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非洲爪蛙(Xenopus laevis)和熱帶爪蛙(Xenopus tropicalis)是經典的模式動物，廣泛應用於胚胎學，發育生物學和人類疾病模型研究領域。然而，由於缺乏有效地同源重組技術和胚胎幹細胞誘導技術，在上述兩種模式動物中很難通過定點突變研究特定基因的功能。這些技術瓶頸制約其在遺傳學研究方面的應用。近來，通過鋅指核酸酶(ZFNs)和類轉錄激活因子效應物核酸酶(TALENs)介導的特異性基因突變技術成功地應用於各種模式動物模型，包括：線蟲、斑馬魚和大鼠。鋅指核酸酶和TALENs核酸酶都是由可編碼設計的DNA結合功能域和非特異性FokI核酸酶的功能域組成的人工構建DNA核酸酶。結合到相鄰DNA位點的鋅指核酸酶或TALENs核酸酶的單體通過FokI功能域結合形成二聚體，從而啟動其核酸酶活性，在預設靶點產生DNA雙鏈斷裂 (double-strand breaks)。通常，非同源末端連接(NHEJ)在修復DNA雙鏈斷裂過程中會導致缺失或者插入突變(indel)。在此研究中，我們在世界上首次利用TALENs核酸酶在爪蛙胚胎中高效誘導產生體細胞突變。我們優化了Golden Gate TALENs核酸酶的拼接方法，使其便於體外RNA轉錄和顯微注射到爪蛙胚胎中。我們還利用基於聚合酶鏈式反應(PCR)的檢測方法，用於檢測基因突變效率。我們設計八對TALENs核酸酶，它們分別靶向識別爪蛙中八個基因。試驗結果表明它們全部都能夠在爪蛙胚胎中誘導基因突變，突變率最高達百分之九十五點七(95.7%)。我們進一步證明，TALENs核酸酶誘導產生的突變可以通過生殖細胞高效地傳遞給F1代的爪蛙。不僅如此，我們還嘗試運用最新的RNA介導的基因組編輯方法(CRISPR)在小鼠誘導多功能幹細胞(iPSCs)模型中研究基因修正。初步結果顯示, 我們設計的TALENs核酸酶和CRISPR可以在小鼠誘導多功能幹細胞中有效地產生基因突變。 / 綜上所述，我們在世界上首次在爪蛙中報導了運用TALENs核酸酶進行反向遺傳學研究。利用TALENs核酸酶誘導突變的方法簡單但高效。我們的實驗結果表明TALENs核酸酶是一種在爪蛙中進行基因編輯或者基因敲除的有效工具。 / Xenopus laevis and Xenopus tropicalis are classical and powerful animal models widely used in the study of embryonic development and human disease modeling. However, due to the lack of methodologies for homologous recombination and embryonic stem cell derivation, it is difficult to perform specific gene targeting inthese two models, which has impeded their use in genetic studies for decades. Recently, site-specific gene targeting by using either zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) has been successfully applied in various animal models including C. elegans, zebrafish, and rat. Both ZFNs and TALENs are engineered DNA nucleases that consist of a custom-designed DNA-binding domain and a nonspecific nuclease domain derived from FokI endonuclease. Binding of adjacent ZFNs or TALENs allows dimerization of the endonuclease domains, leading to double-strand breaks at the predetermined site. These double-strand DNA breaks are frequently repaired through non-omologous end joining (NHEJ), resulting in deletion or insertion (indel) mutations. Here we reported that TALENs can induce somatic mutations in Xenopus embryos with reliably high efficiency and that such mutations are heritable through germline transmission. We modified the Golden Gate method for TALEN assembly to make the product suitable for in vitro RNA transcription and microinjection into Xenopus embryos, and designed a reliable PCR-based assay for the evaluation of gene disruption efficiency. Totally, eight pairs of TALENs were constructed to target eight different Xenopus genes, and all resulted in indel mutations with high efficiencies of up to 95.7% at the targeted loci according to our PCR-based assay. Furthermore, mutations induced by TALENs were highly efficiently passed through the germline to F1 frogs. Moreover, we tried to employ newly published RNA-mediated genome editing tool, clustered regularly interspaced palindromic repeat (CRISPR), to study gene correction in mice induced pluripotent stem cells (iPSCs) model. Our preliminary data showed that TALENs and CRISPR we constructed can efficiently introduce mutations at predetermined sites in mice iPSCs. / So far, our result is the first report to perform specific reverse genetic via TALENs in Xenopus. Together with simple and reliable approaches for detecting TALEN-induced mutations, our results indicate that TALENs are an effective tool for targeted gene editing/knockout in Xenopus. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lei, Yong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 146-171). / Abstracts also in Chinese. / Abstract --- p.I / Acknowledgements --- p.V / Statement --- p.VI / Abbreviation --- p.VII / Contents --- p.IX / Chapter 1. --- Research Background --- p.1 / Chapter 1.1 --- Introduction of reverse genetics --- p.1 / Chapter 1.2 --- Introduction of Xenopus tropicalis --- p.3 / Chapter 1.3 --- Xenopus tropicalis as an animal model for reverse genetics --- p.6 / Chapter 1.4 --- Zinc finger nucleases (ZFNs) --- p.10 / Chapter 1.5 --- Transcription activator-like effector nucleases (TALENs) --- p.29 / Chapter 1.6 --- Clustered regularly interspaced short palindromic repeats (CRISPR) --- p.40 / Chapter 2. --- Objective --- p.52 / Chapter 3. --- Methods and Materials --- p.54 / Chapter 3.1 --- Materials --- p.54 / Chapter 3.1.1 --- Reagents --- p.54 / Chapter 3.1.2 --- Vectors --- p.55 / Chapter 3.1.3 --- Primers --- p.57 / Chapter 3.2 --- Molecular Biology --- p.59 / Chapter 3.2.1 --- Preparation of chemical competent E.coli. --- p.59 / Chapter 3.2.2 --- Transformation --- p.60 / Chapter 3.2.3 --- Mini-preparation of plasmid --- p.61 / Chapter 3.2.4 --- Midi-preparation of plasmid --- p.62 / Chapter 3.2.5 --- Tissue RNA extraction and purification --- p.63 / Chapter 3.2.6 --- Reverse-transcription polymerase chain reaction --- p.64 / Chapter 3.2.7 --- Polymerase chain reaction (PCR) --- p.64 / Chapter 3.2.8 --- PCR/Gel extraction --- p.65 / Chapter 3.2.9 --- Synthesis of mRNA for microinjection --- p.65 / Chapter 3.2.10 --- Synthesis of DIG-labeled anti-sense RNA probe --- p.65 / Chapter 3.2.11 --- Subcloning --- p.66 / Chapter 3.2.12 --- TA cloning --- p.67 / Chapter 3.3 --- Xenopus embryo manipulation --- p.67 / Chapter 3.3.1 --- Xenopus maintenance and handling --- p.67 / Chapter 3.3.2 --- Embryos collection and handing --- p.68 / Chapter 3.3.3 --- Microinjection --- p.69 / Chapter 3.3.4 --- lacZ staining --- p.70 / Chapter 3.3.5 --- Whole-mount in situ hybridization (WHISH) --- p.70 / Chapter 3.4 --- Gene disruption via TALENs --- p.76 / Chapter 3.4.1 --- Extraction and normalization of TALEN assembly vectors --- p.76 / Chapter 3.4.2 --- TALENs assembly with Golden Gate method. --- p.78 / Chapter 3.4.3 --- Synthesize TALEN mRNAs in vitro. --- p.84 / Chapter 3.4.4 --- Microinjection of TALENs into Xenopus embryos and evaluation of gene disruption efficiency --- p.84 / Chapter 3.5 --- CRISPR gRNA synthesis --- p.90 / Chapter 3.6 --- T7E1 assay for mutagenesis detection --- p.94 / Chapter 3.7 --- Tissue culture of mouse induced pluripotent stem cells (iPSCs). --- p.94 / Chapter 3.7.1 --- STO feeder cell collection --- p.94 / Chapter 3.7.2 --- iPSCs passage --- p.95 / Chapter 3.7.3 --- List of tissue culture medium --- p.96 / Chapter 4. --- Results --- p.97 / Chapter 4.1 --- TALENs induce targeted gene disruption in Xenopus embryos --- p.97 / Chapter 4.2 --- Phenotypes of somatic mutations induced by TALENs in X. tropicalis --- p.113 / Chapter 4.3 --- Mutations induced by TALENs were heritable in X. tropicalis --- p.117 / Chapter 4.4 --- TALENs and CRISPR-Cas induced gene disruption in mouse induced pluripotent stem cells (iPSCs) --- p.119 / Chapter 4.5 --- The application of TALE-based regulator --- p.124 / Chapter 4.6 --- List of TALENs, TALE-activator, and CRISPR-Cas vectors --- p.125 / Chapter 5. --- Discussion --- p.131 / Chapter 5.1 --- TALENs induce somatic and heritable mutagenesis in Xenopus --- p.131 / Chapter 5.2 --- Engineered endonucleases are valuable tools in the study of reverse genetics --- p.136 / Chapter 5.3 --- Potential applications of ZFPs and TALEs in genetic manipulation --- p.139 / Chapter 5.4 --- Prospects of genome editing approaches in the therapies of human diseases --- p.141 / Chapter 6. --- References --- p.146 / Chapter 7. --- Publications --- p.172

