Identification of a Long Non-Coding RNA Regulator of Liver Carcinoma

Total Page:16

File Type:pdf, Size:1020Kb

Identification of a Long Non-Coding RNA Regulator of Liver Carcinoma Rybakova et al. Cell Death and Disease (2021) 12:178 https://doi.org/10.1038/s41419-021-03453-w Cell Death & Disease ARTICLE Open Access Identification of a long non-coding RNA regulator of liver carcinoma cell survival Yulia Rybakova 1,2, John T. Gonzalez 1, Roman Bogorad 1, Vikash P. Chauhan1,YizeL.Dong3,4, Charles A. Whittaker1, Timofei Zatsepin 2, Victor Koteliansky2 and Daniel G. Anderson 1,5,6,7 Abstract Genomic studies have significantly improved our understanding of hepatocellular carcinoma (HCC) biology and have led to the discovery of multiple protein-coding genes driving hepatocarcinogenesis. In addition, these studies have identified thousands of new non-coding transcripts deregulated in HCC. We hypothesize that some of these transcripts may be involved in disease progression. Long non-coding RNAs are a large class of non-coding transcripts which participate in the regulation of virtually all cellular functions. However, a majority of lncRNAs remain dramatically understudied. Here, we applied a pooled shRNA-based screen to identify lncRNAs essential for HCC cell survival. We validated our screening results using RNAi, CRISPRi, and antisense oligonucleotides. We found a lncRNA, termed ASTILCS, that is critical for HCC cell growth and is overexpressed in tumors from HCC patients. We demonstrated that HCC cell death upon ASTILCS knockdown is associated with apoptosis induction and downregulation of a neighboring gene, protein tyrosine kinase 2 (PTK2), a crucial protein for HCC cell survival. Taken together, our study describes a new, non-coding RNA regulator of HCC. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Introduction dysregulated in HCC samples compared to healthy tis- Liver cancer is one of the leading causes of cancer sues4. Further Gene Ontology analysis of these genes mortality worldwide, accounting for more than 700,000 identified several gene networks associated with HCC deaths per year1. Hepatocellular carcinoma (HCC) is the progression. Among them were upregulation of cell pro- most frequent subtype of liver cancer. Despite recent liferation, downregulation of apoptosis, loss of hepatocyte progress in HCC treatment it remains one of the deadliest differentiation, immunosuppression, and activation of types of cancer1,2. Notably, the incidence of HCC has been proteins acting at an epigenetic level4. Comprehensive increasing in recent decades, making HCC one of the genomic profiling of patient HCC samples and their fastest-growing causes of death worldwide3. This poor comparison with healthy tissues have helped uncover prognosis underlines the need for new effective therapies. molecular changes promoting the above phenotypic fea- – Better understanding of the molecular mechanisms reg- tures of HCC5 9. Among them, mutations leading to ulating HCC progression may yield new potential drug activation of the WNT signaling pathway were most targets. common5,6, implicating the WNT pathway as a major A meta-analysis of human HCC datasets revealed 935 driver of hepatocarcinogenesis7. Moreover, activation of genes for which RNA expression was significantly the WNT pathway is associated with an immunosup- pressive microenvironment, another hallmark of HCC progression8,9, which emphasizes the role of WNT path- Correspondence: Daniel G. Anderson ([email protected]) way activity in HCC progression. Other common muta- 1David H. Koch Institute for Integrative Cancer Research, Massachusetts tions affected the TERT promoter, TP53, genes regulating Institute of Technology, Cambridge, MA 02142, USA 2 cell cycle, PI3K-AKT-mTOR signaling and cell differ- Skolkovo Institute of Science and Technology, Moscow 121205, Russia 5,6 Full list of author information is available at the end of the article entiation . Notably, up to 50% of clinical HCC samples Edited by B. Rotblat © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Rybakova et