DOCSLIB.ORG

Transposon Mutagenesis Identifies Genes That Transform Neural Stem

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Transposon Mutagenesis Identifies Genes That Transform Neural Stem Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells Hideto Kosoa,b, Haruna Takedaa,c, Christopher Chin Kuan Yewa, Jerrold M. Warda, Naoki Nariaid, Kazuko Uenod, Masao Nagasakid, Sumiko Watanabeb, Alistair G. Ruste, David J. Adamse, Neal G. Copelanda,f, and Nancy A. Jenkinsa,f,1 aDivision of Genetics and Genomics, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore 138673; bDivision of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan; cDepartment of Microbiology, University of Tokyo, Tokyo 113-0033, Japan; dDepartment of Integrative Genomics, Tohoku Medical Megabank Organization, Tohoku University, Sendai 980-8579, Japan; eExperimental Cancer Genetics, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, United Kingdom; and fCancer Research Program, Methodist Hospital Research Institute, Houston, TX 77030 AUTHOR SUMMARY Glioblastoma multiforme (GBM) is the by random chance, and therefore are most common form of malignant brain most likely to harbor genes responsible cancer in adults, whose mean survival is for cancer progression. We identified 1 y. Evidence indicates the presence of 140 and 148 CIS genes in immortalized self-renewing, stem-like cells within hu- cells and tumors, respectively (Fig. P1). man gliomas (1). Neural stem cells Thirty-four genes were common to (NSCs) are considered the cell of origin both immortalized cells and tumors, of GBM, the normal cell that acquires the and the rest were specific to either, first glioma-promoting mutation(s), suggesting different mechanisms for im- because the adult brain has very few mortalization and tumor formation. proliferating cells capable of accumulat- Analysis of CIS genes from immortalized ing mutations required for gliomagenesis. lines identified signaling pathways that To identify these genetic alterations, we involved cytoskeletal organization and used mobile DNA segments called transcriptional regulation, which play transposons that can affect gene function key roles in regulating self-renewal by inserting in or near genes, thus pro- vs. differentiation of NSCs. In contrast, Fig. P1. Transformation of NSCs into cancer- viding an unbiased, high-throughput initiating cells for mesenchymal GBM. Immor- 34 CIS genes, which were common, method for identifying genes important talization-CIS genes (e.g., Gli3, Nf1) promote enriched genes associated with mitosis for cancer (2). Here, we used this ap- immortalization of NSCs. Immortalized astroglial and cell division. proach to identify genes and signaling cells show a large repertoire of insertions re- We also identified genes specificto pathways that are able to transform NSCs presented by different colors. Transposons are tumors. Analysis of these genes associ- into cancer-initiating cells for GBM. continuously jumping to other sites in the genome ated them with the receptor tyrosine The mutagenic Sleeping Beauty (SB) after transplantation, and transplanted cells kinase (RTK) signaling pathway, which transposons, which were designed to elicit become cancer-initiating cells by randomly ac- is the most frequently mutated pathway loss-of-function mutations as well as gain- quiring new insertions in tumor CIS genes (e.g., identified in human GBM. The proto- Met, Pdgfrb, Gab1) and then clonally expand to of-function mutations, were mobilized oncogene Met and the tumor suppressor fi form tumors with characteristics of mesenchymal speci cally in the NSC compartment by GBM. Each tumor showed a distinct combination of gene Nf1 are most frequently mutated using genetically engineered mice (3). CIS genes even though all tumors were derived in the mouse tumors. Frequent NF1 NSCs were expanded in vitro, induced to from a single line. Most of the immortalization CIS mutations and increased MET expres- differentiate, and serially passaged to se- genes, with the exception of 34 genes, are lost sion are characteristic of mesenchymal lect for the immortalized cells that exhibit during tumor formation. The 114 tumor CIS genes GBM (4), indicating that the mouse an unlimited proliferative potential in were newly acquired during