DOCSLIB.ORG

SHX001 Storage Temperature –70 C

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read 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. The conversion can be viewed at: largest and most well studied gene families. Protein www.sigma.com/titer. phosphorylation plays an essential role in intercellular communication in eukaryotic organisms by mediating Screens can be quickly carried out using LentiExpress signal transduction during development, transcription, by simply adding cells of interest into each well of the immune response, metabolism, apoptosis, and cell 96-well plate. Pre-arrayed transduction-ready particles differentiation. Aberrant regulation of kinases plays a allow for rapid lenti-shRNA based screening in a variety causal role in many diseases, and the study of these of mammalian cell types. proteins and their functions will contribute to the discovery and development of new therapeutics. To achieve the best transduction efficiency, especially in hard to transduce cell lines, it is strongly MISSION LentiExpress Human Kinases contains recommended that the MISSION LentiExpress ~3,200 different lentiviruses carrying shRNA sequences Optimization Plate, Catalog No. SHXC01, be used prior targeting ~501 human kinase genes, allowing for quick, to screening. This product is intended for rapid high throughput loss-of-function screens. These kinase determination of the optimal cell number (thus MOI) to genes were chosen based on the 2002 paper by use for LentiExpress assays. Manning, G., et al.,1 and the RNAi Consortium. Each gene is represented by a clone set that consists of ~6 individual constructs, or clones, targeting different regions of the gene sequence. Therefore, a range of knockdown efficiencies can be expected, usually with 1 or more constructs from each gene set being >70%. Each shRNA construct has been cloned and sequence verified to ensure a match to the target gene. 2 Reagents/Components Biosafety Features MISSION LentiExpress Human Kinases consist of forty- The lack of generation of replication competent viral one individual 96-well plates. In each plate, 88 wells particles is an important safety feature of MISSION contain 30 L of shRNA Lentiviral Particles or controls shRNA Lentiviral Particles. Safety features are and 8 wells contain 30 L of media only. A CD contains summarized in Table 1. Users should consult and detailed clone position, RefSeq, locus link, gene observe their own institutional guidelines when working description, gene symbol, clone ID, and hairpin with viral systems. sequence. Table 1. Recommended controls for any shRNA experiment are described in the Control Selection Table that Biosafety Features of Third-Generation Lentiviral follows and are closely aligned with the controls Particles suggested in theNature Cell Biology editorial.6 Feature Result Multi-plasmid No single plasmid contains all the Reagents and equipment required, but not provided approach genes necessary to produce Target cells in culture packaged lentivirus. Resultant Complete growth media particles are replication- Hank’s Balanced Salt Solution, Catalog No. H6648 incompetent. 1x Trypsin-EDTA solution, Catalog No. T3924 SIN 3’ LTR Deletion in U3 portion of 3’ LTR 10% Bleach solution eliminates the promoter-enhancer Hexadimethrine bromide, Catalog No. H9268 region, avoids promoter Puromycin (10mg/ml solution), Catalog No. P9620 interference issues, and further Sterile pipette tips negates the possibility of viral Centrifuge with swinging-plate rotor replication, self-inactivating feature Follow distributor’s instructions for culturing and Elimination of the Removes virulence genes which maintaining the target cell line. It is recommended to majority of lentiviral are not necessary for shRNA use the lowest passage # of cells possible for genes ( vpr, vif, packaging systems transduction experiments. vpu and nef) 5 Chimeric 5’ LTR Reduces the chance of insertional Precautions and Disclaimer oncogenesis, allows for tat This product is for R&D use only, not for drug, independent transcription (tat has household, or other uses. Please consult the Material been implicated in various Safety Data Sheet for information regarding hazards carcinomas). and safe handling practices. Though the lentiviral transduction particles produced Storage/Stability are replication incompetent, it is recommended that After receiving immediately store the product at 70 °C. they be treated as Risk Group Level 2 (RGL-2) Avoid freeze/thaw cycles, as this will severely reduce organisms in laboratory handling.7 Follow all published transduction efficiency. RGL-2 guidelines for laboratory handling and waste decontamination. 3 Representative Plate Layout Procedure Overview Lentiviral particles are provided in a pre-arrayed, Step Description ready-to-use format in 96-well plates. The MISSION Thaw the LentiExpress plates containing LentiExpress Human Kinases consists of 41 plates, 1 viral particles for 10 minutes at room wherein each plate contains up to 80 different kinase- temperature specific clones. Each well is pre-diluted to contain Spin plates at 1000 rpm for 1 minute (if 2 approximately 5,000 lentiviral particles in a single possible) reaction volume of 30 L. In addition to the kinase 3 Remove sealer (important to thaw first) shRNA particles (K), each plate includes negative Seed cells into LentiExpress plates with 4 controls to monitor transduction efficiency: media containing hexadimethrine bromide Change media to fresh media 24 hours post 5 1 2 3 4 5 6 7 8 9 10 11 12 transduction A C K K K K K K K K K K C Add fresh media containing puromycin 48 6 B C K K K K K K K K K K C hours post transduction C N K K K K K K K K K K N Grow cells to optimal confluency to perform 7 D N K K K K K K K K K K N assay of choice E M K K K K K K K K K K M F M K K K K K K K K K K M Note: The appropriate concentration of puromycin for G M K K K K K K K K K K M each cell type will vary. If the appropriate concentration H M K K K K K K K K K K M for the desired cell type is unknown, a titration experiment, or kill curve, must be performed. Typically, Wells A1, A12, B1 and B12 contain MISSION pLKO.1- 210 g/ml are sufficient to kill most untransduced puro Control Transduction Particles (C) Catalog No. mammalian cell types. SHC001V, as a negative control. These “empty vector” particles do not contain an shRNA insert and there is no Puromycin Titration: evidence that they activate the RNAi pathway. These Puromycin titration (kill curve) should be performed wells provide positive controls for efficient transduction when working with a new cell type. 4 since the puromycin resistance gene is expressed 1. Plate 1.6 X 10 cells into wells of a 96-well plate under control of the hPGK promoter (as in the kinase with 120 L fresh medium. clones and other controls). 2. The next day add 500–10,000 ng/ml of puromycin to selected wells. Wells C1, C12, D1 and D12 contain MISSION Non- 3. Examine viability every 2 days. Target shRNA Control Transduction Particles (N), 4. Culture for 3 – 14 days depending on the growth Catalog No. SHC002V. This non-targeting shRNA is a rate of the cell type and the length of time that cells useful negative control that will activate RISC and the would typically be under selection during a normal RNAi pathway, but does not target any known human experimental protocol. Replace the media or mouse genes. containing puromycin every 3 days. The minimum concentration of puromycin that causes complete Wells E1, E12, F1, F12, G1, G12, H1 and H12 do not cell death after the desired time should be used for include shRNA lentiviral particles or control lentiviral that cell type and experiment. particles, but contain 30 µL of media (M) and are Note: Excess puromycin can cause many undesired intended for use as a negative control during the phenotypic responses in most cell types. puromycin selection process. 4 Procedure for each 96-well MISSION LentiExpress 4. Transduction Human Kinase shRNA plate a. Mix the cells and then add 70 L of cell suspension (~1000 cells) to each well in the Day 1: Preparations 96-well plate (making sure the virus is already 1. Prepare container with 10% bleach solution for thawed before adding the cells).
Load more

