocols is its ability to rapidly generate functional data for a large number of genes with minimal manpower and cost. One day prior to transfection designated as day one of the screen, 2 9 3 T cells are seated into 96.
Well plates cells are sized, counted, and resuspended to a concentration of 1.5 times 10 to the fifth cells per milliliter. In DMEM containing 14%FBS pour the cells into a sterile reservoir. Place the reservoir six clear bottomed, 96 well plates and a box of filter pipette tips on the robot deck of the automated liquid handler.
Two plates of cells should be seated for each library plate to be screened. Dispense 100 microliters of cells into each well of each plate for a density of 1.5 times 10 to the fourth cells per well. Three library plates will be screened in this demonstration.
Hence six plates of cells are prepared centrifuge the cell plates at 50 times G for two minutes using low ramp speeds to ensure equal cell density throughout each well. This image illustrates how the plated cells should look after the centrifugation. Incubate the cells at 37 degrees Celsius and 10%carbon dioxide overnight.
To prepare the firefly and vanilla reporter plasmids, dilute each to five nanograms per microliter in endotoxin free water in a 15 milliliter conical tube. Combine the dilutions in a ratio of five to three firefly to vanilla plasmid by volume. Pipette the combined plasmids into each well of a 96 well plate dispensing 17 microliters per library plate, plus two to 10 microliters excess into each well cells are transfected on day two of the screen.
To begin this procedure for each library plate mix 107.5 microliters of room temperature op defect with 4.3 milliliters of serum free media, a one to 40 dilution in a polystyrene tube incubate for five minutes at room temperature dispense 44 microliters per library plate into each well of a 96 well polystyrene v bottomed plate. Place the library DNA plate, the diluted opt effect plate, the reporter plasmid plate, and a box of pipette tips on the robot deck aspirate. 17 microliters of reporter plasmids 43 microliters of diluted opt effect and 26 microliters of library DNA inserting a five microliter air gap between each aspiration.
The library DNA should be aspirated last to prevent cross-contamination. Dispense the contents of the tips into a new polystyrene v bottomed plate mix cover and set aside for 20 minutes to allow lipid DNA complexes to form. If transecting multiple library plates, replace the pipette tips and library plate and repeat after 20 minutes, uncover the opt effect DNA mixture plate and place it on the robot deck with two plates of 2 9 3 T cells using a single box of pipette tips.
Aspirate 80 microliters of the opt effect DNA mixture and slowly dispense 40 microliters per well for each plate of cells. Cover the cells after the opt effect DNA mixture has been added to the cells in all plates. Centrifuge the plates at 1, 200 times G for 30 minutes.
Return the plates to the incubator and incubate cells for four to six hours. During this incubation, prepare fresh media by mixing 12 milliliters of DMEM containing 10%FBS and 10 micrograms per milliliter ciprofloxin. For each transfected library plate, pour fresh media into a sterile reservoir.
Next place two boxes of robot tips, a single plate of cells, the reservoir containing fresh media and a waste reservoir onto the robot deck. Then aspirate 100 microliters of media from the cells and dispense into the waste reservoir, partially filled with 70%ethanol. Using fresh tips aspirate 100 microliters of fresh media and slowly dispense onto the cells.
Return the plates to the incubator for 48 hours. The firefly and vanilla luciferase assays are performed. 48 hours after transfect on day four of the screen for each library plate, prepare eight milliliters of three x firefly assay buffer and 12 milliliters of three x vanilla assay buffer from their respective stock solutions dispense 82 microliters of firefly assay buffer into each well of a 96 well V bottomed plate for each library plate to be assayed.
Similarly, dispense 122 microliters of vanilla assay buffer into each well of another 96 well V bottomed plate. For each plate to be assayed, set aside both assay buffers. Place a box of pipette tips, a waste reservoir, and two plates of cells transfected from the same library plate on the robot deck aspirate 60 microliters of media from each plate and discard in the waste reservoir partially filled with 70%ethanol cover plates and set aside.
Next place two boxes of pipette tips. The plate of three x firefly assay buffer and a set of cell plates on the robot deck. Aspirate 40 microliters of three x firefly assay buffer and add it to the first plate of cells, mixing thoroughly.
Switching to new pipette tips. Repeat for the second cell plate. Place the cells at room temperature for 10 minutes to complete lysis.
Record luminescence from each well of each cell plate on the luminometer recording for one second per well. Return plates to the robot deck. Place two boxes of pipette tips the plate of three x assay buffer and a set of cell plates on the robot deck.
Aspirate 60 microliters of three x vanilla assay buffer and add it to the first plate of cells. Mixing thoroughly. Switching to a new box of pipette tips.
Repeat for the second cell plate. Record luminescence from all plates on a luminometer. Recording each well for one second.
Typical luciferase values for a single half plate are shown in these tables. The firefly to renewal luminescence ratio or F two R ratio for each well is calculated by dividing the counts of firefly signal by the counts of ran renewal signal and the results are shown in Panel C.Panel D shows the induction value for each well calculated by dividing the F two R ratio of the well by the average F two R ratio of the plate, excluding the highest and lowest 25%of wells. The induction values should be averaged across replicates for a single transfected library plate.
And genes inducing or repressing alpha synuclein more than threefold are considered hits. Note the 32 fold inducer in well H two in this representative result shown here is a graphical representation of induction values for the single plate with well H two highlighted in blue. Hi.H two is a neuronal transcription factor not previously associated with alpha synuclein expression.
It is important to verify that the screen is performed within the linear range of the reporter assays so that clones that cause increased expression will be measured as increased luciferase activity to this end cells were transfected with increasing amounts of firefly and vanilla luciferase plasmids under the control of the strong H-C-M-V-I-E one promoter and assay. With this protocol, it was observed that firefly activity remains linear through a broad range of values. While the curve for vanilla activity flattens above 15 nanograms per well.
It is also important to perform the assays promptly after the incubation step to avoid non-linearity due to substrate depletion. A time course of decay signal for firefly luciferase assay revealed significant firefly luciferase activity up to one hour after addition of reagents using this protocol, 7, 670 human genes were screened and 68 novel regulators of alpha synuclein were ultimately identified in the representative results shown here. Each horizontal line represents induction ratio of a single gene hit compared to a controlled GFP neutral inducer.
Blue lines represent positive inducers while the sole red line represents a suppressor induction values are calculated as the ratio of alpha synuclein promoter driving firefly luciferase activity to the CMV promoter driving ran luciferase activity of each hit normalized to the same luciferase constructs assay using a GFP inducer Following this procedure. Other methods like quantitative PCR in conjunction with further overexpression and knockdown assays can be performed in order to ensure that identified hits regulate the endogenous target.
