Tsui, Wing Wun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 125-137). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Statement --- p.iii / Abstract --- p.iv / Chinese abstract --- p.vi / Acknowledgements --- p.viii / General abbreviations --- p.X / List of figures --- p.xiv / List of tables --- p.XV / Table of contents --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Literature review on LIM-homeobox genes in mouse development --- p.1 / Chapter 1.1.1 --- LIM-homeobox genes --- p.1 / Chapter 1.1.2 --- Mouse Lhx1 gene and development --- p.5 / Chapter 1.1.3 --- Mouse Lhx5 gene and development --- p.21 / Chapter 1.2 --- Mouse cerebellar Purkinje neurons --- p.26 / Chapter 1.2.1 --- Cerebellar cortex --- p.26 / Chapter 1.2.2 --- Neuronal circuitry and cerebellar functions --- p.29 / Chapter 1.2.3 --- Development of cerebellar Purkinje neurons --- p.29 / Chapter 1.2.3.1 --- Neurogenesis --- p.30 / Chapter 1.2.3.2 --- Migration and positioning --- p.30 / Chapter 1.2.3.3 --- Specification and differentiation --- p.31 / Chapter 1.2.3.4 --- Maturation --- p.31 / Chapter 1.3 --- Green Fluorescent Protein (GFP) and tau protein --- p.32 / Chapter 1.3.1 --- Introduction to tau proteins --- p.32 / Chapter 1.3.2 --- Tau-GFP fusion protein and its application in tracing neuronal projections --- p.33 / Chapter 1.4 --- Project background and aim --- p.34 / Chapter Chapter 2 --- Generation of Lhx1-tau-GFP knock-in mice --- p.38 / Chapter 2.1 --- Introduction --- p.38 / Chapter 2.2 --- Materials for molecular biological work --- p.39 / Chapter 2.2.1 --- Chemicals and kits --- p.39 / Chapter 2.2.2 --- Enzymes --- p.40 / Chapter 2.2.3 --- Plasmid vectors --- p.40 / Chapter 2.2.4 --- Oligonucleotide linkers --- p.41 / Chapter 2.2.5 --- Bacterial strains --- p.41 / Chapter 2.2.6 --- Solutions and media --- p.41 / Chapter 2.2.7 --- Radioactive isotopes and materials for autoradiography --- p.43 / Chapter 2.2.8 --- DNA probes for Southern blot hybridization --- p.43 / Chapter 2.3 --- Materials for cell culture --- p.44 / Chapter 2.3.1 --- "Chemicals, sera and others" --- p.44 / Chapter 2.3.2 --- Culture solutions and media --- p.44 / Chapter 2.3.3 --- Culture cells --- p.45 / Chapter 2.4 --- PCR primers --- p.46 / Chapter 2.5 --- Animals --- p.46 / Chapter 2.6 --- Methods for molecular biological work --- p.46 / Chapter 2.6.1 --- Preparation of plasmid DNA --- p.46 / Chapter 2.6.1.1 --- Miniprep using simple crude method --- p.47 / Chapter 2.6.1.2 --- Miniprep using purification kits --- p.48 / Chapter 2.6.1.3 --- Midiprep using purification kit --- p.50 / Chapter 2.6.2 --- Purification of specific DNA fragments --- p.51 / Chapter 2.6.2.1 --- QIAquick gel extraction kit --- p.51 / Chapter 2.6.2.2 --- QIAquick PCR purification kit --- p.52 / Chapter 2.6.3 --- Subcloning of DNA fragments --- p.53 / Chapter 2.6.3.1 --- Traditional approach based on restriction endonuclease and DNA ligase --- p.53 / Chapter 2.6.3.2 --- Preparation of subcloning inserts and vectors --- p.54 / Chapter 2.6.3.3 --- Two-way ligation of inserts and vectors --- p.55 / Chapter 2.6.4 --- Transformation of competent cells with recombinant DNA --- p.56 / Chapter 2.6.4.1 --- CaCl2 