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High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).
Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.
Therapeutic options in acute myeloid leukemia (AML) have remained unchanged for nearly the past five decades, with cytarabine (AraC) and anthracyclines as the cornerstone for treating the disease. One of the challenges to the success of AML therapy is the resistance of leukemic stem cells to chemotherapy, leading to disease relapse1,2. Epigenetic regulation plays a vital role in cancer pathogenesis and drug resistance, and several epigenetic factors have emerged as promising therapeutic targets3,4,5. Epigenetic regula....
Follow the Institutional Biosafety Committee (IBSC) guidelines and use the proper facility to handle lentivirus (BSL-2). Personnel should be appropriately trained in the handling and disposal of lentivirus. This protocol follows the biosafety guidelines of Christian Medical College, Vellore.
1. Selection of the most potent promoter to obtain persistent and prolonged expression of the shRNAs
NOTE: It is essential to perform a transduction experiment us.......
The overall screening workflow is depicted in Figure 1A. In vitro cytotoxicity of the MV4-11 AraC R (48 h) revealed the IC50 to cytarabine in the MV4-11 AraC R to be higher than the MV4-11 P (Figure 1B). This cell line was used in the study as the model for screening the epigenetic factors responsible for cytarabine resistance.
Figure 2A shows the linearized pZIP vector maps with.......
RNA interference (RNAi) is extensively used for functional genomics studies, which include siRNA and shRNA screening. The benefit of shRNA is that they can be incorporated into plasmid vectors and integrated into genomic DNA, resulting in stable expression and, thus, more prolonged knockdown of the target mRNA. A pooled shRNA library screening is robust and cost-effective compared to the conventional arrayed screens (siRNA). Identifying the essentiality of a specific class of proteins in a genome-wide screen can be cumbe.......
This study is funded in part by a Department of Biotechnology grant BT/PR8742/AGR/36/773/2013 to SRV; and Department of Biotechnology India BT/COE/34/SP13432/2015 and Department of Science and Technology, India: EMR/2017/003880 to P.B. RVS and P.B. are supported by Wellcome DBT India Alliance IA/S/17/1/503118 and IA/S/15/1/501842, respectively. S.D. is supported by the CSIR-UGC fellowship, and S.I. is supported by an ICMR senior research fellowship. We thank Abhirup Bagchi, Sandya Rani, and the CSCR Flow Cytometry Core Facility staff for their help. We also thank MedGenome Inc. for helping with the high-throughput sequencing and data analysis.
....Name | Company | Catalog Number | Comments |
Reagents | |||
100 bp Ladder Hyper Ladder | BIOLINE | BIO-33025 | |
1kb Ladder Hyper Ladder | BIOLINE | BIO-33056 | |
Agarose | Lonza Seachekm | 50004 | |
Betaine (5mM) | Sigma | B03001VL | |
Boric Acid | Qualigens | 12005 | |
Cell culture plasticware | Corning | as appicable | |
Cytosine β-D-arabinofuranoside hydrochloride | Sigma | C1768-500MG | |
DMEM | MP BIO | 91233354 | |
DMSO | Wak Chemie GMBH | Cryosure DMSO 10ml | |
EDTA | Sigma | E5134 | |
Ethidium Bromide | Sigma | E1510-10 mL | |
Fetal Bovine Serum | Thermo Fisher Scientific | 16000044 | |
Gel/PCR Purification Kit | MACHEREY-NAGEL | REFÂ 740609.50 | |
Gibco- RPMI 1640 | Thermo Fisher Scientific | 23400021 | |
Glacial Acetic Acid | Thermo | Q11007 | |
hCMV GFP Plasmid | Transomics | TransOmics Promoter selection KIT | |
hEF1a GFPlasmid | Transomics | TransOmics Promoter selection KIT | |
HEK 293T | ATCC | CRL-11268 | |
HL60 cell line | ATCC | CCL-240 | |
KOD Hot Start Polymerase | Merck | 71086 | |
Molm13 cell line | Ellen Weisberg Lab, Dana Farber Cancer Institute, Boston, MA, USA | Dana Farber Cancer Institute, Boston, MA, USA | |
MV4-11 cell line | ATCC | CRL-9591 | |
Penicillin streptomycin | Thermo Fisher Scientific | 15140122 | |
psPAX2 and pMD2.G | Addgene | Addgene plasmid no.12260 & Addgene plasmid no. 12259 | |
Qubit dsDNA HS Assay Kit | Invitrogen | REF Q32854 | |
SFFVÂ GFP Plasmid | Transomics | TransOmics Promoter selection KIT | |
shERWOOD-UltrmiR shRNA Library from Transomics | Transomics | Cat No. TLH UD7409; Lot No: A116.V 132.14 | |
Trans-IT-LTI Mirus | Mirus | Mirus Catalog Number: MIR2300 | |
Tris | MP Biomedicals | 0219485591 | |
Trypan Blue | Sigma-Aldrich | T8154-100ML | |
Ultra centrifuge Tubes | Beckman Coulter | Â 103319 | |
Equipments | |||
5% CO2 incubator | Thermo Fisher | ||
BD Aria III cell sorter | Becton Dickinson | ||
Beckman Coulter Optima L-100K- Ultracentrifugation | Beckman coulter | ||
Centrifuge | Thermo Multiguge 3SR+ | ||
ChemiDoc Imaging system (Fluro Chem M system) | Fluro Chem | ||
Leica AF600 | Leica | ||
Light Microscope | Zeiss Axiovert 40c | ||
Nanodrop | Thermo Scientific | ||
Qubit 3.0 Fluorometer | Invitrogen | ||
Thermal Cycler | BioRad |
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