The overall goal of the following video is to demonstrate the use of the pooled shRNA epigenetic library to conduct a negative selection screening. This screening enables to identify specific epigenetic targets through sequencing. This procedure could help to identify the epigenetic factors responsible for mediating acquired cytarabine resistance in AML The steps are as follows.
Selection of the most potent promoter to obtain persistent and prolonged expression of the shRNAs. The current protocol focuses on RNAi screening using epigenetic factor shRNA library in cytarabine-resistant MV4-11 cell line. The promoters used for this purpose are human EF-1 alpha, human CMV, and SFFV, promoters that express green fluorescence protein.
Take the cell count using trypan blue exclusion method. Suspend the cells in 10%RPMI medium containing 8 microgram per mL polybrene. Seed 1 million cells in 1.5 ml of medium per well of a six-well plate in triplicates.
Add different volumes of concentrated lentiviruses. For example, lentiviruses, for example, 10, 20, and 40 microliter, depending on the titer of the lentiviruses to each well. Gently swirl the plate to mix the contents.
Perform spinfection by centrifugation at 920 g at 37 degrees centigrade for 90 minutes. Immediately incubate the plates in a carbon dioxide incubator. Observe the GFP at the end of 48 hours and quantify the same at the end of 72 hours.
Next, follow the steps as depicted in the flow chart to choose the promoter showing consistent GFP expression throughout the culture. Preparation of the pooled lentiviral human epigenetic factor shRNA library. Culture 293T cells at a low passage number with a good proliferation rate in three 10-centimeter cell culture dishes with 8ml of 10%DMFM medium in each plate.
Once the cells attain 60%confluency, replace with fresh medium completely. Prepare the transfection mix as outlined that contains serum-free medium, lentiviral packaging plasmid PAX2, envelope plasmid pMD2. G, pooled library plasmids, and finally the transfection reagent.
Tap the mixture to mix it well and incubate at room temperature for 15 minutes. Add the transfection mixture to the 293T cells and mix gently by swirling the plates. Incubate the plates in a carbon dioxide incubator.
Change the medium after 24 hours. After 48 hours, check for GFP expression under a fluorescence microscope to ensure high transfection efficiency into 293T cells. Collect the virus supernatants after 48, 60 and 72 hours and stored them at 4 degree, and add fresh medium, 8ml, at each time point after collecting the virus.
Filter the pooled viruses with 0.4 micron filter. For obtaining a high titer of viruses, concentrate the pooled viruses by ultracentrifugation. Transfer the filtered virus supernatants to 70 Ti tubes, and centrifuge at 18, 000 g for 2 hours.
Use precooled rotor and centrifuge at 4 degree. Take out the tubes from the centrifuge carefully and remove the supernatant completely. Resuspend the pellet gently in 400 microliter of DMEM without FBS and antibiotics using a micropipette, incubate on ice for 1 hour.
Aliquot the viruses and freeze at minus 80 degree. Calculate the concentration of the virus as shown. Estimation of the transduction efficiency of lentiviruses.
Transduce 293T cells with a concentrated virus in different volumes, 2, 4, and 8, to confirm the successful preparation of the lentivirus. Value to concentrated virus to 100X to perform a titration experiment in the target cell line. Seed 1 million target cell line in 1.5 ml of 10%RPMI medium containing 8 microgram per ml polybrene in one well of a six-well plate.
Add four different volumes, 1, 1.5, 2, 2.5 microliter of 100X viruses. Perform spinfection by centrifugation of the plate at 920 g for 90 minutes at 37 degrees centigrade. After 72 hours, measure the percentage of GFP positive cells by flow cytometry.
Determine the volume of the viruses to obtain 30%transduction efficiency. This low transduction efficiency is to ensure single shRNA integration per cell. Transduction of the pooled epigenetic shRNA library in the drug-resistant cell line.
Calculate the number of the cells to be taken for the experiment as below based on the number of viral integrants. Resuspend 11 million cells in 16 ML of 10%RPMI medium. Add polybrene, eight microgram per ml and mix well, and then add the required volume of viruses to obtain 30%transduction efficiency as calculated before.
Seed all the cells on a six-well plate at a density of 1 million cells per 1.5 ml per well. Centrifuge the plate for 90 minutes at 920 g at 37 degrees centigrade. Incubate the plate overnight in a carbon dioxide incubator.
After 24 hours, change the medium and transfer the transduced cells to a T-75 flask. After 48 hours, check the GFP under a fluorescence microscope to ensure successful transduction. After 72 hours, quantitate the GFP by flow cytometry.
Enrichment of the GFP positive cells. Expand the transduced cells by culturing them at a density of 0.5 million per ml for 5 to 7 days, and perform flow sorting with the flow sorting setting set as high purity and low yield. Culture the sorted cells in a 10%RPMI medium.
