Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 9 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Jeanne H. Freeland-Graves. Vita. Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2001. / Härtill 5 uppsatser.

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