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An Investigation of Novel Genetic Tools for the Manipulation of CHO Cell Phenotypes During Recombinant Protein Production

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An Investigation of Novel Genetic Tools for the Manipulation of CHO Cell Phenotypes During Recombinant Protein Production An investigation of novel genetic tools for the manipulation of CHO cell phenotypes during recombinant protein production A thesis submitted for the degree of Ph.D. Dublin City University by Alan Griffith, B.Sc (Biotechnology) The research described in this thesis was performed under the supervision of Dr. Niall Barron and Prof. Martin Clynes. National Institute for Cellular Biotechnology Dublin City University 2015 i This thesis is dedicated to my wonderful family and friends - especially Angela, Gordon, Shane, Lena and Eddie. ii I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of Ph.D is entirely my own work, and that I have exercised the reasonable care to ensure that the work is original, and does not to the best of my knowledge breach any law of copyright, and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my work. Signed: ________________________ (Candidate) ID no: 54343697 Date: ________________________ iii Acknowledgements It’s a great pleasure for me to thank the following people that have made my experience in the NICB an unforgettable one. Their help and support during my 4+ years coupled with their advice and ideas enabled me to obtain my Ph.D. Mostly I’d like to thank my wonderful girlfriend Laura and parents Angela and Gordon, and of course Shane-dog. The understanding from Laura on weekends especially was nothing short of a miracle and help from my parents near the end when times were tough. Same can be said of the boys in Dunluce, C/W and Braintrust. I cannot express how much thanks and praise I can give to my supervisor Dr. Niall Barron, who gave me help and mentored me like no other boss could have done. The only problem being now is that, I will want all my future bosses to be as kind, understanding and as intelligent as him so maybe the standard is set too high. Also thanks for guiding me through the ups and downs over the duration of the project and keeping me focused and driven and of course thanks for the opportunity. And of course special mention to Professor Martin Clynes, who somehow manages to oversee everyone and everything in and outside the NICB building. Thanks for making time to sit down and discuss career plans, paper ideas and discuss other topics like sport. Never once did I feel under pressure regarding the environment and this made life so much easier. Thank you Martin for putting your faith in me from the beginning. It really hits home now that its been such a long time since I emailed you and asked for 6 weeks work experience. Thanks to the Carol, Geraldine and Yvonne on the reception desk for dealing with deliveries and other behind the scenes tasks, and also Gillian for running the prep room like clockwork and making life easier in general. To everyone in the NICB, I think we all have helped each other at one time or another so I won’t mention names. Two big mentions must go to Dr. Olga Piskereva who taught me a lot at the very start of my career in the NICB and many hints and tricks in a molecular sense and I have taken them with me. Furthermore, thanks to Dr. Nga Lao who thought me so much during her short stays, about science and her home in Vietnam, no problem to huge for these two ladies. Finally thanks to my lunch crew, Paul, Shane, Martin, Mark the baker, Gemma, Noelia, Andrew, Edel, Deirdre, all the newbie’s (Chemistry girls Creina, Zara and Andrea - we had some good laughs in the lab) and of course the enigma that is Mary White over in Invent. iv Table of Contents: Abstract 1 Abbreviations 3 Section 1.0: Introduction 7 1.1: Introduction to Biotechnology in Bioprocessing 8 1.2: Chinese Hamster Ovary Cells 9 1.3: Media Optimistation 10 1.4: Strategies in Mammalian Cell Engineering 12 1.4.1: Stable recombinant clone generation in CHO (rCHO) 12 1.4.2: Overexpression engineering 14 1.4.3: Site-specific techniques 16 1.4.3.1: Site-specific nucleases for genome editing 16 1.4.3.2: Cre/LoxP system 19 1.4.4: RNA interference engineering 20 1.4.5: Cis-regulatory elements 23 1.4.5.1: Scaffold/matrix attachment regions (S/MAR) 23 1.4.5.2: Ubiquitous Chromatin Opening Elements 24 1.4.5.3: IRES elements 25 1.4.5.4: Other engineering strategies 26 1.5: The CHO Genome Project 26 1.6: Gene Promoters 28 1.6.1: Types of Promoters 30 1.6.1.1: Constitutive promoters 30 1.6.1.2: Tissue-specific or development-stage-specific promoters 31 1.6.1.3: Inducible promoters 32 1.6.1.4: Synthetic promoters 36 1.7: Promoter engineering 38 1.8: Exogenous Inducer Molecules (IMs) and building a promoter catalogue 39 1.9: Viral versus Endogenous CHO promoters in bioprocessing 