al. Cell Death and Disease (2021) 12:178 Page 2 of 14 reported in different studies have a mutation in chromatin silencing is restricted to the cytoplasm, limiting targeting modifiers5,6, indicating the importance of epigenetic reg- of nuclear transcripts. However, recent studies suggest ulation in HCC development. RNAi presence and activity in the mammalian nucleus as – Besides shedding light on the roles of protein-coding well, although with less efficiency28 30. Clustered regularly genes, integrative genomic studies have revealed that the interspaced short palindromic repeat interference (CRIS- majority (>70%) of transcribed sequences in the human PRi) is another potent technique for lncRNA silen- genome participate in cell function regulation without cing21,23,25. Given that majority of lncRNA promoters is producing a protein10. Long non-coding RNAs (lncRNAs) poorly annotated and lncRNAs often overlap with are defined as non-coding transcripts longer than 200 protein-coding genes (or their promoters/enhancers), nucleotides and represent a large class of non-coding application of CRISPRi to regulate a lncRNA overlapping elements, comprising more than 50,000 annotated tran- with other transcripts might distort expression of those scripts to date11,12. Pertinently, hundreds of lncRNAs are transcripts, confounding data interpretation31. Thus, in recurrently deregulated in HCC, suggesting potential our screen we have chosen to perturb lncRNA at the roles in hepatocarcinogenesis. Co-expression network transcript level. We performed an shRNA-based pooled analysis determined that these lncRNAs were associated screen to identify lncRNAs essential for the survival of the with cell proliferation, metastasis, immune response, and human HCC cell line HUH7. Based on the lncRNA liver metabolism—hallmarks of HCC progression13,14. expression profile of these cells, we designed a lentiviral While the pathogenic roles of some of these lncRNAs (e.g. shRNA library targeting all identified lncRNAs. Using this HULC, H19, HOTAIR, HOTTIP, DANCR) have already library, we performed a loss-of-function genetic screen been described15, a plurality of lncRNA transcripts remain and found that lncRNA ENST00000501440.1 is critical for largely uncharacterized. Discovery of novel lncRNAs and HUH7 cell growth. We named this lncRNA ASTILCS their intracellular functions promises to expand our (AntiSense Transcript Important for Liver Carcinoma knowledge of HCC cellular physiology and may provide Survival). Importantly, in patient data, ASTILCS is sig- the basis for new therapeutic modalities. nificantly overexpressed in HCC compared to normal Currently, lncRNA functions can hardly be predicted tissues. Further, using gene expression manipulation based on their sequence. Instead, subcellular localization, techniques, we demonstrate that ASTILCS knockdown transcript abundance, and functional genomic screens can results in apoptosis induction and HCC cell death. Finally, help to efficiently narrow down possible lncRNA biolo- we show that ASTILCS knockdown correlates with – gical roles and molecular functions16 19. For instance, downregulation of a neighboring gene expressing Protein lncRNAs located mainly in the nucleus typically function Tyrosine Kinase 2 (PTK2), the silencing of which results as transcription regulators of local genes (in cis) or distant in HCC cell death. genes (in trans)18. Cytoplasmic lncRNAs are more likely to regulate protein production, formation of post- Materials and methods translational modifications, and sequestration of miR- Cell culture NAs or RNA-binding proteins19. Transcript abundance Human HCC HUH7 cell line was a gift from Prof. Jay can provide another hint about lncRNA function. For Horton (UT Southwestern Medical Center). HUH7 and example, low-abundance transcripts tend to function in HEK293ft cell lines were grown in Dulbecco’s modified cis because their low concentration makes diffusion a Eagle’s medium with L-glutamine (DMEM, Gibco™) sup- barrier to activity at long distances from the transcription plemented with 4.5 mg/ml