tumor development. tumors genetically resemble mesen- culture. The frequency of immortaliza- These gene sets are enriched in processes associated chymal GBM. with distinct signaling pathways. tion was higher in cells undergoing active We next examined the combinations SB transposition than in cells that were of CIS genes in tumors derived from not. Gene expression profiling of the immortalized cells showed a single immortalized line. Notably, each tumor showed a distinct that these cells were significantly enriched for genes differentially combination of insertional mutations in RTK pathway genes, expressed in astroglial cells, which represent an immature even though all tumors were derived from a single line, in- stage of the astrocyte lineage. DNA analysis showed that con- dicating that transplanted cells become tumorigenic by acquiring tinuously mobilized transposons generated a large repertoire of immortalized cells with unique combinations of insertions (Fig. P1). More than half of the immortalized lines with active Author contributions: H.K., N.G.C., and N.A.J. designed research; H.K. and H.T. performed SB transposition induced tumors after s.c. transplantation, research; N.N., K.U., M.N., S.W., A.G.R., and D.J.A. contributed new reagents/analytic whereas only one of six immortalized lines that lacked active SB tools; H.K., C.C.K.Y., J.M.W., N.N., and A.G.R. analyzed data; and H.K., N.G.C., and transposition was tumorigenic. Analysis of DNA microarrays N.A.J. wrote the paper. showed that tumors were significantly enriched for genes specific The authors declare no conflict of interest. for the mesenchymal subtype of GBM, consistent with an This is a Contributed submission. astroglial origin for mesenchymal GBM (4). Data deposition: The array data reported in this paper have been deposited in the Gene We next sequenced the transposon insertion sites from Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo/ (accession no. GSE3689). 25 immortalized lines and 67 tumors and identified common 1To whom correspondence should be addressed. E-mail: [email protected]. insertion sites (CISs), which are regions in the genome that See full research article on page E2998 of www.pnas.org. harbor a higher number of transposon insertions than predicted Cite this Author Summary as: PNAS 10.1073/pnas.1215899109. 17746–17747 | PNAS | October 30, 2012 | vol. 109 | no. 44 www.pnas.org/cgi/doi/10.1073/pnas.1215899109 Downloaded by guest on September 24, 2021 new insertions in genes that promote tumor development, fol- These analyses will undoubtedly aid in the identification of PNAS PLUS lowed by clonal expansion and the generation of tumors with potential therapeutic targets for this deadly disease. distinct combinations of mutations in tumor CIS genes (Fig. P1). Functional analysis of the tumor CIS genes identified in our 1. Singh SK, et al. (2004) Identification of human brain tumour initiating cells. Nature 432 screen, such as Met, Pdgfrb, and Gab1, showed that these genes (7015):396–401. play functionally overlapping roles in tumor development. 2. Copeland NG, Jenkins NA (2010) Harnessing transposons for cancer gene discovery. Nat fi Rev Cancer 10(10):696–706. Together, these ndings suggest that tumor formation in this 3. Dupuy AJ, et al. (2009) A modified sleeping beauty transposon system that can be model system mimics the evolutionary processes now thought to used to model a wide variety of human cancers in mice. Cancer Res 69(20): generate many human cancers, in which the pathway to tumor 8150–8156. development has a branched architecture reminiscent of Dar- 4. Verhaak RG, et al.; Cancer Genome Atlas Research Network (2010) Integrated genomic ’ analysis identifies clinically relevant subtypes of glioblastoma characterized by win s iconic evolutionary tree (5). Our dataset of GBM-causing abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17(1):98–110. genes provides a rich resource for cross-species comparative 5. Gerlinger M, et al. (2012) Intratumor heterogeneity and branched evolution revealed analyses of forthcoming human sequencing data from GBMs. by multiregion sequencing. N Engl J Med 366(10):883–892. MEDICAL SCIENCES Koso et al. PNAS | October 30, 2012 | vol. 109 | no. 44 | 17747 Downloaded by guest on September 24, 2021.