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]
  • 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]
  • NMDAR Signaling Facilitates the IPO5-Mediated Nuclear Import of CPEB3 Hsu-Wen Chao1, Yen-Ting Lai1,2, Yi-Ling Lu1, Chi-Long Lin3, Wei Mai3 and Yi-Shuian Huang1,2,*
    8484–8498 Nucleic Acids Research, 2012, Vol. 40, No. 17 Published online 22 June 2012 doi:10.1093/nar/gks598 NMDAR signaling facilitates the IPO5-mediated nuclear import of CPEB3 Hsu-Wen Chao1, Yen-Ting Lai1,2, Yi-Ling Lu1, Chi-long Lin3, Wei Mai3 and Yi-Shuian Huang1,2,* 1Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, 2Graduate Institute of Microbiology, National Taiwan University, Taipei 10051, Taiwan and 3National RNAi Core Facility, Academia Sinica, Taipei 11529, Taiwan Downloaded from https://academic.oup.com/nar/article/40/17/8484/2411581 by guest on 01 October 2021 Received May 1, 2012; Revised May 23, 2012; Accepted May 28, 2012 ABSTRACT controlling gene expression, scaffold molecules, and the proteolyzed intracellular domains of synaptic receptors Cytoplasmic polyadenylation element-binding (4,5). Transcription factors, such as cyclic AMP respon- protein (CPEB)3 is a nucleocytoplasm-shuttling sive element-binding protein (CREB)2, and translation RNA-binding protein and predominantly resides in regulators, such as cytoplasmic polyadenylation element- the cytoplasm where it represses target RNA trans- binding protein (CPEB)3 and CPEB4, move to the nucleus lation. When translocated into the nucleus, CPEB3 after activation of the N-methyl-D-aspartate receptor binds to Stat5b and downregulates Stat5b-depen- (NMDAR) (6–9), suggesting that the nucleocytoplasmic dent transcription. In neurons, the activation of partition of these proteins by specific neuronal signaling N-methyl-D-aspartate receptors (NMDARs) accumu- determines their role in gene expression. lates CPEB3 in the nucleus and redistributes CPEB3 Synapse-to-nucleus communication (i.e. retrograde in the nucleocytoplasmic compartments to control trafficking of protein molecules from synaptic areas to the nucleus) plays an important role in synaptic plasti- gene expression.
    [Show full text]