glucose, 50 µg/ml gentamicin site. Abundant lncRNAs, on the other hand, can achieve sulfate (Sigma), 25 mM HEPES (Gibco™), and 10% heat- high concentrations at multiple target regions, including inactivated fetal bovine serum (FBS, Gibco™). All cells those outside of the nucleus and therefore often function were cultured at 37 °C, 5% CO2. When the cells reached a in trans16,17. Finally, pooled functional genetic screens are 70–80% monolayer, they were detached from the flask a powerful tool allowing for parallel perturbation of using 0.25% Trypsin–EDTA (Gibco™) solution and split multiple genes to select for those that are critical for a 1:10. Concentrations for selection agents were determined phenotype or
Recommended publications
  • A Model for Gene Inactivation by RNA Interference
    Rolle von Chronophin für die Cofilin-vermittelte Aktin-Dynamik in astrozytären Tumorzellen (Role of Chronophin for cofilin-mediated actin dynamics in astrocytic tumour cells) Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Oleg Fedorchenko aus Kimovsk (Russland) Düsseldorf, Juni 2009 Aus dem Institut für Biochemie und Molekularbiologie II der Heinrich-Heine Universität Düsseldorf Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. Antje Gohla Koreferent: Prof. Dr. Lutz Schmitt Tag der mündlichen Prüfung: 22.06.2009 Contents III Contents 1 INTRODUCTION 10 1.1 The eukaryotic cytoskeleton 10 1.2 The regulation of actin cytoskeletal dynamics 10 1.3 The cofilin family of actin regulatory proteins 13 1.4 Characterisation of CIN 15 1.5 Role of the cofilin pathway in tumours 18 1.6 Characterisation of glial tumours 22 2 AIMS OF THE STUDY 25 3 MATERIALS 26 3.1 List of manufacturers and distributors 26 3.2 Chemicals 27 3.3 Reagents for immunoblotting 29 3.4 Reagents for immunohistochemistry 29 3.5 Cell culture, cell culture media and supplements 29 3.6 Cell lines 30 3.7 Protein and DNA standards 30 3.8 Kits 30 3.9 Enzymes 30 3.10 Reagents for microscopy 31 3.11 Solutions and buffers 32 3.12 RNA interference tools 35 3.13 List of primary antibodies 36 4 EXPERIMENTAL PROCEDURES 37 4.1 Transformation of bacteria 37 4.2 Plasmid isolation from E. coli
    [Show full text]
  • SHX001 Storage Temperature –70 C
    MISSION® LentiExpress™ Human Kinases Catalog Number SHX001 Storage Temperature –70 C TECHNICAL BULLETIN Product Description The shRNA sequences are delivered to cells by LentiExpress technology enables lentiviral shRNA replication incompetent lentiviral particles. Unlike screens in an easy, convenient and economical format. murine-based MMLV or MSCV retroviral systems, The technology employs controls and clones of the lentiviral-based particles permit efficient infection and MISSION TRC shRNA libraries which are used in gene integration of the specific shRNA sequence into specific RNA interference (RNAi) in mammalian cells. differentiated and non-dividing cells, such as neurons and dendritic cells2. Self-inactivating replication The MISSION product line includes lentiviral vector- incompetent lentiviral particles are generated in based RNAi libraries against annotated mouse and producer cells (HEK 293T) by co-transfection with human genes. shRNAs that generate siRNAs compatible packaging plasmids3-4 (Catalog Number intracellularly are expressed from amphotropic lentivirus SHP001). In addition, the lentiviral transduction particles, allowing screening in a wide range of particles are pseudotyped with an envelope G mammalian cell types. In these cells, MISSION shRNA glycoprotein from Vesicular Stomatitis Virus (VSV-G), clones permit rapid, cost efficient loss-of-function and allowing transduction of a wide variety of mammalian genetic interaction screens. cells.5 MISSION LentiExpress Human Kinases is a The lentiviral transduction particles are titered via a p24 pre-arrayed set of lentiviral particles allowing for rapid antigen ELISA assay, and pg/ml of p24 are then and effective whole kinome RNAi screening even in converted to transducing units per ml using a difficult to transfect cells. Protein kinases are among the conversion factor.