Load more

Recommended publications
  • Transposon Mutagenesis in Streptomycetes
    Transposon Mutagenesis in Streptomycetes Dissertation zur Erlangung des Grades des Doktors der Naturwissenschaften der Naturwissenschaftlich-Technischen Fakultät III Chemie, Pharmazie, Bio- und Werkstoffwissenschaften der Universität des Saarlandes von Bohdan Bilyk Saarbrücken 2014 Tag des Kolloquiums: 6. Oktober 2014 Dekan: Prof. Dr. Volkhard Helms Berichterstatter: Dr. Andriy Luzhetskyy Prof. Dr. Rolf Müller Vorsitz: Prof. Dr. Claus-Michael Lehr Akad. Mitarbeiter: Dr. Mostafa Hamed To Danylo and Oksana IV PUBLICATIONS Bilyk, B., Weber, S., Myronovskyi, M., Bilyk, O., Petzke, L., Luzhetskyy, A. (2013). In vivo random mutagenesis of streptomycetes using mariner-based transposon Himar1. Appl Microbiol Biotechnol. 2013 Jan; 97(1):351-9. Bilyk, B., Luzhetskyy, A. (2014). Unusual site-specific DNA integration into the highly active pseudo-attB of the Streptomyces albus J1074 genome. Appl Microbiol Biotechnol. Accepted CONFERENCE CONTRIBUTIONS Bilyk, B., Weber, S., Myronovskyi, M., Luzhetskyy, A. Himar1 in vivo transposon mutagenesis of Streptomyces coelicolor and Streptomyces albus. Poster presentation at International VAAM Workshop, University of Braunschweig, September 27-29, 2012. Bilyk, B., Weber, S., Welle, E., Luzhetskyy, A. Himar1 in vivo transposon mutagenesis of Streptomyces coelicolor. Poster presentation at International VAAM Workshop, University of Bonn, September 28 – 30, 2011. Bilyk, B., Weber, S., Welle, E., Luzhetskyy, A. In vivo transposon mutagenesis of streptomycetes using a modified version of Himar1. Poster presentation at International VAAM Workshop, University of Tübingen, September 26 -28, 2010. V TABLE OF CONTENTS SUMMARY XIII 1. INTRODUCTION 15 1.1. Streptomycetes, organisms with outstanding potential 15 1.1.1. Phylogeny of actinomycetes 15 1.1.2. Streptomyces 15 1.1.3. Exploiting the potential of streptomycetes as antibiotic producers.
    [Show full text]
  • Transposon Insertion Mutagenesis in Mice for Modeling Human Cancers: Critical Insights Gained and New Opportunities
    International Journal of Molecular Sciences Review Transposon Insertion Mutagenesis in Mice for Modeling Human Cancers: Critical Insights Gained and New Opportunities Pauline J. Beckmann 1 and David A. Largaespada 1,2,3,4,* 1 Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; [email protected] 2 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA 3 Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA 4 Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA * Correspondence: [email protected]; Tel.: +1-612-626-4979; Fax: +1-612-624-3869 Received: 3 January 2020; Accepted: 3 February 2020; Published: 10 February 2020 Abstract: Transposon mutagenesis has been used to model many types of human cancer in mice, leading to the discovery of novel cancer genes and insights into the mechanism of tumorigenesis. For this review, we identified over twenty types of human cancer that have been modeled in the mouse using Sleeping Beauty and piggyBac transposon insertion mutagenesis. We examine several specific biological insights that have been gained and describe opportunities for continued research. Specifically, we review studies with a focus on understanding metastasis, therapy resistance, and tumor cell of origin. Additionally, we propose further uses of transposon-based models to identify rarely mutated driver genes across many cancers, understand additional mechanisms of drug resistance and metastasis, and define personalized therapies for cancer patients with obesity as a comorbidity. Keywords: animal modeling; cancer; transposon screen 1. Transposon Basics Until the mid of 1900’s, DNA was widely considered to be a highly stable, orderly macromolecule neatly organized into chromosomes.
    [Show full text]
  • Lecture 3 Mutagens and Mutagenesis A. Physical and Chemical
    Lecture 3 Mutagens and Mutagenesis 1. Mutagens A. Physical and Chemical mutagens B. Transposons and retrotransposons C. T-DNA 2. Mutagenesis A. Screen B. Selection C. Lethal mutations Read: 508-514 Figs: 14.23-28 1 Transposon (transposable element) as a mutagen Transposon (or TE): DNA segment that can move from one position to another (1) Retrotransposons Copia Drosophila (LTR-type) Ty1 Yeast (LTR-type) LINEs Human (non-LTR-type) SINEs (Alu) Human (non-LTR-type) (2) Transposons Ac/Ds Maize P-element Drosophila 2 Retrovirus Figure 5-73. The life cycle of a retrovirus. The retrovirus genome consists of an RNA molecule of about 8500 nucleotides; two such molecules are packaged into each viral particle. The enzyme reverse transcriptase first makes a DNA copy of the viral RNA molecule and then a second DNA strand, generating a double-stranded DNA copy of the RNA genome. The integration of this DNA double helix into the host chromosome is then catalyzed by a virus-encoded integrase enzyme. This integration is required for the synthesis of new viral RNA molecules by the host cell RNA polymerase, the enzyme that transcribes DNA into RNA. (From Molecular Biology of the Cell by Alberts et al.) 3 Fig. 14.26 4 5 Figure 5-76. Transpositional site- specific recombination by a nonretroviral retrotransposon. Transposition by the L1 element (red) begins when an endonuclease attached to the L1 reverse transcriptase and the L1 RNA (blue) makes a nick in the target DNA at the point at which insertion will occur. This cleavage releases a 3′-OH DNA end in the target DNA, which is then used as a primer for the reverse transcription step shown.
