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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_327388 |
| Date | January 2011 |
| Contributors | Lau, Yuen Ting., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xvi, 163 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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