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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328571 |
| Date | January 2013 |
| Contributors | Lei, Yong, Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xi, 172 leaves) : ill. (some col.) |
| 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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