Translatomics study of chinese hamster ovary cell cultures

TRANSLATOMICS STUDY OF CHINESE HAMSTER OVARY CELL CULTURES COURTES FRANCK (M.Eng (Biochemical Engineering)), Institut National des Sciences Appliquées de Toulouse, France A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2013 (Blank Page) DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously Franck Courtes 20 June 2013 i (Blank Page) ii Acknowledgements This thesis would not have been possible without the guidance and the help of several individuals who in one way or another contributed and extended their valuable assistance in the preparation and completion of this PhD First and foremost, I would like to express my deepest appreciation to Professor Miranda Yap, Assistant Professor Lee Dong Yup and Doctor Niki Wong for their insightful comments, persistent help and inspiration as I hurdle all the obstacles in the completion of this thesis I sincerely wish a quick recovery to Professor Yap My sincere thanks also go to Assistant Professor Tong Yen Wah, Associate Professor Ren Ee Chee and Associate Professor Lee Yuan Kun members of my thesis Advisory Committee Their continuous support, instructive advices and motivating discussions rendered for the past four years have been instrumental to my research I would like to acknowledge my collaboration with Doctor Bernard Loo and his research officers Sze Wai Ng and Hsueh Lee Lim from the microarrays group of BTI, for guiding the design of microarrays experiments, which were crucial for my PhD Thanks to Doctor Andrea Camattari, I had an opportunity to collaborate with Doctor Leah Vardy, whose contribution to my PhD had been invaluable I am indebted to Leah for welcoming me to her lab, for training me to perform polysome profiling and for always fostering my motivation in research Additionally, I would like to acknowledge Kristin Chong Peini, a research officer from Leah’s group who perfected my polysome profiling skills My sincere gratitude goes to Doctor Muriel Bardor for her indispensable advices and encouragement Muriel has always been not only my great mentor but also a wonderful friend I would like to thank her for being available to iii discuss about sciences as well as personal life and especially for welcoming me to her family in time of need Many thanks to Doctor Yuan Sheng Yang and Doctor Say Kong Ng for their kind consideration and their time to discuss about my project and share with me their valuable recommendations My gratitude goes to Professor Dedon and his graduate student Chen Gu who were kind enough to accept to measure the pseudouridylation content of my RNA samples in their laboratory in the Massachusetts Institute of Technology I also am very thankful for Professor Wei-Shou Hu for his lecture and discussions on CHO cell culture that enlightened my PhD project and research motivations Many thanks to the human resources staffs of BTI, particularly Mei Yi Wong and Siew-Chin Chung, for their persistent and prompt assistance on all the administrative matters during the past years My appreciation also goes to Doctor Vincent Vagenende, Danny Ong, U-ming Lim and Doctor William Chong for having been such great and supportive friends I will always cherish the memories of our joyful times that we spent together in Singapore My everyday life in the animal cell technology group would not have been as pleasant without my PhD colleagues including Janice Tan, Steven Ho and Wanping Loh Thank you for always being willing to discuss technical details and for being my friends Very special thanks go to Janice for all of the special treats and fruits that she shared with me, when I became evidently stressful with my thesis I would like to acknowledge Yasotha Kathiraser, whose endless kindness, simplicity and happiness in life has been a wonderful life lesson to me iv My work on this thesis was supported by the Agency for Science Technology and Research that provided me with the Singapore International