41 1.9.1: Endogenous promoters responsive to temperature inducibility 42 1.9.2: Cirbp – The classical temperature inducible gene 42 1.10: Promoter Analysis – Bioinformatics 44 1.10.1: Identifying and locating transcriptional elements 47 1.11: Engineering of important cellular pathways 50 1.11.1: Engineering of cellular proliferation and cell cycle arrest 50 1.11.2: Programmed cell death (PCD) engineering 53 1.11.3: Effect of low Temperature on culture performance 57 1.12: MicroRNA roles in gene regulation and engineering potential 60 1.12.1: Background and scope 60 1.12.2: The miR-17-92 cluster 64 1.13: XIAP is an important gene in apoptosis regulation 65 1.13.1: Background 65 1.13.2: Structure and Function 66 v 1.14: Identifying miRNAs that regulate bioprocess-relevant phenotypes 67 1.14.1: Methods for identifying mRNA:miRNA interactions 68 1.14.2: MiRNA Pulldown using biotinylated mRNA Capture Technique 72 1.15: Aims of Thesis 74 Section 2.0: Materials and Methods 76 2.1: Ultra Pure Water 77 2.2: Sterilisation 77 2.3: Glassware 77 2.4: Preparation of cell media 77 2.5: Routine Management of cells 78 2.5.1: Precaution measures 78 2.5.2 Sub-culturing of cell lines 78 2.5.2.1 Anchorage dependant cells 78 2.5.2.2 Suspension Cells 79 2.5.3: Cell counting 79 2.5.4: Cryopreservation of cells/Cell freezing 80 2.5.5: Reviving cell lines from cryopreservation/Cell thawing 80 2.5.6: Mycoplasma testing 80 2.6: DNA manipulation and other techniques 81 2.6.1: Designing and Ordering Primers 81 2.6.2: Polymerase Chain Reaction (PCR) 82 2.6.3: Analysis of DNA samples using agarose gel electrophoresis 85 2.6.4: Cloning of DNA: Endonuclease restriction digestion of DNA 85 2.6.4.1: Alkaline Phosphatase (AP) treatment 86 2.6.5: Cloning of DNA: Ligations 87 2.6.6: Transformation into bacterial cells 88 2.6.7: Small scale preparation of plasmid DNA (Miniprep) 89 2.6.8: Large scale preparation of plasmid DNA (Midiprep) 89 2.6.9: Sequencing and Verification 90 2.6.10: Genomic DNA Extraction 90 2.6.11: RNA Extraction 91 2.6.12: Determination of RNA purity with Bioanalyser 92 2.6.13: DNase treatment 93 2.6.14: Reverse Transcription PCR and cDNA generation 94 2.6.15: Real-Time Quantitative PCR (RT-qPCR) 95 2.6.16: Transfection of Mammalian Cells 97 2.7: SEAP assays 98 2.8: Flow cytometry and cell imaging using Guava® Easycyte systems 99 2.9: PCR based miRNA Taqman Low Density Arrays (TLDA) 100 2.9.1: Reverse Transcription 101 2.9.2: PCR using 2x TaqMan® Master mix 102 2.9.3: TLDA data analysis 104 2.10: Functional analysis (FA) of selected gene and miRNA targets 104 2.11: Bradford Assay and Protein Quantification 105 2.12: Western Blotting 106 vi 2.12.1: Western sample preparation 106 2.12.2: Preparation of gel 106 2.12.3: Transfer Preparation 107 2.12.4: Transfer 107 2.12.5: Blocking / Antibodies 107 2.12.6: Antibody detection 108 2.13: Dual-Luciferase Reporter Assay (DLR) 108 2.14: Apoptosis/Nexin® Assay 110 2.14.1: Spent media and sodium butyrate (NaBu) treatment 112 2.15: EPO Assay and Quantification 113 2.16: p27 Assay and Quantification 114 2.17: Other Essential Techniques of Cell Culture 114 2.17.1: Single Cell Cloning via Serial Dilution 114 2.17.2: Cell Sorting / FACS 116 2.18: IgG ELISA Quantitation Set 117 2.19: MicroRNA-mRNA moiety capture using biotinylated oligos 118 2.19.1: Biotinylated DNA Oligo Design 118 2.19.2: miR-Capture full protocol 119 2.20: Bioinformatics and Regulatory Web-based software 122 Section 3.0: Results – Project 1 124 3.1: Identification, Isolation and Verification of CHO gene Promoters 125 3.1.1: Introduction 125 3.1.2: Identification of Temperature-sensitive genes in ‘Omics dataset 127 3.1.2.1: Choosing target genes for study 127 3.1.2.2: Identification of constitutive genes 129 3.1.3: Target gene summary 133 3.1.4: Real Time-qPCR validation of target genes 135 3.1.5: Investigation of Promoter activity versus Transcript stability 144 3.1.6: In silico identification of promoter sequences 150 3.1.7: Workflow of Isolation and Cloning of Gene Promoters 151 3.1.8: Primer Design 153 3.1.9: Promoter amplification and isolation 155 3.1.10: Cloning into Expression Vectors Design/Manipulation 157 3.1.11: Promoter fragments - Initial expression screen 162 3.2: Driving reporter-gene expression in a temperature-dependant manner 165 3.2.1: Introduction 166 3.2.3: Luciferase reporter expression results 177 3.2.4: CMV and SV40: Poor viral controls 181 3.2.5: Driving expression of a bioprocess product (EPO) 186 3.2.6: Driving expression of an cellular engineering transgene (p27) 190 3.2.6.1: Temp responsive p27 expression in ‘attached’ culture 191 3.2.6.2: Temp responsive p27 expression in ‘suspension’ culture 198 3.2.6.3: p27 effect on SEAP activity in CHO-K1-SEAP 205 3.2.7: Promoter – Ranking to GOI 208 vii 3.3: Bioinformatics / In-silico analysis of promoter sequences 210 3.3.1: Introduction
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