    [Show full text]
  • Transposon Based Mutagenesis and Mapping of Transposon Insertion Sites Within the Ehrlichia Chaffeensis Genome Using Semi Random Two-Step Pcr
    TRANSPOSON BASED MUTAGENESIS AND MAPPING OF TRANSPOSON INSERTION SITES WITHIN THE EHRLICHIA CHAFFEENSIS GENOME USING SEMI RANDOM TWO-STEP PCR by VIJAYA VARMA INDUKURI B.S., ANDHRA UNIVERSITY, India, 2008 A THESIS Submitted in partial fulfillment of the requirements for the degree MASTER OF SCIENCE Department of Diagnostic Medicine/Pathobiology College of Veterinary Medicine KANSAS STATE UNIVERSITY Manhattan, Kansas 2013 Approved by: Major Professor Roman Reddy Ganta Copyright VIJAYA VARMA INDUKURI 2013 Abstract Ehrlichia chaffeensis a tick transmitted Anaplasmataceae family pathogen responsible for human monocytic ehrlichiosis. Differential gene expression appears to be an important pathogen adaptation mechanism for its survival in dual hosts. One of the ways to test this hypothesis is by performing mutational analysis that aids in altering the expression of genes. Mutagenesis is also a useful tool to study the effects of a gene function in an organism. Focus of my research has been to prepare several modified Himar transposon mutagenesis constructs for their value in introducing mutations in E. chaffeensis genome. While the work is in progress, research team from our group used existing Himar transposon mutagenesis plasmids and was able to create mutations in E. chaffeensis. Multiple mutations were identified by Southern blot analysis. I redirected my research efforts towards mapping the genomic insertion sites by performing the semi-random two step PCR (ST-PCR) method, followed by DNA sequence analysis. In this method, the first PCR is performed with genomic DNA as the template with a primer specific to the insertion segment and the second primer containing an anchored degenerate sequence segment. The product from the first PCR is used in the second PCR with nested transposon insertion primer and a primer designed to bind to the known sequence portion of degenerate primer segment.
    [Show full text]
  • Potential Whole-Cell Biosensors for Detection of Metal Using Merr Family Proteins from Enterobacter Sp
    micromachines Article Potential Whole-Cell Biosensors for Detection of Metal Using MerR Family Proteins from Enterobacter sp. YSU and Stenotrophomonas maltophilia OR02 Georgina Baya 1, Stephen Muhindi 2, Valentine Ngendahimana 3 and Jonathan Caguiat 1,* 1 Department of Biological and Chemical Sciences, Youngstown State University, Youngstown, OH 44555, USA; [email protected] 2 Department of Biological Sciences, University of Toledo, Toledo, OH 43606, USA; [email protected] 3 Biology Department, Lone Star College-CyFair, 9191 Barker Cypress Rd, Cypress, TX 77433, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-330-941-2063 Abstract: Cell-based biosensors harness a cell’s ability to respond to the environment by repurposing its sensing mechanisms. MerR family proteins are activator/repressor switches that regulate the expression of bacterial metal resistance genes and have been used in metal biosensors. Upon metal binding, a conformational change switches gene expression from off to on. The genomes of the multi- metal resistant bacterial strains, Stenotrophomonas maltophilia Oak Ridge strain 02 (S. maltophilia 02) and Enterobacter sp. YSU, were recently sequenced. Sequence analysis and gene cloning identified three mercury resistance operons and three MerR switches in these strains. Transposon mutagenesis and sequence analysis identified Enterobacter sp. YSU zinc and copper resistance operons, which ap- pear to be regulated by the protein switches, ZntR and CueR, respectively. Sequence analysis and Citation: Baya, G.; Muhindi, S.; reverse transcriptase polymerase chain reaction (RT-PCR) showed that a CueR switch appears to Ngendahimana, V.; Caguiat, J. activate a S. maltophilia 02 copper transport gene in the presence of CuSO4 and HAuCl4·3H2O.