    [Show full text]
  • MISSION Shrna Plasmid DNA (SHCLND)
    MISSION shRNA Plasmid DNA Catalog Number SHCLND Storage Temperature –20 C TECHNICAL BULLETIN Product Description integration of the specific shRNA construct into Small interfering RNAs (siRNAs) processed from short differentiated and non-dividing cells, such as neurons hairpin RNAs (shRNAs) are a powerful way to mediate and dendritic cells, overcoming low transfection and gene specific RNA interference (RNAi) in mammalian integration difficulties when using these cell lines. cells. The MISSION product line is a viral-vector-based RNAi library against annotated mouse and human Each MISSION shRNA clone is constructed within the genes. shRNAs that are processed into siRNAs lentivirus plasmid vector pLKO.1-puro3 or TRC2-pLKO- intracellularly are expressed from amphotropic puro. Both vectors contain the ampicillin and lentivirus particles, allowing screening in a wide range puromycin antibiotic resistance genes for selection of of mammalian cell lines. In these cell lines, MISSION inserts in bacterial or mammalian cells respectively. shRNA clones permit rapid, cost-efficient loss-of- function and genetic interaction screens. A range of knockdown efficiencies can be expected when using multiple clones. This allows one to The TRC1 and TRC1.5 libraries consist of sequence- examine the effect of loss of gene function over a large verified shRNA lentiviral plasmid vectors for mouse series of gene knockdown efficiencies. Each shRNA and human genes cloned into the pLKO.1-puro vector construct has been cloned and sequence verified to (see Figure 1). The TRC2 library consists of ensure a match to the target gene. sequence-verified shRNAs for mouse and human genes in the TRC2-pLKO-puro vector (see Figure 2).
    [Show full text]
  • Aire-Dependent Genes Undergo Clp1
    RESEARCH ARTICLE Aire-dependent genes undergo Clp1- mediated 3’UTR shortening associated with higher transcript stability in the thymus Clotilde Guyon1†, Nada Jmari1†, Francine Padonou1,2, Yen-Chin Li1, Olga Ucar3, Noriyuki Fujikado4‡, Fanny Coulpier5, Christophe Blanchet6, David E Root7, Matthieu Giraud1,2* 1Institut Cochin, INSERM U1016, Universite´ Paris Descartes, Sorbonne Paris Cite´, Paris, France; 2Universite´ de Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, F-44000, Nantes, France; 3Division of Developmental Immunology, German Cancer Research Center, Heidelberg, Germany; 4Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States; 5Ecole Normale Supe´rieure, PSL Research University, CNRS, INSERM, Institut de Biologie de l’Ecole Normale Supe´rieure (IBENS), Plateforme Ge´nomique, Paris, France; 6Institut Franc¸ais de Bioinformatique, IFB-Core, CNRS UMS 3601, Evry, France; 7The Broad *For correspondence: Institute of MIT and Harvard, Cambridge, United States [email protected] †These authors contributed equally to this work Present address: ‡Lilly Abstract The ability of the immune system to avoid autoimmune disease relies on tolerization of Biotechnology Center, Lilly thymocytes to self-antigens whose expression and presentation by thymic medullary epithelial cells Research Laboratories, Eli Lilly (mTECs) is controlled predominantly by Aire at the transcriptional level and possibly regulated at and Company, San Diego, other unrecognized levels. Aire-sensitive gene expression is influenced by several molecular factors, United States some of which belong to the 3’end processing complex, suggesting they might impact transcript Competing interests: The stability and levels through an effect on 3’UTR shortening. We discovered that Aire-sensitive genes authors declare that no display a pronounced preference for short-3’UTR transcript isoforms in mTECs, a feature preceding competing interests exist.
    [Show full text]
  • An Rnai Screen for Aire Cofactors Reveals a Role for Hnrnpl in Polymerase Release and Aire-Activated Ectopic Transcription
    An RNAi screen for Aire cofactors reveals a role for Hnrnpl in polymerase release and Aire-activated ectopic transcription Matthieu Girauda,b, Nada Jmarib, Lina Dua, Floriane Carallisb, Thomas J. F. Nielandc, Flor M. Perez-Campod, Olivier Bensaudee, David E. Rootc, Nir Hacohenc, Diane Mathisa,1, and Christophe Benoista,1 aDivision of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115; bDepartment of Immunology, Institut Cochin, Institut National de la Santé et de la Recherche Médicale (INSERM) U1016, Université Paris Descartes, 75014 Paris, France; cThe Broad Institute of MIT and Harvard, Cambridge, MA 02142; dDepartment of Internal Medicine, Hospital U.M. Valdecilla-Instituto de Formación e Investigación Marqués de Valdecilla, University of Cantabria, 39008 Santander, Spain; and eEcole Normale Supérieure, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8197, INSERM U1024, 75005 Paris, France Contributed by Christophe Benoist, December 19, 2013 (sent for review December 3, 2013) Aire induces the expression of a large set of autoantigen genes in Aire primarily impacts the elongation steps of transcription, in the thymus, driving immunological tolerance in maturing T cells. particular by releasing promoter-bound polymerases that remain To determine the full spectrum of molecular mechanisms underlying paused after abortive initiation (8, 9). Correspondingly, Aire has the Aire transactivation function, we screened an AIRE-dependent been shown to interact with subunits of the key controller of gene-expression system with a genome-scale lentiviral shRNA polymerase release, the positive transcription elongation factor library, targeting factors associated with chromatin architecture/ b (P-TEFb) (8, 10, 11). P-TEFb is recruited to stalled initiation function, transcription, and mRNA processing.