method --- p.56 / Chapter 2.6.4.2 --- Electroporation --- p.57 / Chapter 2.6.5 --- Southern hybridization --- p.59 / Chapter 2.6.5.1 --- Restriction endonuclease digestion and agarose gel electrophoresis --- p.59 / Chapter 2.6.5.2 --- Capillary transfer and fixation of DNA --- p.60 / Chapter 2.6.5.3 --- Radioactive labeling of DNA probe --- p.60 / Chapter 2.6.5.4 --- Purification of radioactive labeled probe for hybridization --- p.61 / Chapter 2.6.5.5 --- Hybridization --- p.61 / Chapter 2.6.5.6 --- Post-hybridization wash and autoradiography for signal detection --- p.62 / Chapter 2.7 --- Methods for generation and analysis of Lhx1-tau-GFP knock-in Mice --- p.63 / Chapter 2.7.1 --- Construction of targeting vector (pLhx1-tauGFP) for gene targeting of Lhx1 locus --- p.63 / Chapter 2.7.2 --- Generation of targeted embryonic stem (ES) cell clones --- p.66 / Chapter 2.7.2.1 --- Preparation of feeder cells --- p.66 / Chapter 2.7.2.2 --- Culture of ES cells on feeder layers and passage --- p.69 / Chapter 2.7.2.3 --- Harvest of cultured ES cells --- p.70 / Chapter 2.7.2.4 --- Preparation of targeting vector for transfection of ES cells --- p.71 / Chapter 2.7.2.5 --- Electroporation for transfection of ES cells --- p.71 / Chapter 2.7.2.6 --- Drug selection for targeted ES cell clones using PNS strategy --- p.72 / Chapter 2.7.2.7 --- Picking and expansion of targeted ES cell clones --- p.72 / Chapter 2.7.2.8 --- Replica plating and freezing of targeted ES cell clones --- p.74 / Chapter 2.7.2.9 --- Genomic DNA extraction from targeted ES cell clones --- p.75 / Chapter 2.7.2.10 --- Screening of homologous recombinants by Southern hybridization analysis --- p.76 / Chapter 2.7.2.11 --- Thawing and expansion of correct targeted ES cell clones --- p.76 / Chapter 2.7.2.12 --- Chromosome counting of ES cells --- p.78 / Chapter 2.7.3 --- Generation of germline chimeric mice --- p.80 / Chapter 2.7.3.1 --- Standard procedure --- p.80 / Chapter 2.7.4 --- Breeding and genotyping of mice --- p.81 / Chapter 2.7.5 --- Imaging of tau-GFP-labelled Purkinje neurons --- p.84 / Chapter 2.7.5.1 --- Animal dissection and tissue preparation --- p.84 / Chapter 2.7.5.2 --- Confocal laser scanning microscopy (CLSM) --- p.84 / Chapter 2.8 --- Results --- p.84 / Chapter 2.8.1 --- Generation of Lhx1 targeting vector (pLhx1-tauGFP) --- p.84 / Chapter 2.8.2 --- Targeted replacement of the mouse Lhx1 coding sequences by tau-GFP genetic reporter --- p.87 / Chapter 2.8.3 --- Germline transmission of Lhx1-tau-GFP allele and generation of Lhx1-tau-GFP knock-in mouse --- p.93 / Chapter 2.8.4 --- Imaging of Lhx1-tau-GFP expressing Purkinje neurons --- p.96 / Chapter 2.9 --- Discussion --- p.98 / Chapter 2.9.1 --- Tau-GFP labeling of Lhx1-expressing Purkinje neurons: implications for real-time live cell imaging --- p.98 / Chapter 2.9.2 --- Use of Lhx1-tau-GFP knock-in mice for study of Lhx1 and Lhx5 functions in Purkinje neurons survival and/or maintenance --- p.99 / Chapter Chapter 3 --- Generation of Lhx5-tau-GFP knock-in allele: alternative approach for real-time tracing of Purkinje neurons --- p.102 / Chapter 3.1 --- Introduction: Recombineering-based approach for DNA subcloning --- p.102 / Chapter 3.1.1 --- λ phage-encoded Red recombination system --- p.102 / Chapter 3.1.2 --- DNA subcloning from bacterial artificial chromosome (BAC) --- p.104 / Chapter 3.2 --- Materials for molecular biological work --- p.105 / Chapter 3.2.1 --- Chemicals and kits --- p.105 / Chapter 3.2.2 --- Enzymes --- p.105 / Chapter 3.2.3 --- Plasmid vectors and BAC DNA --- p.105 / Chapter 3.2.4 --- Bacterial strains --- p.105 / Chapter 3.2.5 --- Solutions and media --- p.106 / Chapter 3.2.6 --- PCR primers --- p.106 / Chapter 3.3 --- Methods for construction of targeting vector for mouse Lhx5 gene --- p.107 / Chapter 3.3.1 --- PCR amplification of homology sequences on BAC DNA --- p.107 / Chapter 3.3.2 --- Synthesis of retrieval arms for recombineering --- p.109 / Chapter 3.3.3 --- DNA sequencing analysis --- p.110 / Chapter 3.3.4 --- Construction of retrieval vector --- p.110 / Chapter 3.3.5 --- Preparation of electrocompetent cells for recombineering --- p.111 / Chapter 3.3.6 --- Recombineering-based retrieval of homology arms --- p.112 / Chapter 3.4 --- Results --- p.113 / Chapter 3.4.1 --- The targeting vector (pLhx5-tauGFP) for mouse Lhx5 gene --- p.113 / Chapter 3.5 --- Discussion --- p.118 / Chapter 3.5.1 --- Use of recombineering-based approach to generate targeting vector --- p.118 / Chapter 3.5.2 --- Further generation of Lhx5-tau-GFP knock-in mice --- p.119 / Chapter Chapter 4 --- Conclusion and future perspectives --- p.120 / Chapter 4.1 --- Conclusion --- p.120 / Chapter 4.2 --- Potential applications of Lhx1-tau-GFP knock-in mice for study of Lhx1 and other gene functions in cerebellum --- p.120 / Chapter 4.3 --- Potential applications of Lhx1-tau-GFP knock-in mice for study of Lhx1 -expressing cells development --- p.122 / References --- p.125

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

by Pui-kuen Lee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 168-182). / ACKNOWLEDGMENT --- p.I / ABSTRACT --- p.II / TABLE OF CONTENTS --- p.IV / ABBREVIATIONS --- p.X / LIST OF FIGURES --- p.XII / LIST OF TABLES --- p.XIV / Chapter CHAPTER1 --- INTRODUCTION AND BACKGROUND --- p.1 / Chapter 1.1 --- ROLE OF CYTOKINES IN MOUSE EMBRYONIC DEVELOPMENT --- p.1 / Chapter 1.1.1 --- Why mouse model --- p.1 / Chapter 1.1.2 --- Embryonic development of mouse --- p.1 / Chapter 1.1.3 --- An overview of cytokines --- p.4 / Chapter a. --- Classes of cytokines --- p.5 / Chapter i) --- Growth factors --- p.5 / Chapter ii) --- Interleukins --- p.7 / Chapter iii) --- Colony-stimulating factors --- p.9 / Chapter iv) --- Interferons --- p.10 / Chapter v) --- Tumor necrosis factor --- p.11 / Chapter b. --- Cytokine networks --- p.12 / Chapter c. --- Role of cytokines in the whole organism --- p.13 / Chapter 1.1.4 --- Cytokine and receptor gene expression in mouse embryonic development --- p.15 / Chapter a. --- Murine embryonic stem cell model --- p.15 / Chapter b. --- Leukemia Inhibitory Factor (LIF) in mouse embryos --- p.16 / Chapter c. --- IL-6 in mouse embryo --- p.19 / Chapter d. --- Ciliary Neurotrophic Factor (CNTF) in mouse embryo --- p.19 / Chapter e. --- TNF-a and TNF-β in mouse embryos --- p.20 / Chapter f. --- TGF-a in mouse embryos --- p.20 / Chapter