DOCSLIB.ORG

Assay for Quantitative Evaluation of Target Site Cleavage by One Or More Crispr-Cas Guide Sequences

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Assay for Quantitative Evaluation of Target Site Cleavage by One Or More Crispr-Cas Guide Sequences (19) *EP003011035B1* (11) EP 3 011 035 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/63 (2006.01) C12N 15/10 (2006.01) (2006.01) (2006.01) 13.05.2020 Bulletin 2020/20 C40B 40/08 C12N 9/22 (21) Application number: 14738672.6 (86) International application number: PCT/US2014/041790 (22) Date of filing: 10.06.2014 (87) International publication number: WO 2014/204723 (24.12.2014 Gazette 2014/52) (54) ASSAY FOR QUANTITATIVE EVALUATION OF TARGET SITE CLEAVAGE BY ONE OR MORE CRISPR-CAS GUIDE SEQUENCES TEST ZUR QUANTITATIVEN BEWERTUNG DER ZIELSTELLENSPALTUNG DURCH EINE ODER MEHRERE CRISPR-CAS FÜHRUNGSSEQUENZEN TEST POUR L’ÉVALUATION QUANTITATIVE DU CLIVAGE DE SITES CIBLES PAR UNE OU PLUSIEURS SÉQUENCES GUIDES DU SYSTÈME CRISPR-CAS (84) Designated Contracting States: (56) References cited: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB • GAJ THOMAS ET AL: "ZFN, TALEN, and GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO CRISPR/Cas-based methods for genome PL PT RO RS SE SI SK SM TR engineering", TRENDS IN BIOTECHNOLOGY, ELSEVIER PUBLICATIONS, CAMBRIDGE, GB, (30) Priority: 17.06.2013 US 201361836123 P vol. 31, no. 7, 9 May 2013 (2013-05-09), pages 12.12.2013 US 201361915397 P 397-405, XP028571313, ISSN: 0167-7799, DOI: 10.1016/J.TIBTECH.2013.04.004 (43) Date of publication of application: • JANSSEN K P ET AL: "Mouse models of 27.04.2016 Bulletin 2016/17 K-ras-initiated carcinogenesis", BBA - REVIEWS ON CANCER, ELSEVIER SCIENCE BV, (73) Proprietors: AMSTERDAM, NL, vol. 1756, no. 2, 25 November • The Broad Institute, Inc. 2005 (2005-11-25), pages 145-154, XP027829577, Cambridge, MA 02142 (US) ISSN: 0304-419X [retrieved on 2005-11-25] • Massachusetts Institute of Technology • B. C. LEWIS: "The c-myc and PyMT oncogenes Cambridge, MA 02139 (US) induce different tumor types in a somatic mouse • The Brigham and Women’s Hospital, Inc. model for pancreatic cancer", GENES & Boston, MA 02115 (US) DEVELOPMENT, vol. 17, no. 24, 15 December 2003 (2003-12-15), pages 3127-3138, XP55049590, (72) Inventors: ISSN: 0890-9369, DOI: 10.1101/gad.1140403 • ZHANG, Feng • L. CONG ET AL: "Multiplex Genome Engineering Cambrige, MA 02139 (US) Using CRISPR/Cas Systems", SCIENCE, vol. 339, • EBERT, Benjamin, Levine no. 6121, 3 January 2013 (2013-01-03), pages Brookline, MA 02445 (US) 819-823, XP055102030, ISSN: 0036-8075, DOI: • HECKL, Dirk 10.1126/science.1231143 29352 Adelheidsdorf (DE) • P. MALI ET AL: "RNA-Guided Human Genome Engineering via Cas9", SCIENCE, vol. 339, no. (74) Representative: Icely, Dominic Michael 6121, 3 January 2013 (2013-01-03), pages 823-826, The IP Asset Partnership Limited XP055111247, ISSN: 0036-8075, DOI: Prama House 10.1126/science.1232033 267 Banbury Road Oxford OX2 7HT (GB) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 3 011 035 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 3 011 035 B1 • LOMBARDO ANGELO ET AL: "Gene editing in • LUKE A. GILBERT ET AL: "CRISPR-Mediated human stem cells using zinc finger nucleases and Modular RNA-Guided Regulation of integrase-defective lentiviral vector delivery", Transcription in Eukaryotes", CELL, vol. 154, no. NATURE BIOTECHNOLOGY, NATURE 2, 1 July 2013 (2013-07-01), pages 442-451, PUBLISHING GROUP, NEW YORK, NY, US, vol. XP055115843, ISSN: 0092-8674, DOI: 25, no. 11, 1 November 2007 (2007-11-01), pages 10.1016/j.cell.2013.06.044 1298-1306, XP002465562, ISSN: 1087-0156, DOI: • MALINA ABBA ET AL: "Repurposing 10.1038/NBT1353 CRISPR/Cas9 for in situ functional assays", 1 • L. CONG ET AL: "Supplementary Material to : December 2013 (2013-12-01), GENES & Multiplex Genome Engineering Using DEVELOPMENT, VOL. 27, NR. 23, PAGE(S) CRISPR/Cas Systems", SCIENCE, vol. 339, no. 2602-2614 ISSN: 0890-9369(print) 6121, 3 January 2013 (2013-01-03), pages 1-25, • N. Manjunath ET AL: "Newer Gene Editing XP002730884, ISSN: 0036-8075, DOI: Technologies toward HIV Gene Therapy", 10.1126/science.1231143 Viruses, vol. 5, no. 11, 14 November 2013 • WANG HAOYI ET AL: "One-Step Generation of (2013-11-14), pages 2748-2766, XP055110267, Mice Carrying Mutations in Multiple Genes by DOI: 10.3390/v5112748 CRISPR/Cas-Mediated Genome Engineering", • Hirotaka Ebina ET AL: "Harnessing the CELL, CELL PRESS, US, vol. 153, no. 4, 2 May CRISPR/Cas9 system to disrupt latent HIV-1 2013 (2013-05-02), pages 910-918, XP028538358, provirus", Scientific Reports, vol. 3, 26 August ISSN: 0092-8674, DOI: 2013 (2013-08-26), XP055110157, DOI: 10.1016/J.CELL.2013.04.025 10.1038/srep02510 • SHALEM OPHIR ET AL: "Genome-Scale • MANNELL HANNA ET AL: "Targeted Endothelial CRISPR-Cas9 Knockout Screening in Human Gene Delivery by Ultrasonic Destruction of Cells", January 2014 (2014-01), SCIENCE Magnetic Microbubbles Carrying Lentiviral (WASHINGTON D C), VOL. 343, NR. 6166, Vectors", PHARMACEUTICAL RESEARCH PAGE(S) 84-87, XP002723432, ISSN: (DORDRECHT), vol. 29, no. 5, May 2012 (2012-05), 0036-8075(print) the whole document pages 1282-1294, ISSN: 0724-8741(print) • HSU PATRICK D ET AL: "Development and Applications of CRISPR-Cas9 for Genome Engineering", CELL, vol. 157, no. 6, 5 June 2014 (2014-06-05), pages 1262-1278, XP028849523, ISSN: 0092-8674, DOI: 10.1016/J.CELL.2014.05.010 2 EP 3 011 035 B1 Description FIELD OF THE INVENTION 5 [0001] The present disclosure generally relates to the delivery, engineering, optimization and therapeutic applications of systems, methods, compositions and vectors used for the control of gene expression involving sequence targeting, such as genome perturbation or gene-editing, that relate to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof. 10 STATEMENT AS TO FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under the NIH Pioneer Award (1DP1MH100706) and NIH (P01 CA108631) grant awarded by the National Institutes of Health. The government has certain rights in the invention. 15 BACKGROUND OF THE INVENTION [0003] Recent advances in genome sequencing techniques and analysis methods have significantly accelerated the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by 20 allowing selective perturbation of individual genetic elements, as well as to advance synthetic biology, biotechnological, and medical applications. Although genome-editing techniques such as designer zinc fingers, transcription activator- like effectors (TALEs), or homing meganucleases are available for producing targeted genome perturbations, there remains a need for new genome engineering technologies that are affordable, easy to set up, scalable, and amenable to targeting multiple positions within the eukaryotic genome. 25 SUMMARY OF THE INVENTION [0004] The CRISPR-Cas system does not require the generation of customized proteins to target specific sequences but rather a single Cas enzyme can be programmed by a short RNA molecule to recognize a specific DNA target. Adding 30 the CRISPR-Cas system to the repertoire of genome sequencing techniques and analysis methods may significantly simplify the methodology and accelerate the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. To utilize the CRISPR-Cas system effectively for genome editing without deleterious effects, it is critical to understand aspects of engineering, optimization and tissue/organ specific delivery of these genome engineering tools, which are aspects of the claimed invention. 