Cheung Chun Kai. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 133-146). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Abstract --- p.iv / 摘要 --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xv / List of Figures --- p.xvi / List of Abbreviations --- p.xxi / Chapter Chapter 1 --- Overview --- p.1 / Chapter Chapter 2 --- Literature Review --- p.3 / Chapter 2.1 --- Historical background --- p.3 / Chapter 2.2 --- Insulin-like growth factor --- p.5 / Chapter 2.2.1 --- Structure and synthesis --- p.5 / Chapter 2.2.2 --- Physiologic role and biological actions --- p.6 / Chapter 2.3 --- Insulin-like growth factor binding protein-3 --- p.8 / Chapter 2.3.1 --- Structure and synthesis --- p.8 / Chapter 2.3.2 --- Physiologic role and biological actions --- p.8 / Chapter 2.4 --- Clinical aspects --- p.10 / Chapter 2.4.1 --- Metabolic effects of IGF-1 --- p.10 / Chapter 2.4.1.1 --- Similarities between IGF-I and insulin --- p.11 / Chapter 2.4.1.2 --- Differences between IGF-I and insulin --- p.13 / Chapter 2.4.2 --- Glucose and protein metabolism --- p.14 / Chapter 2.4.3 --- Therapeutic use of IGF-I --- p.15 / Chapter 2.4.3.1 --- Type 1 diabetes mellitus --- p.16 / Chapter 2.4.3.2 --- Type 2 diabetes mellitus --- p.17 / Chapter 2.4.4 --- Side effects --- p.19 / Chapter 2.5 --- World demands --- p.21 / Chapter 2.5.1 --- Significance of large-scale production --- p.21 / Chapter 2.5.2 --- IGF-I production --- p.21 / Chapter 2.6 --- Plants as bioreactors --- p.24 / Chapter 2.6.1 --- Medical molecular farming --- p.24 / Chapter 2.6.2 --- Advantages of plant bioreactor --- p.24 / Chapter 2.6.3 --- Commercial biopharmaceutical protein --- p.25 / Chapter 2.7 --- Tobacco expression system --- p.26 / Chapter 2.7.1 --- Tobacco model plant --- p.26 / Chapter 2.7.2 --- Transformation methods --- p.26 / Chapter 2.8 --- Hypotheses and aims of study --- p.28 / Chapter Chapter 3 --- Expression of Human IGF-I and IGFBP-3 in Transgenic Tobacco --- p.30 / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Materials and methods --- p.31 / Chapter 3.2.1 --- Chemicals --- p.31 / Chapter 3.2.2 --- Plant materials --- p.31 / Chapter 3.2.3 --- Bacterial strains --- p.32 / Chapter 3.2.4 --- Codon modification of IGF-I and IGFBP-3 cDNAs --- p.32 / Chapter 3.2.5 --- Transient assay to study IGF-I or IGFBP-3 translatability --- p.39 / Chapter 3.2.5.1 --- Construction of chimeric genes for particle bombardment --- p.39 / Chapter 3.2.5.2 --- Particle bombardment of GUS fusion constructs --- p.42 / Chapter 3.2.6 --- Construction of chimeric genes for tobacco transformation --- p.44 / Chapter 3.2.6.1 --- Construction of chimeric genes with different promoters --- p.44 / Chapter 3.2.6.1.1 --- Construction of chimeric gene with CaMV 35S promoter --- p.44 / Chapter 3.2.6.1.2 --- Construction of chimeric genes with phaseolin promoter --- p.46 / Chapter 3.2.6.2 --- Construction of fusion constructs --- p.48 / Chapter 3.2.6.2.1 --- Construction of GUS fusion constructs --- p.48 / Chapter 3.2.6.2.2 --- Construction of LRP fusion constructs --- p.51 / Chapter 3.2.6.3 --- Construction of phaseolin targeting constructs --- p.56 / Chapter 3.2.6.3.1 --- Construction of phaseolin targeting constructs