    [Show full text]
  • Chapter 3 Transposon Mutagenesis of Rhodobacter Sphaeroides
    Chapter 3 Transposon Mutagenesis of Rhodobacter sphaeroides Timothy D. Paustian and Robin S. Kurtz Department of Bacteriology University of Wisconsin–Madison Madison, Wisconsin 53706 (608) 263-4921, [email protected] Robin and Tim are associate faculty and coordinate the instructional labs for the Department of Bacteriology. Tim earned his B.S. in Biochemistry from the University of Wisconsin–Madison and Ph.D. from the Department of Bacteriology at UW–Madison. His interests include curriculum development, computer-aided instruction, and development of computer programs. Robin earned her B.S. and Ph.D. in Bacteriology from UW-Madison. Her interests include instructional lab development, curriculum improvement, and immunology. Robin and Tim have co-authored three lab manuals that are used in the introductory and advanced microbiology laboratories at UW–Madison. Reprinted from: Paustian, T. D., and R. S. Kurtz. 1994. Transposon mutagenesis of rhodobacter sphaeroides. Pages 45-61, in Tested studies for laboratory teaching, Volume 15 (C. A. Goldman, Editor). Proceedings of the 15th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 390 pages. - Copyright policy: http://www.zoo.utoronto.ca/able/volumes/copyright.htm Although the laboratory exercises in ABLE proceedings volumes have been tested and due consideration has been given to safety, individuals performing these exercises must assume all responsibility for risk. The Association for Biology Laboratory Education (ABLE) disclaims any liability with regards
    [Show full text]
  • EZ-Tn5™ Transposon and Transposome Kits Speed Your Metagenomics, Strain Engineering, and Mutagenesis Projects with EZ-Tn5™ Transposomics™
    TRANSPOSON MUTAGENESIS lucigen.com/Transposon-Mutagenesis/ EZ-Tn5™ Transposon and Transposome Kits Speed your metagenomics, strain engineering, and mutagenesis projects with EZ-Tn5™ Transposomics™ Use this powerful system to: • Create mutant libraries for bacterial strain development • Identify essential genes or regulatory elements Exclusively available • Generate random insertional knockout libraries in your favorite bacterial strain thru Lucigen. • Insert promoter sequences for gene expression studies • Sequence large clones and chromosomal DNA easily • Recover and propagate plasmids from diverse bacterial genera Transposomics provides a fast and easy method for generating a library of DNA sequences with insertional mutations, or a library of live mutant bacteria. Many genomics applications bene t from the high e ciency and low bias of the EZ-Tn5 system, including metagenomics, strain develop- ment, functional genomics and large fragment sequencing. Transposomics is easier than chemical mutagenesis, and is a trusted and well-established method for generating mutations across over 60 species of bacteria. With 100’s of publications and a wide array of applications and tools available, you are limited only by your imagination. Method Example Applications Target Transposon Tools Used In Vivo Transposomics Create mutant bacterial libraries and Living bacteria Transposome insertional gene knockouts (genomic DNA) (complex of transposon DNA and EZ-Tn5™ Transposase enzyme) In Vitro Transposomics Insert replication origins into viral or novel Puri ed DNA Transposon DNA sequence + EZ-Tn5™ plasmids (plasmid or genomic DNA) Transposase enzyme What is an EZ-Tn5 Transposome? Transposomes are used for in vivo mutagenesis in a broad EZ-Tn5 Transposome range of bacteria, including Gram positive and Gram negative strains.