g. --- TGF-P in mouse embryos --- p.20 / Chapter h. --- Stem cell factor / c-kit --- p.21 / Chapter i. --- Other cytokines in mouse embryos --- p.22 / Chapter j. --- Cytokine receptors --- p.24 / Chapter 1.2 --- NEUROPOIETIC CYTOKINES --- p.28 / Chapter 1.2.1 --- Family members --- p.28 / Chapter 1.2.2 --- Shared signal transducer gpl30 --- p.29 / Chapter 1.2.3 --- "LIF, CNTF and OSM inhibit differentiation of embryonic stem cells" --- p.31 / Chapter 1.3 --- BIOLOGY OF ONCOSTATIN M (OSM) --- p.33 / Chapter 1.3.1 --- Physical properties of OSM --- p.33 / Chapter 1.3.2 --- Biological activities of OSM --- p.34 / Chapter 1.3.3 --- Molecular aspect of OSM --- p.35 / Chapter 1.4 --- AIMS OF THE STUDY --- p.38 / Chapter CHAPTER2 --- CYTOKINE GFNE EXPRESSION DURING MOUSE EMBRYONIC DEVELOPMENT --- p.40 / Chapter 2.1 --- INTRODUCTION --- p.40 / Chapter 2.1.1 --- Rationale --- p.40 / Chapter 2.1.2 --- Design of primers --- p.43 / Chapter 2.2 --- MATERIALS --- p.44 / Chapter 2.2.1 --- Chemicals and Reagents --- p.44 / Chapter 2.2.2 --- Enzymes --- p.45 / Chapter 2.2.3 --- Buffers --- p.45 / Chapter 2.2.4 --- Solutions --- p.47 / Chapter 2.2.5 --- Probe labeling and detection kits --- p.48 / Chapter 2.2.6 --- Primers and internal probes --- p.49 / Chapter 2.3 --- METHODS --- p.52 / Chapter 2.3.1 --- Preparation of total RNA from mouse embryos at different stages --- p.52 / Chapter a. --- Mice dissection for embryo --- p.52 / Chapter b. --- Guanidinium thiocyanate cell lysate --- p.52 / Chapter c. --- Isolation of RNA by centrifugation through CsCl gradient --- p.53 / Chapter d. --- Spectrophotometric determination of RNA amount --- p.54 / Chapter 2.3.2 --- Preparation of embryo sections --- p.54 / Chapter 2.3.3 --- Primers and internal probes --- p.55 / Chapter 2.3.4 --- Cytokine mRNA Phenotyping by Reverse transcription-Polymerase chain reaction --- p.56 / Chapter a. --- Reverse transcription (First strand cDNA synthesis) --- p.56 / Chapter b. --- Polymerase chain reaction (PCR) --- p.56 / Chapter 2.3.5 --- Analysis of PCR products with agarose gel electrophoresis --- p.57 / Chapter 2.3.6 --- Analysis of PCR products with Southern blotting --- p.58 / Chapter a. --- DNA transfer from gel to nylon membrane --- p.58 / Chapter b. --- Probe labeling --- p.61 / Chapter c. --- Prehybridization --- p.61 / Chapter d. --- Hybridization --- p.62 / Chapter e. --- Detection of DIG-labeled probe --- p.62 / Chapter 2.3.7 --- Cycle titration of PCR and dot blotting of regulatory cytokine mRNA --- p.63 / Chapter a. --- Cycle titration of PCR --- p.63 / Chapter b. --- Dot blotting --- p.63 / Chapter 2.4 --- RESULTS --- p.65 / Chapter 2.4.1 --- Sagittal sections of mouse embryos --- p.65 / Chapter 2.4.2 --- Preparation of total RNA --- p.69 / Chapter 2.4.3 --- Cytokine mRNA phenotyping --- p.71 / Chapter a. --- Southern hybridization for 'no expression' cytokines --- p.74 / Chapter b. --- Consistent' and 'regulatory ´ة cytokines in embryo and placenta --- p.79 / Chapter 2.5 --- DISCUSSION --- p.95 / Chapter 2.5.1 --- Isolation of embryo RNA by guanidinium thiocyanate/ cesium chloride centrifugation --- p.95 / Chapter 2.5.2 --- mRNA Quantitation --- p.96 / Semi-quantitative