35 [0005] There exists a pressing need for alternative and robust systems and techniques for sequence targeting with a wide array of applications. Aspects of this invention address this need and provides related advantages. An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide. The guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence. 40 [0006] In one aspect, the disclosure provides methods for using one or more elements of a CRISPR system. The CRISPR complex provides an effective means for modifying a target polynucleotide. The CRISPR complex has a wide variety of utilities including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucle- otide in a multiplicity of cell types in various tissues and organs. As such the CRISPR complex has a broad spectrum of applications in, e.g., gene or genome editing, gene therapy, generation of cell and animal model systems, drug 45 discovery, drug screening, disease diagnosis, and prognosis. [0007] Aspects of the disclosure relate to the CRISPR-Cas system having the utility of engineering primary cells that recapitulate the genetic complexity of human disease. This complexity arises from the interaction of multiple genetic lesions, which is particularly true in the case of proliferative disorders like cancer. [0008] The disclosure comprehends a method of inducing a proliferative condition in a an organism comprising: isolating 50 a first population of cells from the organism, transducing the first population of cells with a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the poly- nucleotide sequence comprises (a) three or more guide sequences capable of hybridizing to three or more target se- quences in genome of the organism, (b) a tracr mate sequence, and (c) a tracr sequence, and II.
Load more

Recommended publications
  • The Switch from Fermentation to Respiration in Saccharomyces Cerevisiae Is Regulated by the Ert1 Transcriptional Activator/Repressor
    INVESTIGATION The Switch from Fermentation to Respiration in Saccharomyces cerevisiae Is Regulated by the Ert1 Transcriptional Activator/Repressor Najla Gasmi,* Pierre-Etienne Jacques,† Natalia Klimova,† Xiao Guo,§ Alessandra Ricciardi,§ François Robert,†,** and Bernard Turcotte*,‡,§,1 ‡Department of Medicine, *Department of Biochemistry, and §Department of Microbiology and Immunology, McGill University Health Centre, McGill University, Montreal, QC, Canada H3A 1A1, †Institut de recherches cliniques de Montréal, Montréal, QC, Canada H2W 1R7, and **Département de Médecine, Faculté de Médecine, Université de Montréal, QC, Canada H3C 3J7 ABSTRACT In the yeast Saccharomyces cerevisiae, fermentation is the major pathway for energy production, even under aerobic conditions. However, when glucose becomes scarce, ethanol produced during fermentation is used as a carbon source, requiring a shift to respiration. This adaptation results in massive reprogramming of gene expression. Increased expression of genes for gluconeogenesis and the glyoxylate cycle is observed upon a shift to ethanol and, conversely, expression of some fermentation genes is reduced. The zinc cluster proteins Cat8, Sip4, and Rds2, as well as Adr1, have been shown to mediate this reprogramming of gene expression. In this study, we have characterized the gene YBR239C encoding a putative zinc cluster protein and it was named ERT1 (ethanol regulated transcription factor 1). ChIP-chip analysis showed that Ert1 binds to a limited number of targets in the presence of glucose. The strongest enrichment was observed at the promoter of PCK1 encoding an important gluconeogenic enzyme. With ethanol as the carbon source, enrichment was observed with many additional genes involved in gluconeogenesis and mitochondrial function. Use of lacZ reporters and quantitative RT-PCR analyses demonstrated that Ert1 regulates expression of its target genes in a manner that is highly redundant with other regulators of gluconeogenesis.
