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Mechanisms underlying the self-renewal characteristic and cardiac differentiation of mouse embryonic stem cells.

Ng, Sze Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 110-124). / Abstract also in Chinese. / Thesis Committee --- p.i / Acknowledgements --- p.ii / Contents --- p.iii / Abstract --- p.vii / 論文摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xiii / List of Tables --- p.xvii / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Embryonic Stem Cells (ESCs) --- p.1 / Chapter 1.1.1 --- What are ESCs and the characteristics of ESCs --- p.1 / Chapter 1.1.1.1 --- Pluripotent markers --- p.2 / Chapter 1.1.1.2 --- Germ layers' markers --- p.3 / Chapter 1.1.2 --- Mouse ESCs (mESCs) --- p.4 / Chapter 1.1.2.1 --- mESCs co-culture with mitotically inactivated mouse embryonic fibroblast (MEF) feeder layers --- p.4 / Chapter 1.1.2.2 --- Feeder free mESCs --- p.4 / Chapter 1.1.3 --- Promising uses of ESCs and their shortcomings --- p.5 / Chapter 1.1.4 --- Characteristics of ESC-derived cardiomyocytes (ESC-CMs) --- p.6 / Chapter 1.2 --- Cardiovascular diseases (CVD) --- p.7 / Chapter 1.2.1 --- Background --- p.7 / Chapter 1.2.2 --- Current treatments --- p.8 / Chapter 1.2.3 --- Potential uses of ESC-CMs for basic science research and therapeutic purposes --- p.9 / Chapter 1.2.4 --- Current hurdles in application of ESC-CMs for clinical uses --- p.10 / Chapter 1.3 --- Cardiac gene markers --- p.13 / Chapter 1.3.1 --- Atrial-specific --- p.13 / Chapter 1.3.2 --- Ventricular-specific --- p.19 / Chapter 1.4 --- Lentiviral vector-mediated gene transfer --- p.27 / Chapter 1.5 --- Cell cycle in ESCs --- p.29 / Chapter 1.5.1 --- Cell cycle --- p.29 / Chapter 1.5.2 --- Characteristics of cell cycle in ESCs --- p.30 / Chapter 1.6 --- Potassium (K+) channels --- p.31 / Chapter 1.6.1 --- Voltage gated potassium (Kv) channels --- p.32 / Chapter 1.6.2 --- Role of Kv channels in maintenance of membrane potential --- p.32 / Chapter 1.7 --- Objectives and significances --- p.33 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.35 / Chapter 2.1 --- Mouse embryonic fibroblast (MEF) culture --- p.35 / Chapter 2.1.1 --- Derivation of MEF --- p.3 5 / Chapter 2.1.2 --- MEF culture --- p.37 / Chapter 2.1.3 --- Irradiation of MEF --- p.37 / Chapter 2.2 --- mESC culture and their differentiation --- p.38 / Chapter 2.2.1 --- mESC culture --- p.38 / Chapter 2.2.2 --- Differentiation of mESCs --- p.39 / Chapter 2.3 --- Subcloning --- p.40 / Chapter 2.3.1 --- Amplification of Irx4 --- p.40 / Chapter 2.3.2 --- Purification of DNA products --- p.41 / Chapter 2.3.3 --- Restriction enzyme digestion --- p.42 / Chapter 2.3.4 --- Ligation of Irx4 with iDuet101A vector --- p.43 / Chapter 2.3.5 --- Transformation of ligation product into competent cells --- p.43 / Chapter 2.3.6 --- Small scale preparation of bacterial plasmid DNA --- p.44 / Chapter 2.3.7 --- Confirmation of positive clones by restriction enzyme digestion --- p.45 / Chapter 2.3.8 --- DNA sequencing of the cloned plasmid DNA --- p.45 / Chapter 2.3.9 --- Large scale preparation of target recombinant expression vector --- p.45 / Chapter 2.4 --- Lentiviral vector-mediated gene transfer to mESCs --- p.47 / Chapter 2.4.1 --- Lentivirus packaging --- p.47 / Chapter 2.4.2 --- Lentivirus titering --- p.48 / Chapter 2.4.3 --- Multiple transduction to mESCs --- p.48 / Chapter 2.4.4 --- Hygromycin selection on mESCs --- p.49 / Chapter 2.5 --- Selection of stable clone --- p.49 / Chapter 2.5.1 --- Monoclonal establishment and clone selection --- p.49 / Chapter 2.6 --- Differentiation of cell lines after selection --- p.50 / Chapter 2.7 --- Gene expression study on control and Irx4-overexpressed mESC lines --- p.50 / Chapter 2.8 --- Analysis of mESCs at different phases of the cell cycle --- p.55 / Chapter 2.8.1 --- Go/Gi and S phase synchronization --- p.55 / Chapter 2.8.2 --- Cell cycle analysis by propidium iodide (PI) staining followed by flow cytometric analysis --- p.55 / Chapter 2.8.3 --- Gene expression study by qPCR of Kv channel isoforms --- p.56 / Chapter 2.8.4 --- Membrane potential measurement by membrane potential-sensitive dye followed by flow cytometry --- p.57 / Chapter 2.9 --- Apoptotic study --- p.58 / Chapter 2.10 --- Determination of pluripotent characteristic of mESCs --- p.59 / Chapter 2.10.1 --- Expression of germ layers' markers by qPCR --- p.59 / Chapter 2.10.2 --- Differentiation by hanging drop method and suspension method --- p.61 / Chapter CHAPTER THREE --- RESULTS --- p.62 / Chapter 3.1 --- mESC culture --- p.62 / Chapter 3.1.1 --- Cell colony morphology of feeder free mESCs --- p.62 / Chapter 3.2 --- Subcloning --- p.63 / Chapter 3.2.1 --- PCR cloning of Irx4 --- p.63 / Chapter 3.2.2 --- Restriction digestion on iDuet101A --- p.64 / Chapter 3.2.3 --- Ligation of Irx4 to iDuet101A backbone --- p.66 / Chapter 3.2.4 --- Confirmation of successful ligation --- p.67 / Chapter 3.3 --- Lentivirus packaging --- p.68 / Chapter 3.3.1 --- Transfection --- p.68 / Chapter 3.4 --- Multiple transduction of mESCs and hygromycin selection of positively-transduced cells --- p.69 / Chapter 3.5 --- FACS --- p.70 / Chapter 3.6 --- Irx4 and iduet clone selection --- p.71 / Chapter 3.7 --- Characte rization of mESCs after clone selection --- p.74 / Chapter 3.7.1 --- Immunostaining of pluripotent and differentiation markers --- p.74 / Chapter 3.8 --- Differentiation of cell lines after selection --- p.77 / Chapter 3.8.1 --- Size of EBs of the cell lines during differentiation --- p.77 / Chapter 3.9 --- Gene expression study by qPCR --- p.79 / Chapter 3.10 --- Kv channel expression and membrane potential of mESCs at Go/Gi phase and S phases --- p.84 / Chapter 3.10.1 --- Expression of Kv channels subunits at G0/Gi phase and S phase --- p.86 / Chapter 3.10.2 --- Membrane potential at Go/Gi phase and S phase --- p.87 / Chapter 3.11 --- Effects of TEA+ on feeder free mESCs --- p.89 / Chapter 3.11.1 --- Apoptotic study --- p.89 / Chapter 3.11.2 --- Expression of germ layers´ة markers --- p.91 / Chapter 3.11.3 --- Embryo id bodies (EBs) measurement after differentiation --- p.92 / Chapter CHAPTER FOUR --- DISCUSSION --- p.95 / Chapter 4.1 --- Effect of overexpression of Irx4 on the cardiogenic potential of mESCs --- p.95 / Chapter 4.2 --- Role of Kv channels in maintaining the chacteristics of mESCs --- p.99 / Chapter 4.2.1 --- Inhibition of Kv channels led to a redistribution of the proportion of cells in different phases of the cell cycle: importance of Kv channels in cell cycle progression in native ESCs --- p.99 / Chapter 4.2.2 --- Inhibition of Kv channels led to a loss of pluripotency at molecular and functional levels: importance of Kv channels in the fate determination of mESCs --- p.102 / Chapter 4.3 --- Insights from the present investigation on the future uses of ESCs --- p.105 / Conclusions --- p.108 / References --- p.110

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326807
Date January 2009
ContributorsNg, Sze Ying., Chinese University of Hong Kong Graduate School. Division of Life Sciences.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xvii, 124 leaves : ill. (some col.) ; 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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