    [Show full text]
  • Systematic Investigation of Genetic Vulnerabilities Across Cancer Cell Lines Reveals Lineage-Specific Dependencies in Ovarian Cancer
    Systematic investigation of genetic vulnerabilities across cancer cell lines reveals lineage-specific dependencies in ovarian cancer Hiu Wing Cheunga,b,1, Glenn S. Cowleyb,1, Barbara A. Weirb,1, Jesse S. Boehmb,1, Scott Rusinb, Justine A. Scottb, Alexandra Eastb, Levi D. Alib, Patrick H. Lizotteb, Terence C. Wongb, Guozhi Jiangb, Jessica Hsiaob, Craig H. Mermela,b,c, Gad Getzb, Jordi Barretinaa,b, Shuba Gopalb, Pablo Tamayob, Joshua Gouldb, Aviad Tsherniakb, Nicolas Stranskyb, Biao Luob, Yin Rend, Ronny Drapkine,f, Sangeeta N. Bhatiab,d,g,h,i, Jill P. Mesirovb, Levi A. Garrawaya,b,c,g, Matthew Meyersona,b,c,e, Eric S. Landerb,2, David E. Rootb,2, and William C. Hahna,b,c,g,2 aDepartment of Medical Oncology, cCenter for Cancer Genome Discovery and fCenter for Molecular Oncologic Pathology, Dana-Farber Cancer Institute, Boston, MA 02115; bBroad Institute of Harvard and MIT, Cambridge, MA 02142; dHarvard-MIT Division of Health Sciences and Technology and hElectrical Engineering and Computer Science, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139; Departments of ePathology and gMedicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; and iHoward Hughes Medical Institute, Chevy Chase, MD 20815 Contributed by Eric S. Lander, June 13, 2011 (sent for review April 13, 2011) A comprehensive understanding of the molecular vulnerabilities A major feature of ovarian cancers is recurrent regions of of every type of cancer will provide a powerful roadmap to guide copy-number alteration (1). A small number of these recurrent therapeutic approaches. Efforts such as The Cancer Genome Atlas genomic events harbor known oncogenes and tumor suppressor Project will identify genes with aberrant copy number, sequence, genes, such as MYC, CCNE1, and RB (2).
    [Show full text]
  • The Rnai Consortium
    The RNAi Consortium Section II: Preparation of Transfection-Quality DNA for the TRC Library Introduction: We have used two methods for 96-well plate-based transfection-grade DNA preparations: 1.) The commercial plate-based PureLink prep from Invitrogen. 2.) A TRC-developed protocol that uses commercial low-grade Whatman prep with a subsequent magnetic bead purification step. A DNA preparation of pLKO.1 by either of these methods exhibited high transfection efficiencies in 293T cells, comparable to the transfection efficiencies achieved with maxi-prepped DNA. The PureLink method is more expensive but provides more consistent well-to-well yields than the current version of the bead- based protocol. (1) Commercial DNA Prep Instructions: We used the Invitrogen PureLink kit according to manufacturers instructions except that we grew cells as described in Section III.A and performed an additional heating step described in Section III.C3 (see below). (2) TRC-Developed DNA Prep Instructions: We developed a low-cost method using Whatman 96-well filter plates to extract crude DNA followed by Agencourt magnetic particles for purification. This protocol is currently being modified to decrease well- to-well variation in yields. The current Whatman/magnetic bead method is described below. Whatman/Magnetic Bead DNA Prep and Modifications to Invitrogen PureLink Prep 1. Materials 1. 2.2ml Deep well plate filled with 1.2ml TB (terrific broth) containing 100 ug/ml Ampicillin (Marsh Abgene, Cat# DW 9622). 2. Round-bottom plates (Costar, Corning, Cat# 3795). 3. Gas permeable seals (Marsh BioProduct, Cat# AB-0718). 4. Filter plates (ISC BioExpress, Cat# t-3180-1.) 5.