PCR --- p.98 / Chapter 2.5.3 --- Cytokine mRNA phenotyping by RT-PCR --- p.99 / Chapter a. --- Reverse Transcription --- p.99 / Chapter b. --- GAPDH as a control for normalization --- p.100 / Chapter c. --- PCR for cytokine transcripts --- p.101 / Chapter 2.5.4 --- Cytokines and receptors in embryonic development --- p.103 / Chapter 2.5.4.1 --- Cytokines in hematopoietic development of mouse fetus --- p.104 / Chapter 2.5.4.2 --- Other cytokines --- p.113 / Chapter 2.5.5 --- Expression Pattern in placenta: maternal and fetal communication --- p.116 / Chapter CHAPTER3 --- MOLECULAR ANALYSTS OF MOUSE ONCOSTATIN M --- p.117 / Chapter 3.1 --- INTRODUCTION --- p.117 / Chapter 3.2 --- MATERIALS --- p.121 / Chapter 3.2.1 --- Chemicals and Reagents --- p.121 / Chapter 3.2.2 --- Enzymes --- p.121 / Chapter 3.2.3 --- Buffers --- p.122 / Chapter 3.2.4 --- Solutions --- p.122 / Chapter 3.2.5 --- Culture media --- p.124 / Chapter 3.2.6 --- Competent cell --- p.125 / Chapter 3.2.7 --- DNA materials --- p.125 / Chapter 3.2.8 --- Primers --- p.126 / Chapter 3.3 --- METHODS --- p.127 / Chapter 3.3.1 --- Primers and internal probes --- p.127 / Chapter 3.3.2 --- Cloning of human Oncostatin M exon 2 and exon 3 by PCR --- p.127 / Chapter 3.3.3 --- Subcloning of human OSM exons 2 and 3 into pUC18 --- p.128 / Chapter a. --- Preparation of human OSM exons and plasmid --- p.128 / Chapter i) --- Purification of PCR products --- p.128 / Chapter ii) --- T4 DNA polymerase ´بblunt-end´ة reaction for PCR products --- p.129 / Chapter iii) --- Sma I digestion of pUC18 --- p.129 / Chapter b. --- Ligation --- p.129 / Chapter c. --- Preparation of competent cell --- p.130 / Chapter d. --- Transformation --- p.131 / Chapter e. --- Screening of recombinants by PCR --- p.131 / Chapter f. --- Screening of recombinants by restriction enzyme digestion --- p.132 / Chapter i) --- Preparation of plasmids --- p.132 / Chapter ii) --- Double restriction enzymes digestion of pUC18 --- p.133 / Chapter 3.3.4 --- Verification of the clones of human OSM exons 2 and 3 by cycle sequencing --- p.135 / Chapter 3.3.5 --- Purification of human OSM exons from plasmid for making probe --- p.136 / Chapter 3.3.6 --- Southern blotting --- p.136 / Chapter a. --- Probe making and labeling --- p.136 / Chapter b. --- Preparation of mouse genomic DNAs --- p.137 / Chapter c. --- DNA transfer --- p.138 / Chapter i) --- Digestion of genomic DNA with restriction endonucleases --- p.138 / Chapter ii) --- Gel electrophoresis and DNA blotting --- p.139 / Chapter d. --- Hybridization --- p.139 / Chapter 3.4 --- RESULTS --- p.142 / Chapter 3.4.1 --- Cloning of human OSM exon 2 and exon 3 by PCR --- p.142 / Chapter 3.4.2 --- Subcloning of human OSM exons 2 and 3 into pUC18 --- p.142 / Chapter a. --- Screening of recombinants by PCR --- p.142 / Chapter b. --- Screening of recombinants by restriction enzymes digestion --- p.143 / Chapter 3.4.3 --- Sequence of subcloned exons 2 and3 --- p.147 / Chapter 3.4.4 --- Southern hybridization --- p.149 / Chapter a. --- Genomic DNA preparation --- p.149 / Chapter b. --- Digestion of genomic DNAs --- p.151 / Chapter c. --- Hybridization signal --- p.154 / Chapter 3.5 --- DISCUSSION --- p.158 / Chapter 3.5.1 --- Cross-species hybridization --- p.158 / Chapter 3.5.2 --- Hybridization of human OSM exon fragments against mouse genome --- p.158 / Chapter a. --- hOSM exon 2 as probe --- p.158 / Chapter b. --- hOSM exon 3 as probe --- p.160 / Chapter c. --- Feasibility of using hOSM as probe for fishing out the mOSM gene --- p.160 / Chapter d. --- The cloning of mouse OSM by Yoshimura's group --- p.161 / Chapter CHAPTER4 --- CONCLUSION --- p.162 / Chapter 4.1 --- SUMMARY OF CYTOKINE AND CYTOKINE RECEPTOR GENES EXPRESSION DURING EMBRYONIC DEVELOPMENT --- p.162 / Chapter 4.2 --- FURTHER STUDIES OF THE CYTOKINE ACTIONS ON EMBRYOGENESIS --- p.165 / Chapter 4.3 --- MOLECULAR ANALYSIS OF MOUSE OSM GENE --- p.167 / REFERENCES --- p.168

Abstract not available

Cited2 is a member of the Cited gene family, which has no homology to any other genes. It encodes a transcriptional co-factor that is expressed during early heart formation (cardiogenesis). Embryos lacking Cited2 display a range of cardiac defects including bilaterally identical atria, aortic arch abnormalities, rotation of the aorta and pulmonary artery, and malseptation of the cardiac chambers. The latter results in communication between the aorta and pulmonary artery, the aorta and both ventricles, and the atria and ventricles (with themselves and each other). Cardiogenesis is complex, and requires many different cell types and processes to occur correctly. Some of these cells and processes are external to the primary heart. For example, once the initial muscle cells of the heart form a tube, cells from other regions such as the secondary heart field (adjacent mesoderm) and cardiac neural crest (ectoderm) migrate into this tube, and are required for the formation of additional muscle cells and septa. Furthermore, cardiogenesis also requires correct left-right patterning of the embryo to be established prior to heart formation. To understand the developmental origins of the cardiac defects observed in Cited2-null embryos, the expression pattern of Cited2 and the anatomy of Cited2-null embryo hearts were studied. Subsequently, the expression of genes required for left-right patterning were studied in both Cited2-null and Cited2 conditionally-deleted embryos. This demonstrated that Cited2 may be required in many, possibly all, of the processes required for cardiogenesis. Next this study focused on the role of Cited2 in patterning the left-right axis of the embryo. Firstly, Cited2 was found to regulate the expression of the master regulator of left-right patterning (Nodal). Secondly, Cited2 was shown to regulate the expression of the left-specific transcription factor Pitx2 independently of Nodal. Thirdly, gene expression and conditional deletions of Cited2 suggested that Cited2 might regulate left-right patterning in the paraxial mesoderm, a tissue which has not previously been shown to regulate the left-right axis in the mouse. Lastly, an argument is made suggesting the possibility that all the cardiac defects found in Cited2-null embryos may directly or indirectly stem from a failure of correct left-right patterning.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 261-288).