    [Show full text]
  • Transposon Mutagenesis of Drosophila (P Element/Polymerase Chain Reaction/Sib Selection/Singed/Transposon Mutagenesis) KIM Kaisert and STEPHEN F
    Proc. Nati. Acad. Sci. USA Vol. 87, pp. 1686-1690, March 1990 Genetics "Site-selected" transposon mutagenesis of Drosophila (P element/polymerase chain reaction/sib selection/singed/transposon mutagenesis) KIM KAISERt AND STEPHEN F. GOODWIN Department of Genetics, University of Glasgow, Church Street, Glasgow G11 5JS, Scotland Communicated by Seymour Benzer, December 4, 1989 (receivedfor review September 11, 1989) ABSTRACT Despite the wide range of techniques that can desired characteristics merely by identifying at each stage the be brought to bear on the study of basic processes in Dro- sub-population in which at least one individual is laying eggs sophila, there are still deficiencies in our armory. One of these with the appropriate chromosomal configuration. is an ability to select mutants in cases where the gene is known and has been cloned, but where we are ignorant of the associated phenotype. We describe here a solution to this MATERIALS AND METHODS problem as applied to a model system, the singed (sn) locus. Flies and DNAs. CFL3 and CFL5 (sn strains, ref. 4), Our method is a combination of classical genetics and molec- Birm-2; ry5 and w; SbP[ry'A2,3]/TM6 (5), ir2 and Harwich ular biology: sib selection plus the polymerase chain reaction. (strong P strains; ref. 6) and P-cytotype balancer strains were We have used the method to isolate rare individuals with obtained from J. Paterson and K. O'Hare (Imperial College P-element-induced alleles of sn merely by recognition of the of Science, Technology and Medicine, London), as was the DNA structures induced at the locus by transposon insertion.
    [Show full text]
  • Transposon Mutagenesis Reveals Fludarabine Resistance Mechanisms in Chronic Lymphocytic Leukemia
    Published OnlineFirst March 8, 2016; DOI: 10.1158/1078-0432.CCR-15-2903 Biology of Human Tumors Clinical Cancer Research Transposon Mutagenesis Reveals Fludarabine Resistance Mechanisms in Chronic Lymphocytic Leukemia Tatjana Pandzic1, Jimmy Larsson1, Liqun He1, Snehangshu Kundu1, Kenneth Ban1,2, Muhammad Akhtar-Ali1, Anders R. Hellstrom€ 1, Anna Schuh3, Ruth Clifford3, Stuart J. Blakemore4, Jonathan C. Strefford4, Tycho Baumann4, Armando Lopez-Guillermo5, Elias Campo6, Viktor Ljungstrom€ 1, Larry Mansouri1, Richard Rosenquist1, Tobias Sjoblom€ 1, and Mats Hellstrom€ 1 Abstract Purpose: To identify resistance mechanisms for the chemo- transposon-targeted genes had previously been implicated in therapeutic drug fludarabine in chronic lymphocytic leukemia treatment resistance based on somatic mutations seen in patients (CLL), as innate and acquired resistance to fludarabine-based refractory to fludarabine-based therapy. Functional characteriza- chemotherapy represents a major challenge for long-term disease tion of these genes supported a significant role for ARID5B and control. BRAF in fludarabine sensitivity. Finally, pathway analysis of Experimental Design: We used piggyBac transposon-mediated transposon-targeted genes and RNA-seq profiling of fludara- mutagenesis, combined with next-generation sequencing, to bine-resistant cells suggested deregulated MAPK signaling as identify genes that confer resistance to fludarabine in a human involved in mediating drug resistance in CLL. CLL cell line. Conclusions: To our knowledge, this is the first forward Results: In total, this screen identified 782 genes with trans- genetic screen for chemotherapy resistance in CLL. The screen poson integrations in fludarabine-resistant pools of cells. One of pinpointed novel genes and pathways involved in fludarabine the identified genes is a known resistance mediator DCK (deox- resistance along with previously known resistance mechanisms.
