High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

This experiment screens chemical libraries for compounds that modulate post transcriptional control mechanisms via the three prime untranslated region of transcripts. First, identify the three prime untranslated region of interest in the targeted gene. Clone this three prime UTR into El luciferase control reporter plasmid to create El luciferase three prime UTR chimeric transcript.

Then stably. Integrate the two plasmids into a disease relevant cell line seed the cells carrying the chimeric transcript in 96. Well plates treat them with a library of compounds for 24 hours and then identify the molecules that affect luciferase activity.

Cherry pick the selected molecules for further validation screening, and next, treat both the control cells and the cells carrying luciferase three prime UTR chimeric transcript with a cherry picked library subset. Results will differentiate if the effect of a compound is mediated via the three prime untranslated region or via other vector sequences. Unlike the most reporter based screening essays.

Assays that focus on transcription regulation, these report cell based assay focus on identification of compounds that modulate mRNA transcript levels via three prime and translated region. Harvest the stably transfected neuroblastoma cells by trypsin Asian. Dilute the cells to a concentration of two times 10 to the fifth cells per milliliter.

In RPMI containing 10%FBS and 1%L-glutamine for 27 plates. Prepare at least 250 milliliters of cell suspension, taking into account liquid handling, trough dead volumes, and repeated pipetting steps. Then pour the cell suspension into a sterile reservoir and place it on the robot desk.

Load nine barcoded white flat. Bottom 96 well plates and a box of 200 microliter sterile pipette tips on the robot desk. Mix the cell suspension, then dispense 75 microliters of cells into each well of nine plates.

Cover the plates and allow the cells to sediment to the bottom of the well for 30 minutes in order to minimize edge effects. Then place all nine plates at 37 degrees Celsius and 5%carbon dioxide and incubate for 24 hours. Similarly, proceed with the remaining plates nine at a time.

Thaw the compound library plates at room temperature. Fill one trough with sterile PBS and another one with 0.5%DMSO in PBS solution and place them on the robot desk. For each library plate.

Prepare and label accordingly. One dilution plate. Now fill each well of columns, two to 11 of the dilution plates with 398 microliters of sterile PBS.

Then fill columns one and 12 with 50 microliters of 0.5%DMSO in sterile PBS. Next load the two dilution plates. Two library plates, two boxes of 50 microliters sterile tips and six cells plates on the robot desk.

Now uncover the cell plates, pipette two microliters of the 10 millimolar stock solution from one of two library plates in the corresponding dilution plate and mix well. Using the same set of tips. Transfer three microliters of the 50 micromolar dilution into the three barcoded white 96 well plates containing the cells.

Replace the full set of tips and repeat cell inductions for the second library plate. Cover the cultures and incubate for 24 hours. Then repeat the compound dilution and cell induction process for the remaining library plates.

Equilibrate the reconstituted luciferase reagent of choice to room temperature. Place the luciferase reservoir on the robot desk and pour reagent into it. Remove nine plates of induced cultures from the incubator.

Place them on the robot desk, uncover and equilibrate to room temperature. Then add 50 microliters of the luciferase reagent per well between the plates. Introduce a delay equal to the luminescence reading time of one plate.

After a 30 minute incubation. First record the barcode of the plate in order to associate it with the corresponding data file. Next, place the first plate in the luminometer and measure luminescence.

After a brief shaking step, repeat the luminescence readings for all plates of inductions. Then collect the remaining Lucifer race reagent and store it at negative 20 degrees Celsius. Once the experimental data of the primary screening are processed, select the hits to confirm compound specificity by a counter screening cherry.

Pick the selected hits and create a layout for the new screening plate, including positions for controls. Then from the aliquots stored in single 2D barcoded tubes, create new HIIT plates. Following the same protocol is for the screening.

Prepare 3 96 well plates of each stable cell line per hi plate. The next day, treat the cells with library compounds. Use each hi plate to treat three plates of CHP 1 34 micken, three prime UTR and three plates with control cells at two micromolar working concentration in 24 hours.

Measure luciferase activity in all CHP 1 34 plates, the three prime untranslated and control sets. As with the data analysis in the primary screening, normalize experimental data from treated wells to the average of on plate vehicle treated controls. Then calculate the mean and standard deviation over the replicates for each compound tested compute.

The fold change of luciferase activity in CHP 1 34 McIn three prime UTR versus CHP 1 34. Control cells select arbitrary full change cutoffs of greater than 1.5 and significance P values of less than 0.01. This experiment screened a 2000 compound library for potential modulators of post transcriptional control mechanisms exerted through the three prime UTR of the mic and oncogene the results of luciferase signals of a single out of 25 representative library plate displayed as a percentage of vehicle treated.

Controls show that the majority of compounds elicited no apparent effect on the three prime UTR. The hits were selected where the mean value and standard deviation were computed over all assay values compounds reducing luciferase activity below 20%are most likely indicative of pronounced cellular toxicity of the compound. This graph shows representative results for counter screening of four selected compounds designated WX, Y, and Z.Both compounds W and X result in increased luciferase expression from the oncogene three prime UTR reporter as indicated by fold change difference and T-test Note that while effects of both compounds W and X are truly dependent on three prime UTR compound W has no significant effect on cell viability.

While compound X displays some cytotoxic activity as indicated by normalized luciferase signal in CHP 1 34 control cells, neither compound Y nor compound Z at two micromolar concentration display A three prime UTR dependent effect on luciferase levels. The decreased luciferase activity detected in primary screening upon treatment with compound Y is rather a consequence of cytotoxicity. In the case of compound Z, the increased luciferase activity is due to unspecific in terms of three prime UTR activation of viral promoter.

Since an equivalent upregulation is observed in both control and untranslated sets In this high throughput assay, remember that validating the primary heat in the control cells is critical for discrimination of the compounds that act truly through the three prime untranslated region from those compounds that operate via promoter.