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The splotch (sp) mutation has been identified as a mutation in the paired box gene, Pax-3. Heterozygous mice carrying this mutation are phenotypically normal, with the exception of a white spot on their bellies. Homozygous embryos do not live to birth, and suffer from a wide range of developmental defects, all of which result from delayed neural tube closure, or inadequate neural crest cell migration. Most notably, homozygotes have an increased rate of spina bifida with or without exencephaly. Retinoic acid (RA), which has been shown to be very important in the development of a number of systems, was shown to cause a selective mortality of the homozygous splotch embryos when administered maternally at day 9 p.c. (Moase and Trasler, 1987). Since cellular retinoic acid binding protein (CRABP) is localized to the tissues which are affected by both the splotch gene, and retinoic acid teratogenesis, its expression patterns in the developing splotch embryo were examined. It was found that the distribution of CRABP mRNA is normal, but its expression levels are excessive in splotch homozygous day 9 mouse embryos.

No description available.

The striped-mouse, Rhabdomys pumilio, is widely distributed throughout southern Africa within a variety of habitats and rainfall regimes. It is found at sea level in the Eastern and Western Cape regions and at altitudes above 2700 m in the Drakensberg mountains. The attraction of R.pumilio to cultivated land and crops has resulted in extensive damage to plants and cultivated crops. A study of the genetic variation between populations of R.pumilio from different regions of southern Africa was undertaken by protein electrophoresis and randomly amplified polymorphic DNA using the polymerase chain reaction (PCR-RAPD). A cytogenetic study was also undertaken. The mean heterozygosity (H=0.074) for R.pumilio was more than twice that estimated for mammals (H=0.036) while the mean percent polymorphism (P=16.1%) was only slightly higher than the mean percent polymorphism obtained for mammals (P=14.7%). The highest heterozygosities were recorded in the Potchefstroom (H=0 .145) and Zimbabwe (H=0 .118) samples and the lowest mean heterozygosity was recorded in the peninsular Western Cape (H=0. 032). A mean Fst value of 0.459 was obtained, suggesting a high degree of genetic differentiation between the samples of R.pumilio but the negative Fis (-0.01) value emphasized that R.pumilio retained an outbreeding population structure. The similarity coefficient between the samples of R.pumilio using PCR-RAPD's ranged between 0.471 and 0.853 and substantiated the argument for genetic divergence between the samples of R.pumilio. An isolation by distance model for the population genetic structure of R.pumilio was supported by the allozymes (r=0.58, p<0.00l) and PCR-RAPD's (0.75, p<0.00l). Temperature and rainfall also had an influence on the allelic frequency distribution of certain loci of R.pumilio. Rogers (1972) genetic similarity varied between 0.796 and 0.988 while the values for Nei's (1978) unbiased genetic distance varied between 0.000 and 0.189 for the different samples of R.pumilio. Subgrouping of the KwaZulu-Natal samples, the peninsular Western Cape and Eastern Cape samples of R.pumilio was evident with the allozymes. With the PCR-RAPD' s the Zimbabwe sample showed the least similarity to the other samples with a KwaZulu-Natal/Potchefstroom subgroup separating from the less well defined Eastern Cape and Western Cape subgroup. Cytogenetic studies of specimens of R.pumilio from some of the localities in southern Africa revealed a chromosomal number of 2n=48 , while the Potchefstroom and Zimbabwe specimens displayed a chromosomal number of 2n=46. Homology in G-and C-banding was recorded. The allozymes, PCR-RAPD's and chromosomal studies suggested subspecies status for the Zimbabwe population of R.pumilio. The Potchefstroom sample displayed a greater genetic similarity to the remaining South African samples of R.pumilio than the Zimbabwe samples and therefore could not be considered for subspecies status. Although the South African samples of R.pumilio displayed a certain degree of genetic divergence, it was insufficient to warrant subspecies status although evolution in this direction was suggested. / Thesis (Ph.D.)-University of Natal, Durban, 1999.