    [Show full text]
  • Universidade Estadual De Campinas Instituto De Biologia
    UNIVERSIDADE ESTADUAL DE CAMPINAS INSTITUTO DE BIOLOGIA VERÔNICA APARECIDA MONTEIRO SAIA CEREDA O PROTEOMA DO CORPO CALOSO DA ESQUIZOFRENIA THE PROTEOME OF THE CORPUS CALLOSUM IN SCHIZOPHRENIA CAMPINAS 2016 1 VERÔNICA APARECIDA MONTEIRO SAIA CEREDA O PROTEOMA DO CORPO CALOSO DA ESQUIZOFRENIA THE PROTEOME OF THE CORPUS CALLOSUM IN SCHIZOPHRENIA Dissertação apresentada ao Instituto de Biologia da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do Título de Mestra em Biologia Funcional e Molecular na área de concentração de Bioquímica. Dissertation presented to the Institute of Biology of the University of Campinas in partial fulfillment of the requirements for the degree of Master in Functional and Molecular Biology, in the area of Biochemistry. ESTE ARQUIVO DIGITAL CORRESPONDE À VERSÃO FINAL DA DISSERTAÇÃO DEFENDIDA PELA ALUNA VERÔNICA APARECIDA MONTEIRO SAIA CEREDA E ORIENTADA PELO DANIEL MARTINS-DE-SOUZA. Orientador: Daniel Martins-de-Souza CAMPINAS 2016 2 Agência(s) de fomento e nº(s) de processo(s): CNPq, 151787/2F2014-0 Ficha catalográfica Universidade Estadual de Campinas Biblioteca do Instituto de Biologia Mara Janaina de Oliveira - CRB 8/6972 Saia-Cereda, Verônica Aparecida Monteiro, 1988- Sa21p O proteoma do corpo caloso da esquizofrenia / Verônica Aparecida Monteiro Saia Cereda. – Campinas, SP : [s.n.], 2016. Orientador: Daniel Martins de Souza. Dissertação (mestrado) – Universidade Estadual de Campinas, Instituto de Biologia. 1. Esquizofrenia. 2. Espectrometria de massas. 3. Corpo caloso.
    [Show full text]
  • Protein Kinase A-Mediated Septin7 Phosphorylation Disrupts Septin Filaments and Ciliogenesis
    cells Article Protein Kinase A-Mediated Septin7 Phosphorylation Disrupts Septin Filaments and Ciliogenesis Han-Yu Wang 1,2, Chun-Hsiang Lin 1, Yi-Ru Shen 1, Ting-Yu Chen 2,3, Chia-Yih Wang 2,3,* and Pao-Lin Kuo 1,2,4,* 1 Department of Obstetrics and Gynecology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan; [email protected] (H.-Y.W.); [email protected] (C.-H.L.); [email protected] (Y.-R.S.) 2 Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan; [email protected] 3 Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan 4 Department of Obstetrics and Gynecology, National Cheng-Kung University Hospital, Tainan 704, Taiwan * Correspondence: [email protected] (C.-Y.W.); [email protected] (P.-L.K.); Tel.: +886-6-2353535 (ext. 5338); (C.-Y.W.)+886-6-2353535 (ext. 5262) (P.-L.K.) Abstract: Septins are GTP-binding proteins that form heteromeric filaments for proper cell growth and migration. Among the septins, septin7 (SEPT7) is an important component of all septin filaments. Here we show that protein kinase A (PKA) phosphorylates SEPT7 at Thr197, thus disrupting septin filament dynamics and ciliogenesis. The Thr197 residue of SEPT7, a PKA phosphorylating site, was conserved among different species. Treatment with cAMP or overexpression of PKA catalytic subunit (PKACA2) induced SEPT7 phosphorylation, followed by disruption of septin filament formation. Constitutive phosphorylation of SEPT7 at Thr197 reduced SEPT7-SEPT7 interaction, but did not affect SEPT7-SEPT6-SEPT2 or SEPT4 interaction.
