by Liang Yiu-hon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 104-117). / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / ABBREVIATIONS --- p.iii / AMINO ACIDS SHORTHAND --- p.v / TABLE OF CONTENTS --- p.vi-x / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- The Discovery of IGFs --- p.1 / Chapter 1.3 --- The Growth Promoting Actions of IGFs --- p.3 / Chapter 1.4 --- Molecular Biology of IGFs in Mammals --- p.6 / Chapter 1.4.1 --- IGF Genes and Transcripts --- p.6 / Chapter 1.4.2 --- Regulation of IGF Gene Expression --- p.8 / Chapter 1.5 --- IGF Binding Proteins --- p.11 / Chapter 1.5.1 --- Regulation of IGF Action by IGF Binding Proteins --- p.11 / Chapter 1.6 --- The Insulin and IGF Receptors --- p.13 / Chapter 1.6.1 --- IGF-I Receptor --- p.13 / Chapter 1.6.2 --- IGF-II Receptor --- p.13 / Chapter 1.6.3 --- Insulin/IGF-I Hybrid Receptor --- p.15 / Chapter 1.7 --- IGF in Mammalian Fetal Growth --- p.17 / Chapter 1.8 --- The Role of IGFs in Fish --- p.19 / Chapter 1.9 --- Aims of the Present Study --- p.26 / Chapter CHAPTER 2 --- GENERAL METHODOLOGY / Chapter 2.1 --- Materials --- p.27 / Chapter 2.2 --- Methods --- p.32 / Chapter 2.2.1 --- Gene Clean --- p.32 / Chapter 2.2.1A --- Gene Clean by Glassmilk Method --- p.32 / Chapter 2.2.1B --- Gene Clean by Sephaglas´ёØ BandPrep Kit --- p.32 / Chapter 2.2.2 --- Preparation of Radioactive Nucleic Acid Probes --- p.33 / Chapter 2.2.3 --- Sephadex G-50 Spun-column Chromatography --- p.33 / Chapter 2.2.4 --- Small Scale Alkali Preparation of Plasmid DNA --- p.34 / Chapter 2.2.5 --- Large Scale Preparation of Plasmid DNA 36 - using Wizard Maxiprep Kit (Promega) / Chapter 2.2.6 --- DNA Sequencing using T7 DNA Polymerase Sequencing Kit (Pharmacia) --- p.37 / Chapter 2.2.7 --- Restriction Enzyme Digestion --- p.38 / Chapter 2.2.8 --- Agarose Gel Electrophoresis --- p.39 / Chapter 2.2.9 --- Dephosphorylation of Linearized Plasmid DNA --- p.39 / Chapter 2.2.10 --- Ligation of Foreign DNA with Linearized Plasmid --- p.40 / Chapter 2.2.11 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.40 / Chapter 2.2.12 --- Blotting : Transfer of DNA to Nylon Membrane --- p.41 / Chapter 2.2.12A --- Capillary Transfer of DNA to Nylon Membrane in 10X SSC --- p.41 / Chapter 2.2.12B --- Capillary Transfer of DNA to Nylon Membrane under Alkaline Condition --- p.42 / Chapter CHAPTER 3 --- SCREENING OF COMMON CARP LIVER cDNA LIBRARY / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.45 / Chapter 3.2.1 --- Materials --- p.45 / Chapter 3.2.2 --- Methods --- p.48 / Chapter 3.2.2.1 --- Preparation of the Plating Host --- p.48 / Chapter 3.2.2.2 --- Phage Titering --- p.48 / Chapter 3.2.2.3 --- Primary Screening of Common Carp Liver cDNA Library --- p.48 / Chapter 3.2.2.4 --- Purification of the Positive Clone --- p.49 / Chapter 3.2.2.5 --- Checking the Insert Size of the Positive Clone --- p.50 / Chapter 2.2.2.6 --- In vivo Excision to Release Phagemid from the Phage vector --- p.51 / Chapter 3.2.2.7 --- Plasmid Minipreparation of the Positive Clone --- p.52 / Chapter 3.2.2.8 --- Restriction Enzyme Digestion to Release the Insert --- p.52 / Chapter 3.2.2.9 --- Large Scale Plasmid Preparation of the Positive Clone --- p.53 / Chapter 3.2.2.10 --- DNA Sequencing of the Positive Clone --- p.53 / Chapter 3.2.2.11 --- Restriction Mapping of the Positive Clone --- p.53 / Chapter 3.2.2.12 --- Subcloning of the Positive Clone into Plasmid Vectors --- p.53 / Chapter 3.2.2.13 --- DNA Sequencing of the Subclones --- p.54 / Chapter 3.3 --- Results and Discussion --- p.55 / Chapter CHAPTER 4 --- RNA ASSAY USING REVERSE TRANSCRIPTION- POLYMERASE CHAIN REACTION / Chapter 4.1 --- Introduction --- p.70 / Chapter 4.2 --- Materials and Methods --- p.71 / Chapter 4.2.1 --- Materials --- p.71 / Chapter 4.2.2 --- Methods --- p.72 / Chapter 4.2.2.1 --- Tissue Preparation --- p.72 / Chapter 4.2.2.2 --- Total RNA Extraction --- p.72 / Chapter 4.2.2.3 --- Electrophoresis of RNA in Agarose Gel Containing Formaldehyde --- p.73 / Chapter 4.2.2.4 --- First Strand cDNA Synthesis --- p.74 / Chapter 4.2.2.5 --- IGF-I Specific PCR --- p.75 / Chapter 4.2.2.6 --- Preparation of Carp IGF Conserve Region --- p.75 / Chapter 4.2.2.7 --- Southern Hybridization of PCR Products --- p.76 / Chapter 4.3 --- Results and Discussion --- p.76 / Chapter CHAPTER 5 --- GENOMIC SOUTHERN ANALYSIS / Chapter 5.1 --- Introduction --- p.80 / Chapter 5.2 --- Materials and Methods --- p.81 / Chapter 5.2.1 --- Materials --- p.81 / Chapter 5.2.2 --- Methods --- p.82 / Chapter 5.2.2.1 --- Preparation of Genomic DNA from Carp Testis --- p.82 / Chapter 5.2.2.2 --- Restriction Enzyme Digestion of Genomic DNA --- p.82 / Chapter 5.2.2.3 --- Southern Blotting of the Digested Genomic DNA --- p.83 / Chapter 5.2.2.4 --- Preparation of the Carp IGF-I Specific Probe --- p.83 / Chapter 5.2.2.5 --- Genomic Southern Hybridization --- p.83 / Chapter 5.3 --- Results and Discussion --- p.84 / Chapter CHAPTER 6 --- THE SEARCH OF OTHER IGF cDNA SUBTYPES IN COMMON CARP / Chapter 6.1 --- Introduction --- p.88 / Chapter 6.2 --- Materials and Methods --- p.89 / Chapter 6.2.1 --- Materials --- p.89 / Chapter 6.2.2 --- Methods --- p.91 / Chapter 6.2.2.1 --- Screening using a Conserve Region cDNA Probe of Carp IGF-I --- p.91 / Chapter 6.2.2.2 --- PCR using IGF-I Specific Primers --- p.92 / Chapter 6.2.2.3 --- PCR Using T3 and T7 Primers --- p.92 / Chapter 6.2.2.4 --- Southern Blot Analysis of T3 and T7 PCR Products of cDNA Insert --- p.93 / Chapter 6.2.2.5 --- DNA Sequencing of Positive Clones --- p.94 / Chapter 6.3 --- Results and Discussion --- p.94 / Chapter CHAPTER 7 --- GENERAL DISCUSSION AND CONCLUSION --- p.99 / REFERENCES --- p.104-117
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_320664 |
| Date | January 1995 |
| Contributors | Liang, Yiu-hon., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Publisher | Chinese University of Hong Kong |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, x, 117 leaves : ill. (some mounted) ; 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/) |
Page generated in 0.0025 seconds