Lin, Ka Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 108-122). / Abstracts in English and Chinese. / Abstract (In English) --- p.ii / Abstract (In Chinese) --- p.iii / Acknowledgment --- p.V / Abstracts of Publications --- p.vi / Abbreviations --- p.viii / List of Figures --- p.ix / List of Table --- p.X / Contents --- p.xi / Chapter Chapter I --- Introduction --- p.Page / Chapter 1.1 --- Pluripotent Stem Cell --- p.1 / Chapter 1.1.1 --- Characteristic of pluripotent stem cells --- p.1 / Chapter 1.1.2 --- Origin of pluripotent stem cells --- p.1 / Chapter 1.1.2.1 --- Embryonic carcinoma cells --- p.2 / Chapter 1.1.2.2 --- Embryonic stem cells --- p.2 / Chapter 1.1.2.3 --- Epiblast stem cells --- p.2 / Chapter 1.1.2.4 --- Embryonic germ cells and adult germline stem cells --- p.3 / Chapter 1.1.2.5 --- Induced pluripotent stem cells --- p.3 / Chapter 1.1.3 --- Pluripotency in Embryonic Stem Cells --- p.4 / Chapter 1.1.3.1 --- Extrinsic signal governing pluripotency --- p.5 / Chapter 1.1.3.1.1 --- LIF signaling --- p.5 / Chapter 1.1.3.1.2 --- BMP signaling --- p.6 / Chapter 1.1.3.1.3 --- Other signaling pathways --- p.6 / Chapter 1.1.3.2 --- Intrinsic sternness factors --- p.7 / Chapter 1.1.3.2.1 --- Oct4 Expression in Embryonic Stem cells --- p.7 / Chapter 1.1.3.2.2 --- Sox-2 Expression in Embryonic Stem Cells --- p.8 / Chapter 1.1.3.2.3 --- Nanog Expression in Embryonic Stem Cells --- p.9 / Chapter 1.1.3.2.4 --- "Transcriptional Regulation of Oct-4, Nanog and Sox-2 in Embryonic Stem Cells" --- p.10 / Chapter 1.1.3.2.5 --- Others pluripotent genes --- p.11 / Chapter 1.1.3.2.5.1 --- Utfl --- p.11 / Chapter 1.1.3.2.5.2 --- Rexl --- p.11 / Chapter 1.1.3.2.5.3 --- Esrrb --- p.12 / Chapter 1.1.3.2.5.4 --- Eras --- p.12 / Chapter 1.1.3.2.5.5 --- Tell --- p.12 / Chapter 1.1.3.2.5.6 --- Dnm3tl --- p.13 / Chapter 1.1.3.2.5.7 --- Dppa3 --- p.13 / Chapter 1.1.3.2.5.8 --- Dppa4 --- p.14 / Chapter 1.1.3.2.5.9 --- Dppa5 --- p.14 / Chapter 1.1.3.2.5.10 --- Klf2 --- p.15 / Chapter 1.2 --- Somatic cell reprogramming --- p.16 / Chapter 1.2.1 --- Definition of reprogramming --- p.16 / Chapter 1.2.2 --- The history of reprogramming --- p.16 / Chapter 1.2.2.1 --- Reprogramming by nuclear transfer --- p.17 / Chapter 1.2.2.2 --- Reprogramming by fusion with ES or EC cells --- p.18 / Chapter 1.2.2.3 --- Reprogramming with defined factor --- p.19 / Chapter 1.3 --- Induced pluripotent stem cells --- p.20 / Chapter 1.3.1 --- Transcription factor used for reprogramming to iPS cells --- p.20 / Chapter 1.3.1.1 --- Klf4 --- p.20 / Chapter 1.3.1.2 --- c-Myc --- p.21 / Chapter 1.3.2 --- Cornerstone of iPSC generation --- p.22 / Chapter 1.3.3 --- Major events in the reprogramming process --- p.23 / Chapter 1.3.4 --- Gene delivery systems for ips cell generation --- p.26 / Chapter 1.3.5 --- Culture system for embryonic stem cells and iPSC --- p.28 / Chapter 1.3.4.1 --- Feeder and serum used cell culture system --- p.28 / Chapter 1.3.4.2 --- Serum-free culture condition --- p.29 / Chapter 1.3.5 --- Differentiation potential of iPSC --- p.30 / Chapter 1.3.5.1 --- In vitro stringency tests --- p.30 / Chapter 1.3.5.2 --- In vivo stringency test --- p.30 / Chapter 