    [Show full text]
  • C3orf70 Is Involved in Neural and Neurobehavioral Development
    pharmaceuticals Article C3orf70 Is Involved in Neural and Neurobehavioral Development Yoshifumi Ashikawa 1, Takashi Shiromizu 1, Koki Miura 1, Yuka Adachi 1, Takaaki Matsui 2, Yasumasa Bessho 2, Toshio Tanaka 3 and Yuhei Nishimura 1,* 1 Department of Integrative Pharmacology, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan; [email protected] (Y.A.); [email protected] (T.S.); [email protected] (K.M.); [email protected] (Y.A.) 2 Gene Regulation Research, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Nara 630-0192, Japan; [email protected] (T.M.); [email protected] (Y.B.) 3 Department of Systems Pharmacology, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan; [email protected] * Correspondence: [email protected] Received: 10 October 2019; Accepted: 15 October 2019; Published: 16 October 2019 Abstract: Neurogenesis is the process by which undifferentiated progenitor cells develop into mature and functional neurons. Defects in neurogenesis are associated with neurodevelopmental and neuropsychiatric disorders; therefore, elucidating the molecular mechanisms underlying neurogenesis can advance our understanding of the pathophysiology of these disorders and facilitate the discovery of novel therapeutic targets. In this study, we performed a comparative transcriptomic analysis to identify common targets of the proneural transcription factors Neurog1/2 and Ascl1 during neurogenesis of human and mouse stem cells. We successfully identified C3orf70 as a novel common target gene of Neurog1/2 and Ascl1 during neurogenesis. Using in situ hybridization, we demonstrated that c3orf70a and c3orf70b, two orthologs of C3orf70, were expressed in the midbrain and hindbrain of zebrafish larvae.
    [Show full text]
  • Analyzing the Genes Related to Alzheimer's Disease Via a Network
    Hu et al. Alzheimer's Research & Therapy (2017) 9:29 DOI 10.1186/s13195-017-0252-z RESEARCH Open Access Analyzing the genes related to Alzheimer’s disease via a network and pathway-based approach Yan-Shi Hu1, Juncai Xin1, Ying Hu1, Lei Zhang2* and Ju Wang1* Abstract Background: Our understanding of the molecular mechanisms underlying Alzheimer’s disease (AD) remains incomplete. Previous studies have revealed that genetic factors provide a significant contribution to the pathogenesis and development of AD. In the past years, numerous genes implicated in this disease have been identified via genetic association studies on candidate genes or at the genome-wide level. However, in many cases, the roles of these genes and their interactions in AD are still unclear. A comprehensive and systematic analysis focusing on the biological function and interactions of these genes in the context of AD will therefore provide valuable insights to understand the molecular features of the disease. Method: In this study, we collected genes potentially associated with AD by screening publications on genetic association studies deposited in PubMed. The major biological themes linked with these genes were then revealed by function and biochemical pathway enrichment analysis, and the relation between the pathways was explored by pathway crosstalk analysis. Furthermore, the network features of these AD-related genes were analyzed in the context of human interactome and an AD-specific network was inferred using the Steiner minimal tree algorithm. Results: We compiled 430 human genes reported to be associated with AD from 823 publications. Biological theme analysis indicated that the biological processes and biochemical pathways related to neurodevelopment, metabolism, cell growth and/or survival, and immunology were enriched in these genes.