    [Show full text]
  • MISSION Shrna Control Vectors (SHC001, SHC002, SHC003
    MISSIONÒ shRNA Plasmid DNA Control Vectors Catalog Numbers SHC001, SHC002, SHC003, SHC004, SHC005, SHC007, SHC008, SHC009, SHC010, SHC011, SHC012, SHC013, SHC014, SHC015, SHC016, SHC201, SHC202, SHC203, SHC204, SHC216, SHC312, SHC314, SHC317, SHC332, SHC334, and SHC337 Storage Temperature –20 °C TECHNICAL BULLETIN Product Description When conducting experiments using MISSION shRNA RNA interference (RNAi) is a powerful gene-specific clones, proper controls are a key element of silencing mechanism in mammalian cells. The experimental design to permit accurate interpretation MISSIONÒ product line is a viral-vector-based RNAi of knockdown results and provide assurance of the library against annotated mouse and human genes. specificity of the response observed. The MISSION shRNAs that are processed into siRNAs intracellularly Control Vectors are lentiviral-based vectors that are are delivered by amphotrophic lentivirus particles, useful as both positive and negative controls in allowing screening in a wide range of mammalian cell experiments using the MISSION shRNA library. The types. In these cells, MISSION shRNA clones permit DNA format controls may be used in direct transfection rapid, cost-efficient loss-of-function and genetic of target cells or they may also be used to create interaction screens. replication-incompetent viral particles. Target cell lines may be transfected with the purified Sigma’s recommended controls for any shRNA plasmid for transient or stable gene silencing experiment are provided in the Control Selection (puromycin selection). In addition, self-inactivating Table and are closely aligned with the controls replication incompetent viral particles can be produced suggested in the Nature Cell Biology editorial.6 Please in packaging cells (HEK293T) by co-transfection with consult the Control Selection Table to select the compatible packaging plasmids, found in MISSION controls that are most appropriate for your shRNA Lentiviral Packaging Mix, Catalog Number SHP001.1,2 experiments.
    [Show full text]
  • A Comprehensive Platform for Highly Multiplexed Mammalian Functional
    Ketela et al. BMC Genomics 2011, 12:213 http://www.biomedcentral.com/1471-2164/12/213 METHODOLOGYARTICLE Open Access A comprehensive platform for highly multiplexed mammalian functional genetic screens Troy Ketela1†, Lawrence E Heisler1†, Kevin R Brown1, Ron Ammar1,2, Dahlia Kasimer1, Anuradha Surendra1, Elke Ericson1, Kim Blakely1,2, Dina Karamboulas1, Andrew M Smith1,2, Tanja Durbic1, Anthony Arnoldo1, Kahlin Cheung-Ong1, Judice LY Koh1, Shuba Gopal3, Glenn S Cowley3, Xiaoping Yang3, Jennifer K Grenier3, Guri Giaever1,2,4, David E Root3*, Jason Moffat1,2* and Corey Nislow1,2* Abstract Background: Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP) is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens. Results: Experiments with specially constructed lentiviral-based plasmid pools containing ~78,000 shRNAs demonstrated that the GMAP is capable of deconvolving genome-wide shRNA “dropout” screens. Further experiments with a larger, ~90,000 shRNA pool demonstrate that equivalent results are obtained from plasmid pools and from genomic DNA derived from lentivirus infected cells. Parallel testing of large shRNA pools using GMAP and next-generation sequencing methods revealed that the two methods provide valid and complementary approaches to deconvolution of genome-wide shRNA screens.