After 72 hours, perform the post-sorting estimation of the percentage of the GFP positive cells to ensure more than 95%sorting efficiency. Dropout screening to identify epigenetic factors mediating drug resistance. Culture the shRNA library-transduced cells in 10%RPMI for up to 5 days.
Centrifuge 10 million cells in duplicates. Discard the supernatant and store the pellets at minus 80 degree. These samples will serve as the baseline reference for the epigenetic shRNA library.
Culture the remaining transduced cells as duplicates, R1 and R2, each maintained at a cell count that provides a 500X representation of the library. Treat one of the duplicates with the drug, 10 micromolar cytarabine, and the other without the drug treatment. Change the medium for the flasks with, and without the drug, every 72 hours.
Repeat the medium change three times for a cumulative drug exposure of 9 days. After 9th day of drug treatment, check the viability of the cells by the trypan blue exclusion method. Spin down the remaining viable cells, wash with sterile PBS and centrifuge at 280 g for 5 minutes at room temperature.
Discard the supernatant and store the pellets at minus 80 for DNA extraction. Amplification of the integrated shRNAs by PCR. Extract DNA from the transduced baseline cells, treated and untreated cells, followed by checking the concentration using fluorometer, calculate the amount of DNA required as shown and subject the samples to the first round of PCR.
The PCR reaction mixture is depicted in table 2 with the thermal cycler conditions. Set up multiple reactions containing 850 nanogram DNA per tube for a total of 43 reactions. Pool the PCR products and purify them.
Elute the products in 50 microliter buffer and quantify them, and finally, store at minus 20. For second round PCR, use reverse index primers and the reagents are tabulated in table 4. Set up four reactions with 500 nanogram of the primary PCR product in a total reaction volume of 50 microliter for each sample along with the negative control.
Load the entire product for electrophoresis using 2%TBE agarose gel and confirm the band size with one KB molecule ladder. Visualize the PCR products on the gel documentation system. Excise the specific band and purify using a gel purification kit.
Calculations for the purification with the kit are provided in table 3. Elute in a final volume of 30 microliter of elution buffer with the approximate concentration of each eluent around 80 to 90 nanogram per microliter. Next generation sequencing and data analysis.
The gel-purified products are subjected to next-generation sequencing to obtain read counts for the depleted shRNA needs. Trim the adaptor sequences, align the filtered reads to the reference sequences, load the files using SAMtools to obtain alignment summary. Load the trimmed fastQ files in CRISPRCloud2 and perform the analysis as per instructions.
Click on the link provided for the CRISPRCloud2. Select the screen type as Survival and Dropout screens. Set the number of groups and type the name for each group.
Upload the reference library as the FASTA file format. Data uploaded is processed and the results are available in the given URL. Representative data.
This figure shows MV4-11 parental and resistant cells treated with increasing cytarabine concentration, 0.1 micromolar to 1, 000 micromolar, and viability assessment by MTT assay. This figure shows representative flow plots of the GFP quantified by flow cytometry at the end of 72 hours for human EF-1 alpha, human CMV, and SFFV promoters. Human CMV shows our heterogeneous peak while human EF-1 alpha and SFFV show a single homogeneous peak.
This figure shows the bar graph for the three different promoter efficiencies in MV4-11. SFFV-driven GFP showed silencing of the GFP in prolonged culture. hEF-1 alpha-driven GFP cells showed sustained expression post sorting of these cells.
The left panel shows the transfection efficiency of the pooled shRNA library transfected in 293T at the end of 48 hours under fluorescence microscopy. The right panel shows the transduction efficiency of the pool virus in 293T cells with varying virus volumes, 2, 4, and 8 microliter. This figure represents the transduction efficiency in MV4-11 resistant cell line with varying volumes of viruses, like 1, 1.5, 2, 2.5 microliter to achieve 30%transduction efficiency.
This figure shows the sorting of the GFP positive cells with 30%transduction efficiency with high purity and low yield sorting setting. The figure shows schematic illustration of drug treatment for the GFP positive sorted cells for a prolonged cytarabine exposure of 9 days, followed by checking the viability and collecting the cells for DNA. This figure shows the binding regions of the primer used in first and second round of PCR.
This figure illustrates the band size of the PCR product at the end of the first round PCR, 397 base pair, and the second round PCR product 399 base pair, which was gel-eluted, purified, and given for NGS. This figure illustrates the representation of the enriched or depleted shRNAs targeting the epigenetic factors that could mediate cytarabine resistance in AML.Conclusion. This protocol highlights the importance of targeted knockdown screening to interrogate both essential and non-essential genes systematically and demonstrates the usage of this approach as a functional platform, allowing identification of targets responsible for drug resistance.