    [Show full text]
  • HPV E2, E4, E5 Drive Alternative Carcinogenic Pathways in HPV Positive Cancers
    Oncogene (2020) 39:6327–6339 https://doi.org/10.1038/s41388-020-01431-8 ARTICLE HPV E2, E4, E5 drive alternative carcinogenic pathways in HPV positive cancers 1,2 3 3 4,5 4 6 Shuling Ren ● Daria A. Gaykalova ● Theresa Guo ● Alexander V. Favorov ● Elana J. Fertig ● Pablo Tamayo ● 1,7 1 1 1 1 1 Juan Luis Callejas-Valera ● Mike Allevato ● Mara Gilardi ● Jessica Santos ● Takahito Fukusumi ● Akihiro Sakai ● 1 1 1 6 6 1 1 Mizuo Ando ● Sayed Sadat ● Chao Liu ● Guorong Xu ● Kathleen M. Fisch ● Zhiyong Wang ● Alfredo A. Molinolo ● 1 6 3 1,8 J. Silvio Gutkind ● Trey Ideker ● Wayne M. Koch ● Joseph A. Califano Received: 19 February 2020 / Revised: 19 July 2020 / Accepted: 13 August 2020 / Published online: 26 August 2020 © The Author(s) 2020. This article is published with open access Abstract The dominant paradigm for HPV carcinogenesis includes integration into the host genome followed by expression of E6 and E7 (E6/E7). We explored an alternative carcinogenic pathway characterized by episomal E2, E4, and E5 (E2/E4/E5) expression. Half of HPV positive cervical and pharyngeal cancers comprised a subtype with increase in expression of E2/E4/ E5, as well as association with lack of integration into the host genome. Models of the E2/E4/E5 carcinogenesis show p53 1234567890();,: 1234567890();,: dependent enhanced proliferation in vitro, as well as increased susceptibility to induction of cancer in vivo. Whole genomic expression analysis of the E2/E4/E5 pharyngeal cancer subtype is defined by activation of the fibroblast growth factor receptor (FGFR) pathway and this subtype is susceptible to combination FGFR and mTOR inhibition, with implications for targeted therapy.
    [Show full text]
  • Construction of an Mirna‑Gene Regulatory Network in Colorectal Cancer Through Integrated Analysis of Mrna and Mirna Microarrays
    MOLECULAR MEDICINE REPORTS 18: 5109-5116, 2018 Construction of an miRNA‑gene regulatory network in colorectal cancer through integrated analysis of mRNA and miRNA microarrays JUN HU, XIN YUE, JIANZHONG LIU and DALU KONG Department of Colorectal Cancer Surgery, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy of Tianjin, Tianjin's Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, P.R. China Received December 19, 2017; Accepted August 8, 2018 DOI: 10.3892/mmr.2018.9505 Abstract. The aim of the present study was to identify poten- Introduction tial biomarkers associated with colorectal cancer (CRC). The GSE32323 and GSE53592 mRNA and microRNA (miRNA) Colorectal cancer (CRC) is a common malignancy that ranks expression profiles were selected from the Gene Expression as the second leading cause of cancer-associated mortality in Omnibus database. The differentially expressed genes (DEGs) men and women in the USA (1). Despite improvements in CRC and differentially expressed miRNAs (DEMs) in CRC tissue therapy, CRC remains a major public health problem, and it is samples compared with surrounding control tissue samples estimated that there are 1,000,000 individuals suffering from (DEGs-CC), and DEGs in cells treated with 5-aza-2'-de- CRC worldwide, with the mortality rate is as high as ~50% in oxycitidine compared with untreated cells (DEGs-TC) certain developed countries (2). The tumor stage is the most were identified with the Limma package. The Database for important prognostic indicator for CRC. However, the tumors Annotation, Visualization and Integrated Discovery was used are often diagnosed at an intermediate or late stage, and the to conduct the functional and pathways enrichment analysis.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • New Targets of Urocortin-Mediated Cardioprotection
