by Ng Wing Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 156-174). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.ix / List of Figures --- p.xiii / List of Tables --- p.xvi / List of Graphs --- p.xvii / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.3 / Chapter 2.1 --- Human granulocyte colony-stimulating factor (hG-CSF) --- p.3 / Chapter 2.1.1 --- Historical background --- p.3 / Chapter 2.1.2 --- Physiological Roles --- p.5 / Chapter 2.1.3 --- Molecular properties --- p.8 / Chapter 2.1.4 --- Biochemical properties --- p.9 / Chapter 2.1.5 --- Comparison to G-CSF of other species --- p.11 / Chapter 2.1.6 --- Biological Activities --- p.12 / Chapter 2.1.7 --- Clinical Applications --- p.14 / Chapter 2.1.7.1 --- Clinical use in myelosuppressive chemotherapy and neutropenic fever --- p.14 / Chapter 2.1.7.2 --- Clinical use in bone marrow transplantation (BMT) and peripheral blood progenitor cell (PBPC) transplantation --- p.14 / Chapter 2.1.7.3 --- Clinical use in HIV infection --- p.16 / Chapter 2.1.7.4 --- Clinical use in diabetes mellitus --- p.17 / Chapter 2.1.7.5 --- Clinical use in severe chronic neutropenia --- p.18 / Chapter 2.1.7.6 --- Future prospects --- p.18 / Chapter 2.1.7.7 --- Dosages and adverse effects --- p.19 / Chapter 2.1.8 --- Economic value --- p.20 / Chapter 2.2 --- Plant as bioractor --- p.20 / Chapter 2.2.1 --- Medical molecular farming --- p.20 / Chapter 2.2.2 --- Commercial biopharmaceutical proteins --- p.25 / Chapter 2.2.3 --- Transgenic plants producing hematopoietic growth factors --- p.25 / Chapter 2.2.3.1 --- Granulocyte-macrophage colony-stimulating factor (GM-CSF) --- p.26 / Chapter 2.2.3.2 --- Interleukin-2 (IL-2) --- p.28 / Chapter 2.3 --- Rice as expression system --- p.29 / Chapter 2.3.1 --- Characteristics --- p.29 / Chapter 2.3.2 --- Advantages of using rice as bioreactor --- p.30 / Chapter 2.3.3 --- Previous studies --- p.31 / Chapter 2.3.4 --- Transformation method --- p.33 / Chapter 2.3.5 --- Super-binary vector --- p.34 / Chapter 2.4 --- Strategies for enhancing protein expression level --- p.36 / Chapter 2.4.1 --- Vacuolar targeting --- p.36 / Chapter 2.4.1.1 --- Protein targeting signals --- p.38 / Chapter 2.4.1.2 --- Binding protein of 80kDa (BP-80) --- p.39 / Chapter 2.4.1.3 --- a-Tonoplast intrinsic protein (α-TIP) --- p.39 / Chapter 2.4.1.4 --- Receptor homology region-transmembrane domain-Ring H2 motif (RMR) --- p.40 / Chapter 2.4.2 --- Fusion with glutelin in rice --- p.41 / Chapter 2.5 --- Hypotheses and aims of this study --- p.43 / Chapter Chapter 3 --- Materials and Methods --- p.45 / Chapter 3.1 --- Introduction --- p.45 / Chapter 3.2 --- Chemicals --- p.45 / Chapter 3.3 --- Bacterial strains --- p.46 / Chapter 3.4 --- Chimeric genes construction --- p.46 / Chapter 3.4.1 --- Protein targeting constructs --- p.51 / Chapter 3.4.2 --- Enterokinase site constructs --- p.60 / Chapter 3.4.3 --- Glutein signal peptide constructs --- p.65 / Chapter 3.4.4 --- Glutelin fusion constructs --- p.70 / Chapter 3.4.5 --- Sequence fidelity of chimeric genes --- p.77 / Chapter 3.4.6 --- Cloning of chimeric genes into rice super-binary vector --- p.77 / Chapter 3.5 --- Rice transformation --- p.79 / Chapter 3.5.1 --- Plant materials --- p.79 / Chapter 3.5.2 --- Agrobacterium transformation --- p.79 / Chapter 3.5.3 --- A grobacterium-mediated transformation of rice --- p.79 / Chapter 3.6 --- Transgenic expression --- p.81 / Chapter 3.6.1 --- Extraction of leaf genomic DNA --- p.81 / Chapter 3.6.2 --- Synthesis of DIG-labeled double-stranded DNA probe --- p.82 / Chapter 3.6.3 --- Southern blot analysis --- p.83 / Chapter 3.6.4 --- Extraction of total RNA from immature rice seeds --- p.84 / Chapter 3.6.5 --- Northern blot analysis --- p.85 / Chapter 3.6.6 --- Protein extraction --- p.86 / Chapter 3.6.7 --- Tricine SDS-PAGE --- p.86 / Chapter 3.6.8 --- Western blot analysis --- p.87 / Chapter 3.6.9 --- Enterokinase digestion of EK fusion proteins --- p.88 / Chapter 3.7 --- Confocal immunoflorescence studies of rhG-CSF in rice grain --- p.89 / Chapter 3.7.1 --- Preparation of sample sections --- p.89 / Chapter 3.7.2 --- Double-labeling of fluorescence probes --- p.89 / Chapter 3.7.3 --- Image collection --- p.90 / Chapter 3.8 --- Functional analysis of rhG-CSF --- p.91 / Chapter 3.8.1 --- Culture of NFS-60 cells --- p.91 / Chapter 3.8.2 --- MTT cell proliferation assay --- p.92 / Chapter 3.9 --- Bacterial expression of anti-hG-CSF --- p.93 / Chapter 3.9.1 --- pET expression in E. coli --- p.93 / Chapter 3.9.2 --- Purification of His-hG-CSF --- p.97 / Chapter 3.9.3 --- Immunization of rabbits --- p.97 / Chapter Chapter 4 --- Results --- p.99 / Chapter 4.1 --- Construction of chimeric genes for rice transformation --- p.99 / Chapter 4.2 --- "Rice transformation, selection and regeneration" --- p.103 / Chapter 4.3 --- Southern blot analysis --- p.105 / Chapter 4.4 --- Northern blot analysis --- p.109 / Chapter 4.5 --- Western blot analysis --- p.114 / Chapter 4.6 --- Enterokinase digestion of EK fusion proteins --- p.125 / Chapter 4.7 --- Confocal immunofluorescence studies of rhG-CSF in transgenic rice grain --- p.128 / Chapter 4.8 --- Functional analysis of rhG-CSF --- p.132 / Chapter 4.9 --- Bacterial expression of anti-hG-CSF --- p.135 / Chapter 4.9.1 --- Expression and purification of recombinant His-hG-CSF in E. coli --- p.135 / Chapter 4.9.2 --- Titer and specificity of the anti-serum --- p.137 / Chapter Chapter 5 --- Discussion --- p.139 / Chapter 5.1 --- Introduction --- p.139 / Chapter 5.2 --- Fusion of hG-CSF with protein sorting determinants --- p.141 / Chapter 5.3 --- Fusion of hG-CSF with rice glutelin --- p.145 / Chapter 5.4 --- Glutelin signal peptide --- p.146 / Chapter 5.5 --- O-glycosylation --- p.148 / Chapter 5.6 --- Enterokinase digestion --- p.148 / Chapter 5.7 --- Expression level of rhG-CSF --- p.149 / Chapter 5.8 --- Functional analysis of rhG-CSF --- p.151 / Chapter 5.9 --- Future perspectives --- p.151 / Chapter Chapter 6 --- Conclusion --- p.155 / References --- p.156