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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_327367 |
| Date | January 2011 |
| Contributors | Tsui, Wing Wun., 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, xxiii, 137 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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