Tse Chui-ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 130-146). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / 論文撮要 --- p.iii / List of Figures and Tables --- p.iv / Abbreviations --- p.vi / Table of contents --- p.vii / Chapter Chapter I --- Introduction --- p.1 / Chapter 1.1 --- Literature review --- p.1 / Chapter 1.1.1 --- An overview of IGFs --- p.3 / Chapter 1.1.2 --- Molecular biology of IGFs --- p.5 / Chapter 1.1.2.1 --- IGF-I and IGF-II genes and mRNAs --- p.5 / Chapter 1.1.2.2 --- Amino acid sequences of IGF-II --- p.8 / Chapter 1.1.2.3 --- Imprinting of IGF-II --- p.12 / Chapter 1.1.3 --- IGF distribution in tissues and body fluids --- p.14 / Chapter 1.1.3.1 --- IGF in serum --- p.14 / Chapter 1.1.3.2 --- IGF binding proteins --- p.16 / Chapter 1.1.4 --- IGF receptors --- p.19 / Chapter 1.1.4.1 --- Structures of the IGF receptors --- p.20 / Chapter 1.1.4.2 --- Ligand binding of the IGF receptors --- p.21 / Chapter 1.1.4.3 --- Signal transduction and biological response --- p.22 / Chapter 1.1.5 --- Biological effects of IGF --- p.24 / Chapter 1.1.6 --- Expression of recombinant IGF --- p.28 / Chapter 1.2 --- Rationale and Objective --- p.29 / Chapter Chapter II --- Methodology --- p.33 / Chapter 2.1 --- Design of degenerate primers --- p.33 / Chapter 2.2 --- Cloning --- p.35 / Chapter 2.2.1 --- DNA extraction from agarose gel --- p.35 / Chapter 2.2.2 --- Linearization and dephosphorylation of plasmid DNA --- p.35 / Chapter 2.2.3 --- Blunt-end ligation of amplicon with linearized plasmid --- p.36 / Chapter 2.2.4 --- T/A ligation of amplicon with linearized plasmid --- p.37 / Chapter 2.2.5 --- Sticky end ligation of foreign DNA with linearized plasmid --- p.37 / Chapter 2.2.6 --- Preparation of competent of E. coli stain DHI5α cells --- p.38 / Chapter 2.2.7 --- Transformation of plasmid vector into competent cells (heat-shock/ electroporation) --- p.39 / Chapter 2.2.8 --- "Spread single colony, PCR check clone and inoculation" --- p.40 / Chapter 2.2.9 --- Small scale alkali preparation of plasmid DNA --- p.41 / Chapter 2.2.10 --- Large scale preparation of plasmid DNA --- p.41 / Chapter 2.2.11 --- Nucleotide sequencing --- p.41 / Chapter 2.2.11.1 --- Manual sequencing --- p.41 / Chapter 2.2.11.2 --- PCR sequencing --- p.43 / Chapter 2.3 --- Northern blot --- p.45 / Chapter 2.4 --- Preparation of radio-labeled probe and hybridization of radio-labeled probe to nylon immobilized nucleic acid --- p.46 / Chapter 2.5 --- RACE --- p.48 / Chapter 2.5.1 --- Design of gene-specific primer --- p.51 / Chapter 2.5.2 --- First strand cDNA synthesis --- p.51 / Chapter 2.5.3 --- TdT tailing of cDNA --- p.52 / Chapter 2.6 --- Poly-A tract extraction --- p.53 / Chapter 2.7 --- Tissue distribution of mRNA --- p.53 / Chapter 2.7.1 --- Tissue preparation --- p.53 / Chapter 2.7.2 --- Total RNA extraction --- p.54 / Chapter 2.7.3 --- Formaldehyde agarose gel electrophoresis of RNA --- p.54 / Chapter 2.8 --- RNAse protection assay --- p.55 / Chapter 2.8.1 --- Antisense probe generation --- p.56 / Chapter 2.8.2 --- Preparation of the sample RNA --- p.58 / Chapter 2.8.3 --- Hybridization --- p.58 / Chapter 2.8.4 --- RNase digestion of hybridized probe and sample RNA --- p.59 / Chapter 2.8.5 --- Preparation of radioactive marker --- p.60 / Chapter 2.8.6 --- Separation and detection of protected fragments --- p.60 / Chapter 2.8.7 --- Data processing and statistical analysis --- p.61 / Chapter 2.9 --- Injection of GH --- p.62 / Chapter 2.10 --- Recombinant protein expression --- p.62 / Chapter 2.10.1 --- Plasmid construction --- p.62 / Chapter 2.10.2 --- Expression --- p.63 / Chapter 2.11 --- Resolution of proteins on SDS-PAGE --- p.63 / Chapter 2.12 --- Purification --- p.64 / Chapter 2.13 --- Western transfer --- p.64 / Chapter 2.14 --- Immunodetection --- p.65 / Chapter Chapter III --- Results & Discussion --- p.67 / Chapter 3.1 --- Isolation and characterization of IGF-II cDNA and its gene organization --- p.67 / Chapter 3.1.1 --- Introduction --- p.67 / Chapter 3.1.2 --- Results --- p.68 / Chapter 3.1.2.1 --- Generation of a fragment of the common carp IGF-II cDNA by PCR --- p.68 / Chapter 3.1.2.2 --- Isolation of the full length common carp IGF-II cDNA by RACE. --- p.69 / Chapter 3.1.2.3 --- Nucleotide sequence analysis --- p.74 / Chapter 3.1.2.4 --- Relationship of common carp IGF-II to common carp IGF-I and insulin --- p.78 / Chapter 3.1.2.5 --- Confirmation of the presence of IGF-II in common carp --- p.79 / Chapter 3.1.2.6 --- Multiple mRNA forms of common carp IGF-I and IGF-II --- p.80 / Chapter 3.1.2.7 --- Gene organization of the common carp IGF-II gene --- p.83 / Chapter 3.1.3 --- Discussion --- p.86 / Chapter 3.2 --- Tissue specific distribution of IGF-I and IGF-II mRNA and their hormonal regulation --- p.90 / Chapter 3.2.1 --- Introduction --- p.90 / Chapter 3.2.2 --- Results --- p.94 / Chapter 3.2.2.1 --- RNase protection assay measurement of tissue mRNA levels in juvenile and adult common carp --- p.94 / Chapter 3.2.2.2 --- Expression of IGF-II mRNA during larval development --- p.99 / Chapter 3.2.2.3 --- Effect of GH on IGF-I and IGF-II mRNA levels in brain and Hver of juvenile common carp --- p.102 / Chapter 3.2.3 --- Discussion --- p.106 / Chapter 3.3 --- Recombinant common carp IGF-II expressed in E. coli --- p.110 / Chapter 3.3.1 --- Introduction --- p.110 / Chapter 3.3.2 --- Results --- p.112 / Chapter 3.3.2.1 --- Product of recombinant common carp IGF-II --- p.112 / Chapter 3.3.2.2 --- Purification of common carp IGF-II --- p.115 / Chapter 3.3.2.3 --- Immunodetection --- p.117 / Chapter 3.3.3 --- Discussion --- p.118 / Chapter Chapter IV --- General conclusion --- p.120 / Appendix: Reagents --- p.124 / Reference list --- p.130
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_323472 |
| Date | January 2001 |
| Contributors | Tse, Chui-ling., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, x, 146 leaves : ill. ; 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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