Graduate Award, the Bioprocessing Technology Institute that provided me with necessary facilities for carrying out this research work, and the National University of Singapore Last but not least, I am heartily thankful to my beloved wife for her endless love, care, understanding, moral support and inspiration during my research Thank you for showing me what love means every day I am also thankful to my family especially my parents Alain and Genevieve as well as my beloved aunts and uncles, who sacrificed a lot to provide me with a good education and a loving environment to grow up in I am grateful for their infinite love and support throughout everything in my life It has been an immense honor to spend this part of my life in the great city of Singapore and I am looking forward to keeping strong ties with this country v Table of Contents Acknowledgements iii Table of Contents vi Summary xi List of Tables xiii List of Figures xiv Chapter 1: Introduction 1.1 Background 1.2 Thesis objective 1.3 Thesis organization Chapter 2: Literature Review 2.1 Translatome: the missing gap between transcriptome and proteome 2.1.1 Transcriptome: “What seems to happen” 10 2.1.2 Proteome: “What makes it happen” 14 2.1.3 Discrepancy between transcriptome and proteome 17 2.1.4 Translatome: “What cells need to happen” 19 2.2 Translational control 22 2.2.1 Reasons for regulating translational activity 22 2.2.2 Molecular mechanisms of translation 23 2.2.3 Initiation of translation 25 2.2.4 Different mechanisms of translational control 28 2.3 mTOR signaling pathway 32 2.3.1 Organization of mTOR pathway 32 2.3.2 Upstream signaling of mTOR activity 34 vi 2.3.3 Downstream targets of mTOR pathway 35 2.3.4 Rapamycin treatment of mTOR pathway 39 2.4 Small Nucleolar RNAs (snoRNAs) 41 2.4.1 Biogenesis and mode of action of snoRNAs 42 2.4.2 Function of H/ACA box snoRNAs 45 Chapter 3: Materials and Methods 47 3.1 Cell culture 47 3.1.1 Cell lines 47 3.1.2 Cell maintenance 48 3.1.3 Batch cultures 49 3.1.4 Feeding of batch cultures 49 3.1.5 Rapamycin treatment of batch cultures 50 3.1.6 Determination of cell viability 50 3.1.7 Determination of monoclonal antibody titers 50 3.1.8 Measurement of residual glutamine and glucose concentrations 51 3.2 Translation activity 51 3.2.1 Sucrose solutions and gradient preparation 51 3.2.2 Polysome extraction 52 3.2.3 Polysome profiling and fractionation 53 3.2.4 Fraction pooling 54 3.2.5 RNA extraction from pools and purification 55 3.3 Translatome profiling and transcriptome profiling 56 3.3.1 Preparation of labeled cDNA library for microarrays 57 3.3.2 Microarrays procedure 58 3.3.3 Microarray data analysis 60 3.4 Targeted relative quantification of specific RNA level 61 vii 3.4.1 Primers 61 3.4.2 Template DNA 64 3.4.3 Quantitative-RT PCR 70 3.5 mTOR pathway activity 71 3.5.1 Total protein extraction 71 3.5.2 Total protein quantification 71 3.5.3 Immunobloting 71 3.6 Generation of stable CHO U19 pools 72 3.6.1 Construction of expression vector pcDNA3.1/hygro-U19 72 3.6.2 Transfection of CHO M250-9 cell line 78 3.6.3 Selection of stable CHO U19 pool 80 3.7 Quantification of pseudouridine in 28S rRNA 80 3.8 Design of experiment and calculation of variables effect 82 Chapter 4: Developing a Strategy for the Translatomic Analysis of CHO Cells 83 4.1 Introduction 83 4.2 Results and discussion 84 4.2.1 Generation of first translatome data in CHO cells 84 4.2.2 Global translation activity during exponential growth phase 93 4.2.3 Translatome for identifying key growth genes 95 4.2.4 Translational control mechanisms in CHO cells 101 4.3 Summary 106 Chapter 5: Understanding Translational Control Mechanisms of the mTOR Pathway in CHO Cells by Polysome Profiling 107 5.1 Introduction 107 5.2 Results and discussion 109 5.2.1 Targeted inhibition of the mTOR pathway by rapamycin 109 viii Wong DC, Wong KT, Nissom PM, Heng CK, Yap MG 2006b Targeting early apoptotic genes in batch and fed-batch CHO cell cultures