Chan Chung Yiu Patrick. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 134-151). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Abbreviations --- p.xv / Chapter CHAPTER 1 --- Literature review --- p.1 / Chapter 1.1 --- Aquatic heavy metal contaminations --- p.1 / Chapter 1.1.1 --- Biology of heavy metals --- p.1 / Chapter 1.1.2 --- Mechanism of heavy metal toxicity --- p.2 / Chapter 1.2 --- Environmental monitoring of aquatic heavy metal contaminations --- p.4 / Chapter 1.2.1 --- Bioconcentration effects of heavy metals --- p.4 / Chapter 1.2.2. --- The concept of bioindicator in pollution assessment --- p.5 / Chapter 1.3 --- Metallothioneins --- p.7 / Chapter 1.3.1 --- Biological functions of MT and its metal inducibility --- p.7 / Chapter 1.4 --- Zebrafish (Daino reio) as an animal model --- p.11 / Chapter 1.4.1 --- Biology of zebrafish --- p.11 / Chapter 1.4.2 --- Current applications of transgenic zebrafish --- p.12 / Chapter 1.5 --- The use of cell culture systems in toxicology research --- p.13 / Chapter 1.6 --- Project aims --- p.16 / Chapter CHAPTER 2 --- Quantification of zMT mRNA levels using real-time PCR --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.1.1 --- The use of zebrafish embryos in toxicity assessment --- p.17 / Chapter 2.1.2 --- MT mRNA as a bioindicator of metal exposure --- p.18 / Chapter 2.1.3 --- Quantification of gene transcripts by RT-PCR --- p.19 / Chapter 2.1.4 --- Specific aims of this chapter --- p.27 / Chapter 2.2 --- Materials and methods --- p.28 / Chapter 2.2.1 --- Animal --- p.28 / Chapter 2.2.2 --- Cell culture --- p.31 / Chapter 2.2.3 --- General molecular biology techniques --- p.33 / Chapter 2.2.4 --- mRNA quantification by Real-time PCR --- p.35 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Heavy metal toxicity --- p.42 / Chapter 2.3.1.1 --- In vivo metal toxicity in zebrafish adult --- p.43 / Chapter 2.3.1.2 --- In vivo metal toxicity in zebrafish embryos at late epiboly --- p.44 / Chapter 2.3.1.3 --- In vivo metal toxicity in zebrafish eleutheroembryo --- p.46 / Chapter 2.3.1.4 --- In vitro metal toxicity on ZFL cell line --- p.48 / Chapter 2.3.2 --- Optimization of real-time PCR conditions --- p.50 / Chapter 2.3.2.1 --- Construction of relative standard curve --- p.50 / Chapter 2.3.2.2 --- Optimization of primer concentration --- p.52 / Chapter 2.3.2.3 --- Melting curve analysis for PCR specificity --- p.53 / Chapter 2.2.3 --- Quantification of zMT mRNA by Real-time PCR --- p.54 / Chapter 2.2.3.1 --- Relative zMT mRNA induction in zebrafish embryos at late epiboly --- p.54 / Chapter 2.2.3.2 --- Relative zMT mRNA induction in zebrafish eleutherombryos --- p.57 / Chapter 2.2.3.3 --- Relative zMT mRNA induction in SJD. 1 cell line --- p.58 / Chapter 2.2.2.4 --- In vitro zMT mRNA induction in ZFL cell line --- p.62 / Chapter 2.4 --- Discussions --- p.64 / Chapter 2.4.1 --- Change in metal sensitivity during zebrafish embryo development --- p.64 / Chapter 2.4.2 --- Developmental stage-specfic inducibility of zMT gene expression and metal toxicity --- p.65 / Chapter 2.4.3 --- In vitro zMT regulation by heavy metal ions --- p.67 / Chapter 2.4.4 --- The potential use of zMT expression as exposure biomarker --- p.69 / Chapter CHAPTER 3 --- Functional analysis of a cloned zMT-II gene promoter in zebrafish cell-lines: SJD.1 and ZFL --- p.71 / Chapter 3.1 --- Zebrafish MT gene promoter --- p.71 / Chapter 3.1.1 --- The structure of zMT promoter --- p.71 / Chapter 3.1.2 --- Functional analysis of cloned zMT-II promoter region in HepG2 cells --- p.72 / Chapter 3.1.3 --- Specific aims of this chapter --- p.77 / Chapter 3.2 --- Materials and methods --- p.78 / Chapter 3.2.1 --- General molecular biology techniques --- p.78 / Chapter 3.2.2 --- Cell culture --- p.79 / Chapter 3.2.3 --- Transient transfection assay --- p.81 / Chapter 3.3 --- Results --- p.84 / Chapter 3.3.1 --- Metal responsiveness of zMT-II promoter by transient transfection --- p.84 / Chapter 3.3.2 --- Deletion analysis --- p.88 / Chapter 3.3.2.1 --- Deletion analysis of zMT-II gene promoter in SJD. 1 cell line --- p.88 / Chapter 3.3.2.2 --- Deletion analysis of zMT-II gene promoter in ZFL cell line --- p.90 / Chapter 3.4 --- Discussions --- p.91 / Chapter 3.4.1 --- Structure of zMT-II gene promoter --- p.91 / Chapter 3.4.2 --- Metal responsiveness of zMT-II promoter --- p.94 / Chapter 3.4.3 --- Deletion analysis of zMT-IIgene promoter --- p.95 / Chapter CHAPTER 4 --- Transgenic zebrafish model for in vivo zMT gene regulation study --- p.97 / Chapter 4.1 --- Introduction --- p.97 / Chapter 4.1.1 --- Development of transgenic fish --- p.97 / Chapter 4.1.2 --- The principle of gene delivery --- p.98 / Chapter 4.1.3 --- The application of transgenic zebrafish model --- p.99 / Chapter 4.1.4 --- Specific aim of this chapter --- p.101 / Chapter 4.2 --- Materials and methods --- p.102 / Chapter 4.2.1 --- General molecular biology techniques --- p.102 / Chapter 4.2.2 --- Sequence of primers used --- p.103 / Chapter 4.2.3 --- Engineering of constructs for transgenic zebrafish study --- p.103 / Chapter 4.2.4 --- In vitro efficacy test of the GFP constructs --- p.105 / Chapter 4.2.5 --- Gene transfer into zebrafish embryos by electroporation --- p.107 / Chapter 4.2.6 --- Screening of transgenic candidates --- p.108 / Chapter 4.3 --- Results --- p.110 / Chapter 4.3.1 --- Engineering of DNA constructs for transgenic zebrafish production --- p.110 / Chapter 4.3.2 --- In vitro efficacy test of the DNA constructs --- p.111 / Chapter 4.3.3 --- Optimization of electroporation voltage --- p.117 / Chapter 4.4.4 --- Screening of transgenic candidates --- p.118 / Chapter 4.4 --- Discussion --- p.120 / Chapter 4.4.1 --- Potential application of the zMT promoter transgenic mode --- p.120 / Chapter 4.4.2 --- The use of GFP transgenic zebrafish model in developmental gene regulation study --- p.121 / Chapter 4.4.3 --- In vitro efficacy of the GFP constructs --- p.122 / Chapter 4.4.4 --- Transgene expression in zebrafish --- p.122 / Chapter CHAPTER 5 --- General discussion --- p.126 / REFERENCES --- p.134
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324971 |
| Date | January 2004 |
| Contributors | Chan, Chung Yiu Patrick., 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, xvi, 151 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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