1.3.5.3 --- In utero stringency test --- p.31 / Chapter 1.4 --- Mouse Lewis lung carcinoma-D 122 --- p.32 / Chapter 1.5 --- Dendritic cell vaccine in cancer immunotherapy --- p.33 / Chapter 1.5 --- Green Fluorescence protein Reporters --- p.35 / Chapter 1.5.1 --- GFP reporters in embryos and stem cell --- p.35 / Chapter 1.5.2 --- copGFP --- p.35 / Chapter 1.6 --- Aim of study --- p.36 / Chapter Chapter II --- Methods and Materials / Chapter 2.1 --- Materials --- p.37 / Chapter 2.1.1 --- Synthetic oligos used in polymerase chain reaction (PCR) --- p.37 / Chapter 2.1.2 --- DNA clones used in the study --- p.39 / Chapter 2.1.3 --- Materials for DNA manipulation --- p.39 / Chapter 2.1.4 --- Materials for RNA manipulation --- p.39 / Chapter 2.1.5 --- Antibodies --- p.40 / Chapter 2.1.6 --- Kits --- p.41 / Chapter 2.1.7 --- Bacteria strain and culture reagents 41 / Chapter 2.1.8 --- Culture media and reagents --- p.42 / Chapter 2.1.8.1 --- General culture media and reagents --- p.42 / Chapter 2.1.8.2 --- Traditional ES medium --- p.42 / Chapter 2.1.8.3 --- Feeder-free Serum-free ESGRO medium --- p.42 / Chapter 2.1.9 --- Cell lines used --- p.43 / Chapter 2.1.10 --- Instrumentation --- p.43 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Cell culture --- p.44 / Chapter 2.2.1.1 --- Routine cell culture --- p.44 / Chapter 2.2.1.2 --- Resuscitation and culture from frozen stock --- p.44 / Chapter 2.2.1.3 --- Passage of cells --- p.44 / Chapter 2.2.1.4 --- Cryopreservation of cells --- p.45 / Chapter 2.2.1.5 --- Mouse ES cells culture --- p.45 / Chapter 2.2.1.5.1 --- Passage and maintenance of SNL --- p.45 / Chapter 2.2.1.5.2 --- Inactivation and plating of SNLs (Feeder preparation) --- p.45 / Chapter 2.2.1.5.3 --- Cryopreservation (freezing) of SNLs --- p.46 / Chapter 2.2.1.6 --- Mouse ES cells culture in feeder-free culture conditions --- p.46 / Chapter 2.2.1.6.1 --- Preparation of gelatin coated plates --- p.46 / Chapter 2.2.1.6.2 --- Thawing mouse ES cells --- p.46 / Chapter 2.2.1.6.3 --- Passage of mouse ES cells --- p.47 / Chapter 2.2.1.6.4 --- Freezing mouse ES cells --- p.47 / Chapter 2.2.1.7 --- ES cells differentiation-Formation of embryoid bodies (EBs) --- p.47 / Chapter 2.2.1.8 --- Direct differentiation by retinoic acid --- p.48 / Chapter 2.2.1.9 --- Generation of iPS --- p.48 / Chapter 2.2.2 --- Cell transfections --- p.48 / Chapter 2.2.2.1 --- Lipofectamine 2000 transfection --- p.48 / Chapter 2.2.2.2 --- Nucleofection --- p.49 / Chapter 2.2.2.2.1 --- Optimization of nucleofection --- p.49 / Chapter 2.2.2.2.2 --- Nucleofection condition --- p.49 / Chapter 2.2.3 --- Nucleic acid --- p.49 / Chapter 2.2.3.1 --- Genomic DNA isolation --- p.49 / Chapter 2.2.3.2 --- Restriction Enzyme Digestion --- p.50 / Chapter 2.2.3.3 --- RNA and genomic DNA quantification --- p.50 / Chapter 2.2.3.4 --- Reversed transcription polymerase chain reaction (RT-PCR) --- p.50 / Chapter 2.2.3.4.1 --- RNA isolation and Reverse transcription (RT) --- p.50 / Chapter 2.2.3.4.2 --- Polymerase chain reaction (PCR) --- p.51 / Chapter 2.2.3.4.3 --- Real-time polymerase chain reaction (qRT- PCR) --- p.52 / Chapter 2.2.3.5 --- Agarose gel electrophoresis --- p.53 / Chapter 2.2.3.6 --- Genomic PCR for bisulfite sequencing --- p.53 / Chapter 2.2.4 --- Bacteria and Plasmid preparation --- p.54 / Chapter 2.2.4.1 --- Preparation of competent cells --- p.54 / Chapter 2.2.4.2 --- Heat-shock transformation --- p.54 / Chapter 2.2.4.3 --- Midi prep of plasmid --- p.54 / Chapter 2.2.5 --- Cell Staining --- p.55 / Chapter 2.2.5.1 --- Alkaline phosphatase staining --- p.55 / Chapter 2.2.5.2 --- Immunofluorescence --- p.55 / Chapter 2.2.6 --- Flow cytometry --- p.56 / Chapter 2.2.7 --- Animal Handling --- p.56 / Chapter Chapter III --- Results / Chapter 3.1 --- Generation of Nanog-reporter-GFP-D 122 --- p.57 / Chapter 3.2 --- Nucleofection optimization for D122 --- p.60 / Chapter 3.3 --- Generation ofD122-iPS --- p.65 / Chapter 3.3.1 --- Plasmid construct used in the study --- p.65 / Chapter 3.3.2 --- Protocol of D122-iPS generation --- p.67 / Chapter 3.3.3 --- Reprogramming Efficiency of D12´2ؤreNanog cells --- p.69 / Chapter 3.4 --- Expression of pluripotency markers upon reprogramming --- p.70 / Chapter 3.4.1 --- Alkaline Phosphatase staining --- p.70 / Chapter 3.4.2 --- Nanog-GFP expression --- p.72 / Chapter 3.4.3 --- Pluripotency gene expression upon reprogramming --- p.74 / Chapter 3.4.4 --- GFP positive D122 reNanog Colonies --- p.79 / Chapter 3.5 --- Characterization of the D122-iPS-lC --- p.80 / Chapter 3.5.1 --- Morphology of D122-iPS-lC --- p.80 / Chapter 3.5.2 --- Pluripotency gene expression --- p.82 / Chapter 3.5.3 --- Pluripotency markers SSEA-1 and Oct4 --- p.85 / Chapter 3.5.4 --- Bisulfite genomic sequencing --- p.88 / Chapter 3.5.5 --- Differentiation of the D122-iPS-lC --- p.90 / Chapter 3.5.5.1 --- Embryoid body formation by hanging drop --- p.90 / Chapter 3.5.5.2 --- Retinoic acid induced differentiation --- p.92 / Chapter Chapter IV --- Discussion / Chapter 4.1 --- General Discussion --- p.96 / Chapter 4.1.1 --- Cancer immunotherapy and dendritic cells --- p.96 / Chapter 4.1.2 --- Dendritic vaccine and tumor antigen --- p.97 / Chapter 4.1.3 --- Induced pluripotent stem cell technology and dendritic cells --- p.98 / Chapter 4.1.4 --- Tumor antigen presentation and dendritic cells --- p.98 / Chapter 4.1.5 --- D122 and cancer immunotherapy --- p.99 / Chapter 4.1.6 --- Method to introduce transcription factors for reprogramming --- p.100 / Chapter 4.1.7 --- Kinetics of reprogramming --- p.101 / Chapter 4.1.8 --- Culture condition for reprogramming D122_reNanog --- p.102 / Chapter 4.1.9 --- Reprogramming efficiency --- p.103 / Chapter 4.1.10 --- Establishment of D122-iPS-lC --- p.103 / Chapter 4.1.11 --- Differentiation of D122-iPS-1C --- p.104 / Chapter 4.2 --- Future Work --- p.106 / Chapter 4.3 --- Conclusion --- p.107 / Chapter Chapter V --- Bibliography --- p.108 / Appendix --- p.124
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_327564 |
| Date | January 2011 |
| Contributors | Lin, Ka Yin., Chinese University of Hong Kong Graduate School. Division of Chemical Pathology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xv, 123 leaves : ill. (chiefly 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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