Generation of Highly Productive Polyclonal and Monoclonal Tobacco Suspension Lines from a Heterogeneous Transgenic BY-2 Culture Through Flow Cytometric Sorting
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Generation of highly productive polyclonal and monoclonal tobacco suspension lines from a heterogeneous transgenic BY-2 culture through flow cytometric sorting Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften vorgelegt von Diplom-Biologin Janina Kirchhoff aus Dortmund Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer Universitätsprofessor Dr. rer. nat. Dirk Prüfer Tag der mündlichen Prüfung : 30.04.2012 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. TABLE OF CONTENT Table of Content 1 Introduction ............................................................................................................... 1 1.1 Plant cells as production systems for recombinant proteins .......................................... 1 1.1.1 Advantages of plant cells ...................................................................................... 1 1.1.2 Plant-made pharmaceuticals from plant suspension cultures ............................... 3 1.2 Optimization of plant cell culture expression platforms .................................................. 5 1.3 Flow cytometric sorting and its application for plant cells .............................................. 7 1.4 Objective and experimental design of this work ........................................................... 10 2 Materials and Methods ............................................................................................ 13 2.1 Materials ...................................................................................................................... 13 2.1.1 Chemicals and consumables .............................................................................. 13 2.1.2 Equipment and accessories ................................................................................ 13 2.1.3 Reaction kits ........................................................................................................ 14 2.1.4 Antibodies and substrates ................................................................................... 14 2.1.5 Bacterial strains ................................................................................................... 15 2.1.6 Plant suspension cultures ................................................................................... 15 2.1.7 Plasmids .............................................................................................................. 16 2.1.8 Oligonucleotides .................................................................................................. 17 2.1.9 Antibiotics and herbicides ................................................................................... 18 2.1.10 Solutions, media and buffers ............................................................................... 18 2.2 Methods ....................................................................................................................... 18 2.2.1 Cultivation of Agrobacterium tumefaciens ........................................................... 18 2.2.2 Growth and induction of A. tumefaciens for plant cell transformation ................. 19 2.2.3 Cultivation of plant suspension cultures .............................................................. 19 2.2.3.1 Determination of growth parameters ......................................................... 19 2.2.3.2 Fermentation ............................................................................................. 20 2.2.4 Stable transformation of tobacco BY-2 suspension cells .................................... 20 2.2.5 Preparation of protoplasts from BY-2 suspension cultures ................................. 21 2.2.6 Fluorescence staining of BY-2 protoplasts .......................................................... 22 2.2.6.1 Viability staining ......................................................................................... 22 2.2.6.2 Cell wall staining ........................................................................................ 23 2.2.7 Flow cytometric analysis and cell sorting ............................................................ 23 2.2.7.1 Flow cytometric analysis ............................................................................ 23 2.2.7.2 Fluorescence activated cell sorting (FACS) .............................................. 24 2.2.8 Regeneration of FACS selected protoplasts ....................................................... 24 2.2.8.1 Regeneration of sorted protoplasts at low density ..................................... 24 2.2.8.2 Regeneration of single sorted protoplasts (monoclonal cultures) .............. 25 TABLE OF CONTENT 2.2.9 Nomenclature of FACS generated suspension cultures ..................................... 25 2.3 Proteinchemical and immunological methods .............................................................. 26 2.3.1 Extraction of total soluble proteins from BY-2 suspension cultures .................... 26 2.3.2 Discontinuous SDS polyacrylamide gelelectrophoresis (SDS-PAGE) ................ 27 2.3.3 Immunoblot analysis ........................................................................................... 27 2.3.4 Enzyme linked immuno sorbent assay (ELISA) .................................................. 28 2.4 Recombinant DNA methods ........................................................................................ 28 2.4.1 Extraction of genomic DNA from plant material .................................................. 28 2.4.2 Quantitative real time PCR .................................................................................. 28 3 Results .................................................................................................................... 30 3.1 Preparation of tobacco BY-2 protoplasts and generation of suspension cultures ........ 30 3.1.1 Preparation of protoplasts from BY-2 suspension cultures ................................. 30 3.1.2 Optimization of the protoplast regeneration medium and conditions .................. 31 3.1.3 Monitoring of cell wall synthesis of regenerating BY-2 protoplasts ..................... 34 3.1.4 Regeneration of flow cytometrically sorted protoplasts ....................................... 35 3.1.5 Establishment of a feeder cell-based strategy for monoclonal cultures .............. 36 3.2 Proof of feeder cell removal during the regeneration of sorted protoplasts ................. 37 3.3 FACS-based optimization of existing transgenic plant cell lines .................................. 39 3.3.1 Enrichment of highly productive cells through sorting of protoplast pools ........... 39 3.3.2 Growth characteristics of polyclonal sorted cultures ........................................... 42 3.3.3 Recombinant protein production stability in FACS improved suspension cultures ................................................................................................................ 44 3.3.4 Generation of monoclonal cell lines from a heterogeneous transgenic suspension culture .............................................................................................. 45 3.3.5 Growth performance of monoclonal cell cultures ................................................ 49 3.3.6 Stability of recombinant protein Production in monoclonal suspension cultures . 51 3.4 Determination of transgene copy numbers in FACS generated cultures ..................... 52 3.5 Up-scaling and optimization of recombinant protein production of monoclonal cultures ............................................................................................................... 54 3.5.1 Fermentation and recombinant protein production in a monoclonal cell line ...... 54 3.5.2 Cultivation of monoclonal cell lines in nitrate-enriched medium .......................... 56 3.6 Development of a transformation procedure for rapid flow cytometric analysis and sorting of elite transgenic events ............................................................................ 57 3.6.1 Generation of high producing suspension cultures from liquid regenerated transgenic events (accelerated transformation strategy) .................................... 57 3.6.2 Comparative analysis of conventional and accelerated transformation procedures for transgenic cell line generation .................................................... 59 TABLE OF CONTENT 3.6.3 Timelines for FACS-based generation of high recombinant protein producing cell lines .............................................................................................................. 62 4 Discussion ............................................................................................................... 64 4.1 Preparation of tobacco BY-2 protoplasts for flow cytometric sorting and its regeneration ............................................................................................................ 64 4.2 Application of flow cytometric sorting for the establishment of highly productive BY-2 suspension cultures ................................................................................................ 68 4.3 Characterization of FACS-derived polyclonal and monoclonal cultures ...................... 72 4.4 Application of monoclonal BY-2 cultures for the production of M12 antibody .............. 76 4.5 Flow sorting for the selection of highly productive BY-2 cultures early after transformation ........................................................................................................