    69 New targets of urocortin-mediated cardioprotection Sea´n P Barry1, Kevin M Lawrence4, James McCormick1, Surinder M Soond5, Mike Hubank2, Simon Eaton3, Ahila Sivarajah6, Tiziano M Scarabelli7, Richard A Knight1, Christoph Thiemermann6, David S Latchman1, Paul A Townsend8 and Anastasis Stephanou1 1Medical Molecular Biology Unit, 2Department of Molecular Haematology and 3Department of Surgery, Institute of Child Health, University College London, 30 Guilford Street, London, WC1N 1EH, UK 4Department of Cellular Pathology, St George’s, University of London, Cranmer Terrace, Tooting, London, SW17 0RE, UK 5School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK 6St Bartholomew’s and The Royal London School of Medicine and Dentistry, William Harvey Research Institute, Centre for Translational Medicine and Therapeutics, Queen Mary University of London, London, EC1M 7BQ, UK 7Center for Heart and Vessel Preclinical Studies, St John Hospital and Medical Center, Wayne State University School of Medicine, 22201 Moross Road, Detroit, Michigan 48336, USA 8Human Genetics Division, MP808, Southampton General Hospital, University of Southampton, Southampton SO16 6YD, UK (Correspondence should be addressed to S P Barry; Email: [email protected]) Abstract The urocortin (UCN) hormones UCN1 and UCN2 have been shown previously to confer significant protection against myocardial ischaemia/reperfusion (I/R) injury; however, the molecular mechanisms underlying their action are poorly understood. To further define the transcriptional effect of UCNs that underpins their cardioprotective activity, a microarray analysis was carried out using an in vivo rat coronary occlusion model of I/R injury. Infusion of UCN1 or UCN2 before the onset of reperfusion resulted in the differential regulation of 66 and 141 genes respectively, the majority of which have not been described previously.
    [Show full text]
  • A Novel Resveratrol Analog: Its Cell Cycle Inhibitory, Pro-Apoptotic and Anti-Inflammatory Activities on Human Tumor Cells
    A NOVEL RESVERATROL ANALOG : ITS CELL CYCLE INHIBITORY, PRO-APOPTOTIC AND ANTI-INFLAMMATORY ACTIVITIES ON HUMAN TUMOR CELLS A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Boren Lin May 2006 Dissertation written by Boren Lin B.S., Tunghai University, 1996 M.S., Kent State University, 2003 Ph. D., Kent State University, 2006 Approved by Dr. Chun-che Tsai , Chair, Doctoral Dissertation Committee Dr. Bryan R. G. Williams , Co-chair, Doctoral Dissertation Committee Dr. Johnnie W. Baker , Members, Doctoral Dissertation Committee Dr. James L. Blank , Dr. Bansidhar Datta , Dr. Gail C. Fraizer , Accepted by Dr. Robert V. Dorman , Director, School of Biomedical Sciences Dr. John R. Stalvey , Dean, College of Arts and Sciences ii TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………….………v LIST OF TABLES……………………………………………………………………….vii ACKNOWLEDGEMENTS….………………………………………………………….viii I INTRODUCTION….………………………………………………….1 Background and Significance……………………………………………………..1 Specific Aims………………………………………………………………………12 II MATERIALS AND METHODS.…………………………………………….16 Cell Culture and Compounds…….……………….…………………………….….16 MTT Cell Viability Assay………………………………………………………….16 Trypan Blue Exclusive Assay……………………………………………………...18 Flow Cytometry for Cell Cycle Analysis……………..……………....……………19 DNA Fragmentation Assay……………………………………………...…………23 Caspase-3 Activity Assay………………………………...……….….…….………24 Annexin V-FITC Staining Assay…………………………………..…...….………28 NF-kappa B p65 Activity Assay……………………………………..………….…29
    [Show full text]
  • Clinical Validation of the Tempus Xt Next-Generation Targeted Oncology Sequencing Assay