Biotechnol Bioeng 95(3):350-61 Wong N, Yap M 2007 The future of biomanufacturing in Singapore Biotechnol J 2(11):1327-9 Wu G, Xiao M, Yang C, Yu YT 2011 U2 snRNA is inducibly pseudouridylated at novel sites by Pus7p and snR81 RNP EMBO J 30(1):79-89 Wurm F 2004 Production of recombinant protein therapeutics in cultivated mammalian cells Nat Biotechnol 22(11):1393-8 Xu X, Nagarajan H, Lewis NE, Pan S, Cai Z, Liu X, Chen W, Xie M, Wang W, Hammond S and others 2011 The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line Nat Biotechnol 29(8):73541 Yamashita R, Suzuki Y, Takeuchi N, Wakaguri H, Ueda T, Sugano S, Nakai K 2008 Comprehensive detection of human terminal oligo-pyrimidine (TOP) genes and analysis of their characteristics Nucleic acids res 36(11):3707-15 Yang Q, Guan KL 2007 Expanding mTOR signaling Cell Res 17(8):666-81 Yee JC, de Leon Gatti M, Philp RJ, Yap M, Hu WS 2008a Genomic and proteomic exploration of CHO and hybridoma cells under sodium butyrate treatment Biotechnol Bioeng 99(5):1186-204 Yee JC, Gerdtzen ZP, Hu WS 2009 Comparative transcriptome analysis to unveil genes affecting recombinant protein productivity in mammalian cells Biotechnol Bioeng 102(1):246-63 Yee JC, Wlaschin KF, Chuah SH, Nissom PM, Hu WS 2008b Quality assessment of cross-species hybridization of CHO transcriptome on a mouse DNA oligo microarray Biotechnol Bioeng 101(6):1359-65 Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN, Cate JH, Noller HF 2001 Crystal structure of the ribosome at 5.5 A resolution Science 292(5518):883-96 Zhang C, Yang N, Yang CH, Ding HS, Luo C, Zhang Y, Wu MJ, Zhang XW, Shen X, Jiang HL and others 2009 S9, a novel anticancer agent, exerts its anti-proliferative activity by interfering with both PI3K-AktmTOR signaling and microtubule cytoskeleton PLoS One 4(3):e4881 175 Appendices Appendix A - List of publications Courtes F, Lin J, Lim HL, Ng SW, Wong N, Koh G, Vardy L, Loo B, Yap M, Lee DY 2013 Translatome analysis of CHO cells identifies key growth genes - Submitted to the Journal of Biotechnology Courtes F, Vardy L, Wong N, Bardor M, Yap M, Lee DY 2013 Understanding translational control mechanisms of the mTOR pathway in CHO cells by polysome profiling - Submitted to New Biotechnology Journal Courtes F, Gu C, Wong N, Dedon PC, Yap M, Lee DY 2013 28S rRNA is inducibly pseudouridylated by the mTOR pathway translational control in CHO cell cultures - To be submitted to Biotechnology Journal 176 Appendix B - RNA quality assessment via bioanalyzer prior to cDNA library synthesis The RNA integrity number (RIN) ranged from 8.60 to 10 indicating a very high integrity of RNA samples B.1 Pool A - RNA samples 177 B.2 Pool B - RNA samples 178 B.3 Transcriptome - RNA samples 179 Appendix C - cDNA library quality assessment via bioanalyzer The size of cDNA library was on average around 200 bp C.1 Pool A - cDNA samples 180 C.2 Pool B - cDNA samples 181 C.3 Transcriptome - cDNA samples 182 Appendix D - Standard curves for primers efficiency Appendix D.1 Appendix D.2 Appendix D.3 Appendix D.4 183 Appendix D.5 Appendix D.6 Appendix D.7 Appendix D.8 184 Appendix D.9 Appendix D.10 Appendix D.11 Appendix D.12 185 Appendix D.13 Appendix D.14 Appendix D.15 Appendix D.16 186 Appendix D.17 Appendix D.18 Appendix D.19 Appendix D.20 187 Appendix E - Receipt of RNA samples shipment for pseudouridylation analysis in Professor Dedon’s laboratory 188 (Blank Page) 189 ... Batch cultures 49 3.1.4 Feeding of batch cultures 49 3.1.5 Rapamycin treatment of batch cultures 50 3.1.6 Determination of cell viability 50 3.1.7 Determination of. .. Background Chinese hamster ovary (CHO) cells are one of the most commonly used mammalian host cell lines by biopharmaceutical industries for the production of recombinant proteins of therapeutic... Chinese hamster ovary (CHO) cells have been extensively used for understanding cellular mechanisms and for identifying cell engineering targets towards an optimization of cell cultures However,