The debate on genetic testing for Huntington???s disease has been dominated heavily by the bioethical and biomedical discourses. Yet upon analysis, both discourses are highly inadequate for understanding the complexity of the difficult choices people are faced with, and the inter-personal relations that are central to decisions regarding the uptake of genetic tests. The purpose of this thesis is two-fold. Firstly, to conduct a theoretically-informed critical analysis of the existing bioethical discourse on genetic testing for Huntington???s disease, that draws primarily on the work of contemporary feminist thinkers. Secondly, to explore how people with a genetic risk for Huntington???s disease negotiate the available choices between certainty and uncertainty; how they experience the liminality of ???being at risk??? in everyday life; how they manage their social environments; and how they interpret their own situation. The matter of ???choice??? is heightened because of the ready availability of genetic testing for Huntington???s disease, and the moral rhetoric that accompanies the provision of genetic services. Empirically, the research draws on the narratives by eleven people with a family history of Huntington???s disease, through which they discuss their fears of living in the shadow of the fatal disease, and consider their choices on reproduction and genetic testing. Their narratives will be analysed through the work of Foucault and Goffman, as well as a wide range of contemporary sociologists.The thesis being proposed is that decisions on genetic testing cannot be said to be ???individual???, but are instead dispersed among the social relations between the self and others, reflecting and transforming the values, competing desires, and the discourses that are prevalent in their social worlds. This is achieved through the discursive production of a web of narratives through which both individuals and institutions attempt to govern, with varying degrees of success, the implications of this relatively new field of knowledge.