    www.oncotarget.com Oncotarget, 2019, Vol. 10, (No. 24), pp: 2384-2396 Research Paper Clinical validation of the tempus xT next-generation targeted oncology sequencing assay Nike Beaubier1, Robert Tell1, Denise Lau1, Jerod R. Parsons1, Stephen Bush1, Jason Perera1, Shelly Sorrells1, Timothy Baker1, Alan Chang1, Jackson Michuda1, Catherine Iguartua1, Shelley MacNeil1, Kaanan Shah1, Philip Ellis1, Kimberly Yeatts1, Brett Mahon1, Timothy Taxter1, Martin Bontrager1, Aly Khan1, Robert Huether1, Eric Lefkofsky1 and Kevin P. White1 1Tempus Labs Inc., Chicago, IL 60654, USA Correspondence to: Nike Beaubier, email: [email protected] Kevin P. White, email: [email protected] Keywords: tumor profiling, next-generation sequencing assay validation Received: August 03, 2018 Accepted: February 03, 2019 Published: March 22, 2019 Copyright: Beaubier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT We developed and clinically validated a hybrid capture next generation sequencing assay to detect somatic alterations and microsatellite instability in solid tumors and hematologic malignancies. This targeted oncology assay utilizes tumor- normal matched samples for highly accurate somatic alteration calling and whole transcriptome RNA sequencing for unbiased identification of gene fusion events. The assay was validated with a combination of clinical specimens and cell lines, and recorded a sensitivity of 99.1% for single nucleotide variants, 98.1% for indels, 99.9% for gene rearrangements, 98.4% for copy number variations, and 99.9% for microsatellite instability detection. This assay presents a wide array of data for clinical management and clinical trial enrollment while conserving limited tissue.
    [Show full text]
  • Transcriptomic and Proteomic Profiling Provides Insight Into
    BASIC RESEARCH www.jasn.org Transcriptomic and Proteomic Profiling Provides Insight into Mesangial Cell Function in IgA Nephropathy † † ‡ Peidi Liu,* Emelie Lassén,* Viji Nair, Celine C. Berthier, Miyuki Suguro, Carina Sihlbom,§ † | † Matthias Kretzler, Christer Betsholtz, ¶ Börje Haraldsson,* Wenjun Ju, Kerstin Ebefors,* and Jenny Nyström* *Department of Physiology, Institute of Neuroscience and Physiology, §Proteomics Core Facility at University of Gothenburg, University of Gothenburg, Gothenburg, Sweden; †Division of Nephrology, Department of Internal Medicine and Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan; ‡Division of Molecular Medicine, Aichi Cancer Center Research Institute, Nagoya, Japan; |Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; and ¶Integrated Cardio Metabolic Centre, Karolinska Institutet Novum, Huddinge, Sweden ABSTRACT IgA nephropathy (IgAN), the most common GN worldwide, is characterized by circulating galactose-deficient IgA (gd-IgA) that forms immune complexes. The immune complexes are deposited in the glomerular mesangium, leading to inflammation and loss of renal function, but the complete pathophysiology of the disease is not understood. Using an integrated global transcriptomic and proteomic profiling approach, we investigated the role of the mesangium in the onset and progression of IgAN. Global gene expression was investigated by microarray analysis of the glomerular compartment of renal biopsy specimens from patients with IgAN (n=19) and controls (n=22). Using curated glomerular cell type–specific genes from the published literature, we found differential expression of a much higher percentage of mesangial cell–positive standard genes than podocyte-positive standard genes in IgAN. Principal coordinate analysis of expression data revealed clear separation of patient and control samples on the basis of mesangial but not podocyte cell–positive standard genes.
    [Show full text]