without AFVY --- p.56 / Chapter 3.2.6.3.2 --- Construction of phaseolin targeting constructs with AFVY --- p.60 / Chapter 3.2.6.4 --- Cloning of chimeric genes into Agrobacterium binary vector pBI 121 --- p.64 / Chapter 3.2.7 --- Confirmation of sequencing fidelity of chimeric genes --- p.66 / Chapter 3.2.8 --- Transformation of Agrobacterium by electroporation --- p.66 / Chapter 3.2.9 --- Transformation of tobacco --- p.67 / Chapter 3.2.10 --- Selection and regeneration of transgenic tobacco --- p.67 / Chapter 3.2.11 --- GUS assay --- p.68 / Chapter 3.2.12 --- Extraction of leaf genomic DNA --- p.68 / Chapter 3.2.13 --- PCR of genomic DNA --- p.69 / Chapter 3.2.14 --- Synthesis of DIG-labeled double-stranded DNA probe --- p.69 / Chapter 3.2.15 --- Southern blot analysis --- p.70 / Chapter 3.2.16 --- Extraction of total RNA from leaves or developing seeds --- p.70 / Chapter 3.2.17 --- Northern blot analysis --- p.71 / Chapter 3.2.18 --- Extraction of total protein --- p.71 / Chapter 3.2.19 --- Tricine SDS-PAGE --- p.72 / Chapter 3.2.20 --- Western blot analysis --- p.72 / Chapter 3.2.21 --- Enterokinase digestion of fusion protein --- p.73 / Chapter Chapter 4 --- Results --- p.74 / Chapter 4.1 --- Particle bombardment for transient assay --- p.74 / Chapter 4.1.1 --- Construction of GUS fusion genes for particle bombardment --- p.74 / Chapter 4.1.2 --- Transient expression of GUS fusion genes in soybean cotyledons and tobacco leaves --- p.76 / Chapter 4.2 --- Construction of chimeric genes for tobacco transformation --- p.78 / Chapter 4.3 --- "Tobacco transformation, selection and regeneration" --- p.81 / Chapter 4.4 --- Detection of GUS activity --- p.83 / Chapter 4.5 --- Detection of transgene integration --- p.84 / Chapter 4.5.1 --- Extraction of genomic DNA and PCR --- p.84 / Chapter 4.5.2 --- Southern blot analysis --- p.88 / Chapter 4.6 --- Detection of transgene transcription --- p.92 / Chapter 4.6.1 --- Extraction of total RNA --- p.92 / Chapter 4.6.2 --- Northern blot analysis --- p.92 / Chapter 4.7 --- Detection of transgene translation --- p.99 / Chapter 4.7.1 --- Extraction of total protein and Tricine SDS-PAGE --- p.99 / Chapter 4.7.2 --- Western blot analysis --- p.102 / Chapter 4.7.3 --- Enterokinase digestion of fusion protein --- p.109 / Chapter Chapter 5 --- Discussion --- p.111 / Chapter 5.1 --- Codon modification of IGF-I and IGFBP-3 cDNAs --- p.114 / Chapter 5.2 --- Transient expression of IGF-I and IGFBP-3 cDNAs --- p.116 / Chapter 5.3 --- Fusion of IGF-I and IGFBP-3 cDNA with LRP gene --- p.118 / Chapter 5.4 --- Enterokinase digestion --- p.120 / Chapter 5.5 --- Phaseolin targeting signal --- p.122 / Chapter 5.6 --- Gene silencing --- p.124 / Chapter 5.7 --- Future perspectives --- p.128 / Chapter Chapter 6 --- Conclusion --- p.131 / References --- p.133
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324644 |
| Date | January 2004 |
| Contributors | Cheung, Chun Kai., Chinese University of Hong Kong Graduate School. Division of Medical Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xxiv, 146 leaves : ill. (some col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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