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Hormonal regulation and promoter analysis of the follicle-stimulating hormone b-subunit gene (FSHb)of goldfish, carassius auratus.

Ko Nga Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 98-131). / Abstracts in English and Chinese. / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.v / Acknowledgements --- p.vii / Table of Contents --- p.ix / List of Figures --- p.xiv / List of Tables --- p.xvii / Symbols and Abbreviations --- p.xviii / Scientific Names --- p.xxi / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Gonadotropins --- p.1 / Chapter 1.1.1 --- Structure --- p.1 / Chapter 1.1.2 --- Function --- p.3 / Chapter 1.1.3 --- Regulation --- p.5 / Chapter 1.1.3.1 --- Hypothalamic regulators (GnRH) --- p.5 / Chapter 1.1.3.2 --- Endocrine regulators from gonads (steroids) --- p.7 / Chapter 1.1.3.3 --- Paracrine regulators (activin) --- p.9 / Chapter 1.1.4 --- Promoter analysis --- p.9 / Chapter 1.2 --- Activin Family of Growth Factors --- p.12 / Chapter 1.2.1 --- Activin --- p.12 / Chapter 1.2.1.1 --- Structure --- p.12 / Chapter 1.2.1.2 --- Function --- p.13 / Chapter 1.2.1.3 --- Signaling --- p.15 / Chapter 1.2.2 --- Follistatin --- p.16 / Chapter 1.2.2.1 --- Structure --- p.16 / Chapter 1.2.2.2 --- Function --- p.17 / Chapter 1.3 --- Objectives --- p.18 / Chapter Chapter 2 --- Establishment and Characterization of Stable LβT2 Cell Lines Containing and Expressing SEAP Driven by the Goldfish FSHβ Promoter / Chapter 2.1 --- Introduction --- p.29 / Chapter 2.2 --- Materials and Methods --- p.31 / Chapter 2.2.1 --- Construction of expression plasmid --- p.31 / Chapter 2.2.2 --- Cell culture --- p.32 / Chapter 2.2.3 --- Cotransfection of LβT2 cells --- p.32 / Chapter 2.2.4 --- G418 selection of transfected LpT2 cells --- p.33 / Chapter 2.2.5 --- SEAP reporter gene assay --- p.33 / Chapter 2.2.6 --- Cloning of pSEAP/gfFSHβ promoter and pBK- CMV-transfected LβT2 cells by limited dilution --- p.34 / Chapter 2.2.7 --- Extraction of genomic DNA --- p.34 / Chapter 2.2.8 --- Isolation of total RNA --- p.35 / Chapter 2.2.9 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Optimization of G418 concentration for selection --- p.36 / Chapter 2.3.2 --- Expression of SEAP reporter gene by pSEAP/gfFSHβ promoter and pBK-CMV-transfected LβT2 cells --- p.37 / Chapter 2.3.3 --- Establishment of LβT2 cell lines that contain a functional gfFSHp promoter --- p.37 / Chapter 2.3.4 --- Characterization of LβT2#23 that contains a functional gfFSHβ promoter --- p.38 / Chapter 2.4 --- Discussion --- p.39 / Chapter Chapter 3 --- Hormonal Regulation of Goldfish Follicle-Stimulating Hormone β (FSHβ) Promoter Activity in LpT2#23 Cells / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Materials and Methods --- p.54 / Chapter 3.2.1 --- Cell culture --- p.55 / Chapter 3.2.2 --- Drug treatment --- p.56 / Chapter 3.2.3 --- SEAP reporter gene assay --- p.56 / Chapter 3.2.4 --- Isolation of total RNA --- p.57 / Chapter 3.2.5 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.57 / Chapter 3.2.6 --- Data analysis --- p.58 / Chapter 3.3 --- Results --- p.59 / Chapter 3.3.1 --- Effects of goldfish activin on FSHβ promoter --- p.59 / Chapter 3.3.2 --- Blockade of activin effects by follistatin --- p.59 / Chapter 3.3.3 --- Effects of different hormones and steroids on FSHβ promoter --- p.60 / Chapter 3.4 --- Discussion --- p.61 / Chapter Chapter 4 --- Promoter Analysis for the Activin Responsive Element (ARE) in the Goldfish Follicle-Stimulating Hormone β (FSHβ) Gene / Chapter 4.1 --- Introduction --- p.71 / Chapter 4.2 --- Materials and Methods --- p.74 / Chapter 4.2.1 --- Generation of SEAP reporter plasmids containing the gfFSHβ promoter of different lengths --- p.74 / Chapter 4.2.2 --- PCR screening and restriction analysis --- p.75 / Chapter 4.2.3 --- Midiprep --- p.76 / Chapter 4.2.4 --- Cell culture --- p.77 / Chapter 4.2.5 --- Transfection of the pSEAP/gfFSHβ promoter constructs into LβT2 cells --- p.77 / Chapter 4.2.6 --- Activin treatment --- p.77 / Chapter 4.2.7 --- SEAP assay --- p.78 / Chapter 4.3 --- Results --- p.78 / Chapter 4.3.1 --- Subcloning of the gfFSHβ promoter of decreasing length into SEAP reporter vector --- p.78 / Chapter 4.3.2 --- Activin stimulation of the pSEAP/gfFSHβ promoter constucts in LβT2 cells --- p.79 / Chapter 4.4 --- Discussion --- p.80 / Chapter Chapter 5 --- General Discussion / Chapter 5.1 --- Overview --- p.92 / Chapter 5.2 --- Contribution of the present research --- p.95 / Chapter 5.2.1 --- Establishment of stable LβT2 cell lines containing and expressing SEAP driven by gfFSHβ promoter --- p.95 / Chapter 5.2.2 --- Hormonal regulation of the gfFSHβ promoterin LβT2#23 cells --- p.95 / Chapter 5.2.3 --- Identification of the activin responsive element (ARE) on the gfFSHβ promoter --- p.96 / Chapter 5.3 --- Future research direction --- p.96 / References --- p.98

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324010
Date January 2002
ContributorsKo, Nga Ling., Chinese University of Hong Kong Graduate School. Division of Biology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xxi, 131 leaves : ill. ; 30 cm.
RightsUse 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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