Copy of author's previously published work inserted. Bibliography: leaves 193-216. Thesis which examines in detail the genetics of congental ataxias, and early and late onset ataxias.

Bibliography: leaves 116-129.

Interleukin (IL)-3, IL-5 and granulocyte-macrophage-colony-stimulating factor (GM-CSF) are cytokines that affect the growth, survival and differentiation of many cells within the haematopoietic system. The functions of these factors are mediated by membrane bound receptor complexes that are composed of specific ligand binding subunits (α)and a common signal transducing subunit(hβc). Constitutively activated mutants of hβc have been previously identified that are able to confer factor-independent signalling in a number of haematopoietic cell lines (including FDC-P1 and FDB-1). These activated mutants fall into two classes defined by the location of the mutation and their biochemical and leukaemogenic properties. In particular, the transmembrane mutant, V449E, causes an acute myeloid leukaemia in vivo, whereas the extracellular mutants (FI∆ or I374N) cause chronic myeloproliferative disorders. The work described in this thesis used the activated hβc mutants to uncover novel transcriptional events induced by the GM-CSF/IL-3/IL-5 receptor complex and to define pathways associated with proliferation and differentiation. Large-scale gene expression profiling techniques were used to investigate the genes involved in these biological processes in the murine myelomonocytic cell line FDC-P1, and the bi-potent FDB-1 myeloid cell line, which are responsive to IL-3 and GM-CSF. Membrane arrays were used to identify differences in gene expression between I374N and V449E expressing FDC-P1 cells. This technique revealed that the gene Ptpmt1 was differentially expressed between V449E and I374N, which was subsequently confirmed by Northern blotting. This finding suggested that the phosphatase encoded by Ptpmt1 may be involved in the different outcomes induced by these two hβc mutants. Northern analysis also revealed Ptpmt1, Nab1 and Ddx26b to be regulated in response to human GM-CSF in FDC-P1 cells expressing human GM CSFα and hβc. A large-scale cDNA microarray experiment was also performed to identify genes that are selectively expressed during differentiation of FI∆ expressing FDB-1 cells, compared to proliferating V449E expressing FDB-1 cells over 24 hours. A comprehensive analysis approach was adopted to examine the microarray data and identify differentially expressed genes. Among the genes displaying differential expression were Btg1, S100a9, Cd24, and Ltf found to be differentiation-associated and Bnip3, Cd34, Myc, Nucleophosmin, and Nucleostemin found to be proliferation-associated. Hipk1, Klf6, Sp100, and Sfrs3 were also identified as potential transcriptional regulators during growth and differentiation. Northern analysis was used to confirm differences in expression for these 13 genes between FI∆ and V449E expressing FDB-1 cells. Eleven of the 13 genes examined were confirmed to be differentially expressed between FI∆ and V449E expressing FDB-1 cells over 24 hours. Furthermore, six genes (Btg1, Hipk1, Cd24, Cd34, Klf6 and Nucleostemin) examined over 72 hours revealed differences in gene expression at early (6-12 hours) and late (48-72 hours) time points. Cell surface expression of CD24 protein was also shown to be induced upon FI∆ expression or GM-CSF induced differentiation of FDB-1 cells, consistent with elevated levels of Cd24 mRNA in FI∆ cells over time. Based on their confirmed gene expression differences seen on the microarrays and Northern analysis, four genes (Btg1, Cd24, Klf6 and Nucleostemin) were selected for over-expression analysis in FDC-P1 or FDB-1 cells, in order to gain insights into the function of these genes. Optimisation of the retroviral infection process was performed so that the role of these genes in proliferation and differentiation could be investigated in the FDB-1 model. Such preliminary functional experiments in FDB-1 cells will enable prioritisation of the genes for further analysis of their function in primary cells. Thus, the work in this thesis describes the first use of microarrays to identify gene expression differences between hβc mutants with differential activities affecting myeloid growth and differentiation. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1284103 / Thesis (PhD)-- School of Medicine, 2005

