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Pathogenesis of sacral agenesis studied using a mouse model.

by Poon Lit Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 97-113). / Title page --- p.i / Acknowledgements --- p.ii / Table of contents --- p.iii / List of tables --- p.vii / List of figures --- p.viii / Abbreviations --- p.x / Abstract --- p.xi / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- Sacral agenesis --- p.2 / Chapter 1.1.1 --- Skeletal anomalies --- p.3 / Chapter 1.1.2 --- Neurological anomalies --- p.4 / Chapter 1.1.3 --- Other anomalies --- p.5 / Chapter 1.1.4 --- Etiology --- p.5 / Chapter 1.1.5 --- Pathogenetic mechanism of SA --- p.7 / Chapter 1.2 --- Retinoids --- p.8 / Chapter 1.2.1 --- RA in embryonic development --- p.9 / Chapter 1.2.2 --- Teratogenic effect of RA in embryonic development --- p.10 / Chapter 1.3 --- Strategy of the thesis --- p.13 / Chapter Chapter 2: --- General Materials and Methods --- p.15 / Chapter 2.1 --- Mouse maintenance and mating method --- p.16 / Chapter 2.2 --- All-trans RA preparation and injection --- p.16 / Chapter 2.3 --- Dissection of embryos --- p.16 / Chapter 2.4 --- Preparation of histological sections --- p.17 / Chapter 2.4.1 --- Dehydration and embedding --- p.17 / Chapter 2.4.2 --- Sectioning --- p.18 / Chapter 2.4.3 --- Haematoxylin and eosin staining --- p.18 / Chapter 2.5 --- Plasmid preparation --- p.19 / Chapter 2.5.1 --- Competent cells preparation --- p.19 / Chapter 2.5.2 --- Bacterial transformation --- p.19 / Chapter 2.5.3 --- Mini-scale preparation of plasmid DNA --- p.20 / Chapter 2.6 --- In situ hybridization --- p.21 / Chapter 2.6.1 --- Sample preparation --- p.21 / Chapter 2.6.2 --- Probe synthesis --- p.21 / Chapter 2.6.3 --- Hybridization --- p.23 / Chapter 2.6.4 --- Post-hybridization wash and antibody labeling --- p.24 / Chapter 2.6.5 --- Post-antibody wash and colour development --- p.25 / Chapter 2.6.6 --- Embryo powder preparation --- p.25 / Chapter Chapter 3: --- Time and Dose Responses to RA --- p.26 / Chapter 3.1 --- Introduction --- p.27 / Chapter 3.1.1 --- Time response --- p.27 / Chapter 3.1.2 --- Dose response --- p.29 / Chapter 3.2 --- Materials and methods --- p.31 / Chapter 3.2.1 --- Dose response --- p.31 / Chapter 3.2.2 --- Time response --- p.31 / Chapter 3.2.3 --- Skeletal preparations and staining --- p.31 / Chapter 3.2.4 --- Early teratogenic responses to RA --- p.32 / Chapter 3.3 --- Results --- p.33 / Chapter 3.3.1 --- Dose response --- p.33 / Chapter 3.3.1.1 --- Tail length --- p.33 / Chapter 3.3.1.2 --- Vertebral pattern --- p.33 / Chapter 3.3.2 --- Time response --- p.36 / Chapter 3.3.2.1 --- Tail length --- p.36 / Chapter 3.3.2.2 --- Vertebral pattern --- p.36 / Chapter 3.3.2.3 --- Other anomalies associated with human SA --- p.40 / Chapter 3.3.3 --- Early teratogenic responses in RA-treated embryos --- p.41 / Chapter 3.4 --- Discussion --- p.44 / Chapter Chapter 4: --- RA-induced Cell Death in Tail Bud --- p.51 / Chapter 4.1. --- Introduction --- p.52 / Chapter 4.1.1 --- Cell death --- p.52 / Chapter 4.1.2 --- Methods in cell death identification --- p.54 / Chapter 4.2 --- Materials and methods --- p.57 / Chapter 4.2.1 --- Histology of tail bud region --- p.57 / Chapter 4.2.2 --- Detection of internucleosomal DNA fragmentation --- p.57 / Chapter 4.2.2.1 --- Sample collection --- p.57 / Chapter 4.2.2.2 --- DNA extraction --- p.58 / Chapter 4.2.2.3 --- Analysis of DNA fragmentation pattern by ethidium bromide staining --- p.59 / Chapter 4.2.2.4 --- Analysis of DNA fragmentation pattern by autoradiography --- p.59 / Chapter 4.2.3 --- TUNEL --- p.61 / Chapter 4.2.3.1 --- Sample collection / Chapter 57 4.2.3.2 --- In situ end-labeling --- p.62 / Chapter 4.2.4 --- In situ hybridization of Wnt-5A --- p.63 / Chapter 4.2.4.1 --- Sample collection --- p.63 / Chapter 4.2.4.2 --- Probe preparation --- p.64 / Chapter 4.3 --- Results --- p.65 / Chapter 4.3.1 --- Histological examination of cell death in tail bud --- p.65 / Chapter 4.3.2 --- Analysis of DNA fragmentation of tail bud cells --- p.66 / Chapter 4.3.3 --- TUNEL --- p.67 / Chapter 4.3.4 --- Wnt-5A gene expression --- p.68 / Chapter 4.4 --- Discussion --- p.71 / Chapter Chapter 5: --- Retinoic Acid Receptors (RARs) --- p.75 / Chapter 5.1 --- Introduction --- p.76 / Chapter 5.1.1 --- RARs and their isoforms --- p.76 / Chapter 5.1.2 --- Expression pattern of RARs during embryogenesis --- p.77 / Chapter 5.1.3 --- RAR mutants and functional redundancy of RARs --- p.78 / Chapter 5.1.4 --- RARs and SA --- p.79 / Chapter 5.2 --- Materials and methods --- p.80 / Chapter 5.2.1 --- In situ hybridization of RAR-α --- p.80 / Chapter 5.2.1.1 --- Sample collection --- p.80 / Chapter 5.2.1.2 --- Probe preparation --- p.80 / Chapter 5.2.2 --- In situ hybridization of RAR-β --- p.81 / Chapter 5.2.2.1 --- Sample collection --- p.81 / Chapter 5.2.2.2 --- Probe preparation --- p.81 / Chapter 5.2.3 --- In situ hybridization of RAR-γ --- p.82 / Chapter 5.2.3.1 --- Sample collection --- p.82 / Chapter 5.2.3.2 --- Probe preparation --- p.82 / Chapter 5.3 --- Results --- p.83 / Chapter 5.3.1 --- RAR-α gene expression --- p.83 / Chapter 5.3.2 --- RAR-β gene expression --- p.84 / Chapter 5.3.3 --- RAR-γ gene expression --- p.86 / Chapter 5.4 --- Discussion --- p.88 / Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.92 / Chapter 6.1 --- Conclusion and future perspectives --- p.93 / References --- p.97 / Appendices --- p.114 / Figures --- p.118

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_321674
Date January 1996
ContributorsPoon, Lit Man., Chinese University of Hong Kong Graduate School. Division of Basic Medical Sciences.
PublisherChinese University of Hong Kong
Source SetsThe Chinese University of Hong Kong
LanguageEnglish
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xii, 118, [34] 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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