    [Show full text]
  • Piggybac-Based Transposon Mutagenesis and Enhancer Trapping As a Tool for Functional Insect Genomics
    Proceedings of 6th International Fruit Fly Symposium 6–10 May 2002, Stellenbosch, South Africa pp. 419–425 piggyBac-based transposon mutagenesis and enhancer trapping as a tool for functional insect genomics Carsten Horn*, Nils Offen & Ernst A. Wimmer‡ Lehrstuhl Genetik, Universität Bayreuth, Universitätsstrasse 30, NW 1, 95447 Bayreuth, Germany Transgene-based approaches to the sterile insect technique need to provide a combination of conditional female-specific lethality and male sterility.The identification of appropriate cis-regula- tory sequences that mediate female- or testis-specific expression is of key importance to develop such transgenic approaches. Here, we present a transposon mutagenesis system that is suitable for genome-wide trapping of cis-regulatory elements. This system is based on the controlled mobiliza- tion of the broad-range transposable element piggyBac. A ‘jumpstarter’ element expressing the piggyBac transposase gene is used to mobilize a non-autonomous piggyBac-based ‘mutator’ element. This mutator element carries a heterologous transactivator gene that serves as a primary reporter of enhancer activities.The heterologous transactivator then activates a secondary reporter within a ‘responder’ element, which is used for the visible detection of the enhancer activity. To be able to independently follow the different elements, they were marked with discernible spectral variants of the green fluorescent protein or DsRed under control of the widely applicable eye- specific promoter 3×P3.In a pilot screen carried out in Drosophila melanogaster,we observed trans- position events in the progeny of about 72–92% of single male crosses. Owing to the use of broad- range components, this system should allow for large-scale, genome-wide mutagenesis and enhancer trapping in different genetically tractable insects, including pest species of economic importance such as the Mediterranean fruit fly, Ceratitis capitata.
    [Show full text]
  • Promoterless Transposon Mutagenesis Drives Solid Cancers Via Tumor Suppressor Inactivation
    bioRxiv preprint doi: https://doi.org/10.1101/2020.08.17.254565; this version posted August 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation 2 Aziz Aiderus1, Ana M. Contreras-Sandoval1, Amanda L. Meshey1, Justin Y. Newberg1,2, Jerrold M. Ward3, 3 Deborah Swing4, Neal G. Copeland2,3,4, Nancy A. Jenkins2,3,4, Karen M. Mann1,2,3,4,5,6,7, and Michael B. 4 Mann1,2,3,4,6,7,8,9 5 1Department of Molecular Oncology, Moffitt Cancer Center & Research Institute, Tampa, FL, USA 6 2Cancer Research Program, Houston Methodist Research Institute, Houston, Texas, USA 7 3Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 8 Singapore, Republic of Singapore 9 4Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, 10 Maryland, USA 11 5Departments of Gastrointestinal Oncology & Malignant Hematology, Moffitt Cancer Center & Research 12 Institute, Tampa, FL, USA 13 6Cancer Biology and Evolution Program, Moffitt Cancer Center & Research Institute, Tampa, FL, USA 14 7Department of Oncologic Sciences, Morsani College of Medicine, University of South Florida, Tampa, FL, 15 USA. 16 8Donald A. Adam Melanoma and Skin Cancer Research Center of Excellence, Moffitt Cancer Center, Tampa, 17 FL, USA 18 9Department of Cutaneous Oncology, Moffitt Cancer Center & Research Institute, Tampa, FL, USA 19 These authors contributed equally: Aziz Aiderus, Ana M.
    [Show full text]
  • Exploring the Functional Landscape of the Fission Yeast Genome Via Hermes Transposon Mutagenesis
    Exploring the Functional Landscape of the Fission Yeast Genome via Hermes Transposon Mutagenesis Leanne Grech Submitted for the Degree of Doctor of Philosophy (PhD) University College London Primary Supervisor: Professor Jürg Bähler (UCL) January 2018 1 Declaration “I, Leanne Grech, confirm that the work presented in this thesis is my own, and where information has been derived from other sources, I confirm that this has been indicated in the thesis.” 2 Abstract In general, the non-protein-coding dark matter of eukaryotic genomes remains poorly understood. Neither diversity studies, comparative genomics, nor the biochemical outputs allow fine-scale descriptions of the genomic elements that are required for an organism to grow. Transposon mutagenesis offers an alternative approach to locate functional regions. In principle, insertion mutants are created in large pools, and mutants harbouring harmful insertions are quantitatively removed from the population. Subsequent sequencing of mutant libraries should reveal functionality in regions with fewer insertions. Transposon mutagenesis works well in bacteria. We applied the Hermes transposon system to locate functional regions in the Schizosaccharomyces pombe, or fission yeast genome. We created multiple dense insertion libraries, during log phase growth and chronological ageing, achieving a saturating (or near-saturating) insertion density of 1 insertion per 13 nucleotides of the genome for log phase samples. To account for the complexity and stochastic nature of the data, we applied a five-state hidden Markov model (HMM) that includes generalised linear models to account for systematic insertion biases (e.g. nucleosomes). The HMM state provides a semi-quantitative estimate of the functional significance of the genome at single nucleotide-level resolution.
    [Show full text]