    [Show full text]
  • Document Title
    Expression ArrestTM-The RNAi Consortium (TRC) lentiviral shRNA RHS3979, RHS4012, RMM3981, RMM4013 CONTENTS Topic Page Product Description 1 Shipping and Storage 1 Design Information 2 Vector Information 2 Antibiotic Resistance 3 Vector Map- pLKO.1 3 Protocols 4 Protocol I-Replication 4 Protocol II-Plasmid Prep 5 Protocol III-Restriction Digest 6 Protocol IV-Transfection 7 Protocol V-Packaging 10 Protocol VI-Transduction 11 Related Reagents 15 FAQs 16 Troubleshooting 17 Suggested Reading 18 Licensing Information 19 PRODUCT DESCRIPTION The Expression Arrest TRC library is the result of a collaborative research effort based at the Broad Institute of MIT and Harvard, and includes six MIT and Harvard associated research institutions and five international life sciences organizations. The goal of TRC is to create lentiviral shRNA libraries targeting 15,000 human and 15,000 mouse annotated genes with multiple constructs per gene. Open Biosystems has partnered with the TRC to make these shRNA libraries available to researchers worldwide. SHIPPING AND STORAGE Individual constructs are shipped as bacterial cultures of E. coli (DH5α) in LB-Lennox (low salt) broth with 8% glycerol, 100µg/ml carbenicillin. Individual constructs are shipped on wet ice. Collections are shipped in 96 well plate format on dry ice. Individual constructs and collections should be stored at -80oC. Open Biosystems checks all cultures for growth prior to shipment. TO ALLOW ANY CO2 THAT MAY HAVE DISSOLVED INTO THE MEDIA FROM THE DRY ICE IN SHIPPING TO DISSIPATE, PLEASE STORE CONSTRUCTS AT –80°C FOR AT LEAST 48 HOURS BEFORE THAWING. Important Safety Note: Follow NIH guidelines regarding lentiviral production and transduction; follow Biosafety Level 2 (BL2) or BL2+ laboratory criteria.
    [Show full text]
  • RNA Interference Read and Download the Brochure
    MISSION® RNAi Tools Depend on our comprehensive shRNA & miRNA portfolio From screening to validation • Libraries • Panels • Custom cloning The life science business of Merck operates as MilliporeSigma in the U.S. and Canada. Contents Introduction to RNA Interference ...................................................................................3 MISSION® shRNA Library Screening ...............................................................................4 MISSION® TRC Lentiviral shRNA Libraries .......................................................................5 MISSION® shRNA Gene Family Sets ............................................................................5 shRNA LentiPlex® Lentiviral Pooled Libraries .................................................................6 Custom shRNA Pools ................................................................................................6 MISSION® Lentiviral Controls ........................................................................................7 MISSION® shRNA Controls ........................................................................................8 MISSION® Lentiviral Packaging Mix .............................................................................8 Deconvolution .........................................................................................................9 Individual MISSION® shRNA Clones ............................................................................9 MISSION® shRNA Custom Cloning, Lentiviral Manufacturing and DNA Purification
    [Show full text]
  • High-Throughput Rnai Screening in Cultured Cells: a User's Guide
    REVIEWS High-throughput RNAi screening in cultured cells: a user’s guide Christophe J. Echeverri* and Norbert Perrimon‡ Abstract | RNA interference has re-energized the field of functional genomics by enabling genome-scale loss-of-function screens in cultured cells. Looking back on the lessons that have been learned from the first wave of technology developments and applications in this exciting field, we provide both a user’s guide for newcomers to the field and a detailed examination of some more complex issues, particularly concerning optimization and quality control, for more advanced users. From a discussion of cell lines, screening paradigms, reagent types and read-out methodologies, we explore in particular the complexities of designing optimal controls and normalization strategies for these challenging but extremely powerful studies. Ribozyme Tissue culture cells have provided a powerful system mammalian cells. The fast development of these RNAi An RNA molecule with catalytic for studying many fundamental problems in signal tools has been driven by advances in the molecular activity. transduction, cell differentiation and physiology. understanding of the RNAi pathway (BOX 1). However, functional studies in cultured cells were RNAi has accelerated a wide range of small-scale RNAi RNA interference refers to the hampered in the past by the lack of a powerful method gene characterization studies, but arguably the most process by which dsRNA for perturbing gene activities. Several technologies important way in which it has transformed biological molecules silence a target gene designed to knock down gene function, such as those research is by enabling genome-scale screens in cell through the specific based on ribozymes and antisense approaches, showed culture systems.
    [Show full text]