Name: Sim Siow Leng Degree: Master of Engineering (Chemical) Dept: Chemical and Biomolecular Engineering Thesis Title: Effect of Zinc on Insulin-free Hybridoma Culture Abstract The murine hybridoma CRL1606 was found to be dependent on insulin for proliferation in serum-free conditions A novel insulin-free medium, developed by zincsupplementation, could support cell growth and antibody production to the same extent as the original insulin-supplemented medium No adaptation into the new medium was necessary The effects of these media on the transcriptional response of the cells were investigated using a 7524-element microarray based on the Compugen basic mouse oligolibrary Analysis via pathway visualization suggested that zinc could have insulin-mimetic effects in insulin-free, zinc-supplemented CRL1606 culture via the up-regulation of PUR-1 and CD19 (which then imparts survival signals via the Pi3k-dependent pathway), as well as through the downregulation of the apoptotic adaptor FADD (that eventually inhibits apoptosis via the caspase cascade) Taken together, the microarray results suggest that Pi3k may be important in integrating zinc-induced signaling with the insulin-mimetic effects observed in zincsupplemented insulin-free CRL1606 cultures Keywords: Hybridoma, Insulin, Media, Microarray, Transcription, Zinc EFFECT OF ZINC ON INSULIN-FREE HYBRIDOMA CULTURE SIM SIOW LENG 2004 EFFECT OF ZINC ON INSULIN-FREE HYBRIDOMA CULTURE SIM SIOW LENG NATIONAL UNIVERSITY OF SINGAPORE 2004 EFFECT OF ZINC ON INSULIN-FREE HYBRIDOMA CULTURE SIM SIOW LENG (B Eng (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2004 TABLE OF CONTENTS ACKNOWLEDGEMENTS v SUMMARY vi NOMENCLATURE viii LIST OF FIGURES xiv LIST OF TABLES xvii 1 INTRODUCTION 1.1 MONOCLONAL ANTIBODY PRODUCTION 1.2 THESIS SCOPE 1.2.1 Effects of Zinc as an Insulin Substitute 1.2.2 Effects of Zinc and Insulin on Global Genetic Expression 1.2.3 Thesis Organization LITERATURE REVIEW 2.1 MEDIA DEVELOPMENT FOR HYBRIDOMA CELL CULTURE 2.2 INSULIN 2.2.1 Insulin in Cell Culture 2.2.1.1 Insulin in Serum-Free Hybridoma Culture 2.2.1.2 Insulin Degradation During Culture 10 2.2.2 Insulin-Mediated Signaling Pathways 11 2.2.3 The Structure of Insulin 12 2.3 ZINC 2.3.1 16 Major Functions of Zinc in Cell Culture 2.3.1.1 Growth 16 16 i 2.3.1.2 Transcription and Zinc Fingers 16 2.3.1.3 Cytoprotection 18 2.3.1.4 Suppression of Apoptosis 19 2.3.2 Cellular Zinc Sensors 21 2.3.3 Effective Zinc Dosage and Zinc Toxicity in Cell Culture 22 2.4 ZINC – INSULIN INTERACTIONS 2.4.1 Inclusion of Zinc in Insulin Preparations 25 2.4.1.1 Zinc Ions in Insulin Structure 26 2.4.1.2 Survey of Zinc Content in Insulin Preparations 27 2.4.2 Insulin-Mimetic Properties of Zinc 2.5 MICROARRAY 25 29 29 2.5.1 Introduction to Microarray Technology 30 2.5.2 Current Microarray Applications in Bioprocessing 32 MATERIALS AND METHODS 3.1 CELL CULTURE 34 34 3.1.1 Cell Line and Culturing Conditions 34 3.1.2 Experimental Setup and Culture Media 34 3.1.3 Analytical Methods 37 3.1.3.1 Cell Counts and Viability 37 3.1.3.2 Nutrient and Metabolite Concentrations 37 3.1.3.3 Zinc Ion Concentration 38 3.1.3.4 Monoclonal Antibody Concentration 38 3.2 MICROARRAY 39 3.2.1 Initial Preparation 41 3.2.1.2 Preparation of Probe DNA 42 ii 3.2.1.3 3.2.2 Array Design Printing Probe DNA 42 44 3.2.2.1 Microarray Printer 44 3.2.2.2 Printing Pins 45 3.2.2.3 Printing Procedure 46 3.2.2.4 DNA Immobilization on Slides 48 3.2.3 Experimental Setup and RNA Extraction 48 3.2.3.1 Sample Collection 49 3.2.3.2 RNA Extraction 50 3.2.4 Preparation of Target DNA 50 3.2.5 Hybridization 52 3.2.6 Signal Detection and Analysis 54 3.2.6.1 Signal Detection 54 3.2.6.2 Image Analysis 56 3.2.6.3 Data Normalization 58 3.2.7 Data Screening 60 3.2.8 Data Mining 64 3.2.8.1 Functional Classification 65 3.2.8.2 Pathway Analysis 66 RESULTS AND DISCUSSION 68 4.1 INSULIN DOSAGE RESPONSE 68 4.2 EFFECT OF INSULIN WITHDRAWAL 70 4.3 ZINC DOSAGE RESPONSE 75 4.4 COMPARATIVE ANALYSIS OF ‘ZINC’ VS ‘INSULIN’ 85 4.5 COMPARATIVE ANALYSIS OF ‘LOW ZINC’ VS ‘OPTIMAL ZINC’ 96 iii CONCLUSION 104 5.1 CONCLUSIONS 104 5.2 RECOMMENDATIONS FOR FUTURE WORK 104 REFERENCES 108 APPENDIXES 121 APPENDIX A: Cell Culture Inventory List 121 APPENDIX B: Microarray Inventory List 124 APPENDIX C: Saturation of Microarray Data 127 APPENDIX D: Variance of Microarray Data 130 APPENDIX E: Reproducibility of Microarray Data 133 APPENDIX F: Overview of Regulation Profiles (Pre-screened) 140 APPENDIX G: Secondary Expression Data (