Bibliography: leaves 116-129 / iv, 129 leaves, [23] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1987

Includes a copy of an article co-authored by the author during the preparation of this thesis. / Bibliography: leaves 132-148. / iv, 148 leaves : ill. (some col.); 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis describes the screening of the sheep wool follicle for expression of Antennapedia-like homeobox genes by RT-PCR and the isolation and characterisation of the Hoxc-13 gene and a novel hoeobox gene, Barx2. / Thesis (Ph.D.)--Adelaide University, Dept. of Animal Science, 2001

Retinoic acid (RA) exerts important effects in the processes of vertebrate development, cellular growth and differentiation, and homeostasis. However, the mechanisms of action of RA in the control of cellular and developmental processes are incompletely understood, as the retinoid target genes have not been fully characterized. The goal of these studies described herein was to contribute towards a greater understanding of the cellular effects of retinoids through the identification and characterization of an RA-induced gene from mouse P19 embryonal carcinoma cells. The predicted amino acid sequence of GRASP is characterized by several putative protein-protein interaction motifs, suggesting that GRASP may function in cell signaling pathways. Towards the goal of identifying which signaling pathways GRASP may participate in, a yeast two-hybrid screen was performed using GRASP as a bait to identify protein interaction partners. The general receptor for phosphinositides 1 (GRP1), a guanine nucleotide exchange factor for the ADP-ribosylation factor 6 (ARF6) GTPase, was identified as a GRASP interaction partner. GRASP was shown to colocalize with endogenous ARFs in cells and enhance GRP1 association with the plasma membrane, suggesting that GRASP may function as a scaffold protein in the recruitment of GRP1 and ARF6 to plasma membrane loci. Overexpression of GRASP was observed to induce accumulation of GRASP in the endosomal compartment where GTP-binding deficient mutants of ARF6 reside, suggesting that GRASP induced a block in an ARF6 plasma membrane recycling pathway. Coexpression of GRP1, but not a catalytically inactive mutant, dramatically reduced the accumulation of GRASP in this compartment. Furthermore, GRP1 mutants that lack the region of interaction with GRASP failed to prevent accumulation of GRASP in the endosomal compartment, suggesting that GRASP recruits GRP1 to the endosomal compartment where GRP1 stimulates nucleotide exchange on ARF6 and recycling. Results described herein demonstrate that GRASP functions in the ARF6 regulated plasma membrane recycling pathway, and that upon overexpression, induces a block in recycling. Our results suggest a role for GRASP as an adapter or scaffold protein that may link cell surface receptors to the ARF6 recycling pathway, resulting in modulation of signal transduction events at the cell surface. / Graduation date: 2002

Graduation date: 1994