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Simon, Ming-yuen Lee. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 239-257). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Ng Kai-on. / "July 2001." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 162-180). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

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

Cells of the ventrolateral dermomyotome delaminate and migrate into the limb buds where they give rise to all muscles of the limbs. The migratory cells proliferate and form myoblasts, which withdraw from the cell cycle to become terminally differentiated myocytes. The regulatory mechanisms that control the later steps of this myogenic program are not well understood. The homeodomain transcription factor Pitx2 is expressed specifically in the muscle lineage from the migration of precursors to adult muscle. Ablation of Pitx2 results in distortion, rather than loss, of limb muscle anlagen, suggesting that its function becomes critical during the colonization of, and/or fiber assembly in, the anlagen. Microarrays were used to identify changes in gene expression in flow-sorted migratory muscle precursors from Wild type and Pitx2 null mice. Changes in gene expression were observed in genes encoding cytoskeletal, adhesion and fusion proteins which play a role in cell motility and myoblast fusion. We observed decreased cellular motility, disrupted cytoskeleton organization and focal adhesion distribution, decreased fusion of mononucleated myoblasts into multinucleated myotubes and decreased proliferation in presence of Ptix2. These studies suggest that Pitx2 plays a critical role in regulating the timing of myoblast filling the limb anlagen which may have detrimental consequences for higher order muscle architecture. / Graduation date: 2013

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 63-82).

Thesis (Ph. D.)--University of Oklahoma. / Includes bibliographical references.

Homeodomains (HDs) are a large family of DNA-binding domains contained in transcription factors that are most notable for regulating body development and patterning in metazoans. HDs consist of three alpha helices preceded by an N- terminal arm, where the third helix (the recognition helix) and the N-terminal arm are responsible for defining DNA-binding specificity. Here we attempted to engineer the HDs by fully randomizing positions in the recognition helix to specify each of the 64 possible 3’ triplet sites (i.e. TAANNN). We recovered HD variants that preferentially recognize or are compatible with 44 of the possible sites, a dramatic increase from the previously observed range of specificities. Many of these HD variants contain combinations of novel specificity determinants that are uncommon or absent in extant HDs, where these determinants can be grafted into alternate HD backbones with an accompanying alteration in their specificity. The identified determinates expand our understanding of HD recognition, allowing for the creation of more explicit recognition models for this family. Additionally, we demonstrate that HDs can recognize a broader range of DNA sequences than anticipated, thus raising questions about the fitness barrier that restricts the evolution HD-DNA recognition in nature. Finally, these new HD variants have utility as DNA-binding domains to direct targeting of customizable sequence-specific nuclease as demonstrated by site-specific lesions created in zebrafish. Thus HDs can guide sequence-specific enzymatic function precisely and predictably within a complex genome when used in engineered artificial enzymes.

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