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Role of mouse PinX1 in maintaining the characteristics of mouse embryonic stem cells.

Lau, Yuen Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 156-163). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要) --- p.iii / Acknowledgements --- p.iv / Table of content --- p.V / List of figures --- p.ix / List of tables --- p.xiii / List of abbreviations --- p.xiv / Chapter 1 --- INTRODUCTION --- p.Page / Chapter 1.1 --- Embryonic stem cells (ESCs) --- p.1 / Chapter 1.1.1 --- What are ESCs and the characteristics of ESCs --- p.1 / Chapter 1.1.2 --- Promising use of ESCs in drug development and regenerative medicine --- p.1 / Chapter 1.1.3 --- Maintenance of self-renewal and pluripotent properties of ESCs --- p.3 / Chapter 1.2 --- Cell cycle in ESCs --- p.5 / Chapter 1.2.1 --- Cell cycle --- p.5 / Chapter 1.2.2 --- Characteristics of cell cycle of ESCs --- p.6 / Chapter 1.3 --- Telomere --- p.8 / Chapter 1.3.1 --- Telomere structure and the telomeric proteins --- p.8 / Chapter 1.3.2 --- End replication problem --- p.10 / Chapter 1.3.3 --- Telomere dysfunction in cancer and cellular aging --- p.11 / Chapter 1.4 --- Telomerase --- p.12 / Chapter 1.4.1 --- Telomerase and stem cell characteristics --- p.13 / Chapter 1.4.1.1 --- Telomerase and cell proliferation --- p.13 / Chapter 1.4.1.2 --- Telomerase and stem cell differentiation --- p.14 / Chapter 1.4.2 --- Regulation of telomerase expression/ activity --- p.15 / Chapter 1.4.2.1 --- Regulation of telomerase at different levels --- p.15 / Chapter 1.4.2.2 --- Regulation of telomerase activity by cellular components in ESCs --- p.16 / Chapter 1.5 --- PinXl --- p.18 / Chapter 1.5.1 --- Expression of PinXl --- p.18 / Chapter 1.5.2 --- Effects of PinXl on the activities and the sub-cellular localization of telomerase --- p.19 / Chapter 1.5.3 --- Structure-function relationship of PinXl --- p.19 / Chapter 1.5.4 --- Effect of PinXl on the growth rate of normal and cancer cells --- p.21 / Chapter 1.5.5 --- Other functions of PinX 1 V --- p.22 / Chapter 1.5.6 --- Mouse homolog of PinXl and its function in mESCs --- p.23 / Chapter 1.6 --- Aims of this study --- p.24 / Chapter 2 --- METERIALS AND METHODS --- p.Page / Chapter 2.1 --- mESC culture and differentiation --- p.25 / Chapter 2.1.1 --- Cell line --- p.25 / Chapter 2.1.2 --- Irradiation of MEF --- p.25 / Chapter 2.1.3 --- mESC culture --- p.26 / Chapter 2.1.4 --- Differentiation of mESCs --- p.26 / Chapter 2.1.5 --- Establishment and' culture of feeder-free mESCs --- p.28 / Chapter 2.1.6 --- Culture of feeder-free mESCs --- p.28 / Chapter 2.2 --- Trypan Blue Exclusion Assay --- p.29 / Chapter 2.3 --- Sub-cloning --- p.29 / Chapter 2.3.1 --- Amplification of the insert gene by PCR --- p.29 / Chapter 2.3.2 --- Purification of PCR products --- p.31 / Chapter 2.3.3 --- Restriction enzyme digestion --- p.32 / Chapter 2.3.4 --- Ligation of digested insert and vector --- p.33 / Chapter 2.3.5 --- Transformation of ligation product into competent cells --- p.34 / Chapter 2.3.6 --- Confirmation of positive clone by colony PCR --- p.34 / Chapter 2.3.7 --- Small scale preparation of the recombinant plasmid DNA --- p.35 / Chapter 2.3.8 --- Confirmation of positive clone by restriction digestion --- p.36 / Chapter 2.3.9 --- DNA sequencing of the recombinant plasmid DNA --- p.36 / Chapter 2.3.10 --- Large scale preparation of the recombinant plasmid DNA --- p.37 / Chapter 2.4 --- Design of siRNA targeting mPinXl and mPinXlt --- p.38 / Chapter 2.5 --- Transient transfection --- p.38 / Chapter 2.6 --- Cloning of siRNA into shRNA insert in Lentiviral Vector pLVTHM --- p.39 / Chapter 2.7 --- Lentiviral vector-mediated gene transfer to mESCs --- p.42 / Chapter 2.7.1 --- Lentivirus packaging --- p.42 / Chapter 2.7.2 --- Checking of successful transduction by lentivirus in HEK cells --- p.43 / Chapter 2.7.3 --- Multiple transductions to mESCs --- p.43 / Chapter 2.7.4 --- Selection of positive clones --- p.44 / Chapter 2.7.5 --- Monoclonal establishment --- p.44 / Chapter 2.8 --- "Total RNA preparation, Reverse Transcription (RT) and Quantitative Polymerase Chain Reaction (qPCR)" --- p.45 / Chapter 2.9 --- Immunocytochemistry --- p.46 / Chapter 2.10 --- Western Blotting --- p.48 / Chapter 2.10.1 --- Total Protein Extraction vi --- p.48 / Chapter 2.10.2 --- Measurement of Protein Concentration --- p.48 / Chapter 2.10.3 --- SDS-PAGE and chemiluminescent detection --- p.49 / Chapter 2.11 --- Co-immunoprecipitation --- p.51 / Chapter 2.12 --- Telomere Repeat Amplification Protocol (TRAP) Assay --- p.52 / Chapter 2.13 --- Cell cycle analysis --- p.54 / Chapter 2.14 --- MTT assay --- p.54 / Chapter 2.15 --- Statistical analysis --- p.55 / Chapter 3 --- RESULTS --- p.Page / Chapter 3.1 --- mPinXlt was discovered in mESCs --- p.56 / Chapter 3.2 --- mPinXl and mPinXlt were expressed at transcriptional level in the inspected mouse tissues --- p.61 / Chapter 3.3 --- Expression of mPinXl and mPinXlt changed upon differentiation --- p.64 / Chapter 3.4 --- mPinXl and mPinXlt were both located in the nucleolus and the nucleoplasm in undifferentiated mESCs --- p.69 / Chapter 3.5 --- Co-immunoprecipitation (Co-IP) of mPinXl and mPinXlt with mTERT --- p.73 / Chapter 3.6 --- Transient knockdown of mPinXl in mESCs --- p.78 / Chapter 3.6.1 --- Knockdown of mPinXl decreased proliferation but did not change cell viability --- p.79 / Chapter 3.6.2 --- Knockdown of mPinXl decreased telomerase activity --- p.79 / Chapter 3.6.3 --- Knockdown of mPinXl did not change pluripotency --- p.80 / Chapter 3.6.4 --- Knockdown of mPinXl did not affect cell cycle progression --- p.80 / Chapter 3.7 --- Transient knockdown of mPinXlt using siRNA against mPinXlt in mESCs --- p.88 / Chapter 3.8 --- Transient over-expression of mPinXl and mPinXlt in mESCs --- p.90 / Chapter 3.8.1 --- Over-expression of mPinXl and mPinXlt decreased cell proliferation but didn't affect cell viability --- p.91 / Chapter 3.8.2 --- Over-expression of mPinXl increased telomerase activity --- p.92 / Chapter 3.8.3 --- Over-expression of mPinXl and mPinXlt did not affect pluripotency --- p.93 / Chapter 3.8.4 --- Over-expression of mPinXl and mPinXlt did not affect cell cycle progression --- p.93 / Chapter 3.9 --- Stable over-expression and knockdown of mPinXl and mPinXlt in mESCs --- p.103 / Chapter 3.9.1 --- Expression of mPinXl and mPinXlt at mRNA and protein levels in all over-expression stable cell lines --- p.108 / Chapter 3.9.2 --- Expression of mPinXl and mPinXlt at mRNA and protein levels in mPinXl knockdown stable cell lines --- p.113 / Chapter 3.9.3 --- Proliferation of all stable cell lines --- p.116 / Chapter 3.9.4 --- Telomerase activity of all stable cell lines --- p.121 / Chapter 3.9.5 --- Cell cycle distribution of all stable cell lines --- p.123 / Chapter 3.9.6 --- Pluripotency of all stable cell lines --- p.127 / Chapter 3.9.7 --- Differentiation of the stable cell lines --- p.130 / Chapter 3.9.7.1 --- Size of EBs formed from stable cell lines at Day 7 --- p.130 / Chapter 3.9.7.2 --- Beating curves of the stable cell lines derived EBs --- p.130 / Chapter 4 --- DISCUSSIONS --- p.Page / Chapter 4.1 --- mPinXlt gene was detected in mESCs --- p.137 / Chapter 4.2 --- "Presence of mPinXl and mPinXlt in mouse tissues, mESCs and their differentiation derivatives" --- p.138 / Chapter 4.3 --- Differences in expressions of mPinXl and mPinXlt in undifferentiated mESCs and their differentiation derivatives --- p.139 / Chapter 4.4 --- mPinXl and mPinXlt are pre-dominantly localized in the nucleolus --- p.141 / Chapter 4.5 --- mPinXl and mPinXlt interacted with mTERT --- p.143 / Chapter 4.6 --- "Transient knockdown of mPinXl slightly inhibited, while over-expression of mPinXl slightly promoted telomerase activity" --- p.143 / Chapter 4.7 --- Both transient knockdown and over-expression of mPinXl inhibited the growth of mESCs --- p.146 / Chapter 4.8 --- Both stable knockdown and over-expression of mPinXl did not affect cell proliferation and telomerase activity of mESCs --- p.148 / Chapter 4.9 --- Involvement of mPinXl and mPinXlt in the differentiation process of mESCs --- p.149 / Chapter 4.10 --- Regulation of mPinXl gene expression by mPinXlt --- p.151 / Chapter 4.11 --- Future perspectives --- p.152 / Chapter 5 --- CONCLUSION --- p.154 / Chapter 6 --- REFERENCES --- p.156

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_327388
Date January 2011
ContributorsLau, Yuen Ting., 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, xvi, 163 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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