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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃筱鈞 | |
dc.contributor.author | Ming Wang | en |
dc.contributor.author | 王敏 | zh_TW |
dc.date.accessioned | 2021-05-12T09:33:31Z | - |
dc.date.available | 2019-12-17 | |
dc.date.available | 2021-05-12T09:33:31Z | - |
dc.date.copyright | 2018-12-17 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-11-29 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/handle/123456789/1159 | - |
dc.description.abstract | Anti-mitotic chemotherapy is a cancer therapy that kills cells with high division rate. Since abnormal proliferation is one of the most prominent characteristics of can-cer cells, antimitotic drugs are expected to have high selectivity and sensitivity. How-ever, non-specific targeting of hematopoietic stem cells (HSCs) that also divide rapidly is one of the most severe side effects. To overcome this problem, we constructed a synthetic AND genetic logic, utilizing two sensors, cyclin B1 promoter and miR-142-5p binding sites, in mammalian cells. miR-142-5p was chosen as it is present exclusively in hematopoietic lineages. When cells enter G2 phase of cell cycle, cyclin B1 promoter will be active to express Tet-On 3G transactivator protein. In the presence of doxycycline, the transactivator will then activate a toxic gene, here hBax-β or trun-cated caspase-3/7, tagged with miR-142-5p binding sites. In this setting, HSCs, pro-grammed with miR-142 production, toxic gene will be repressed thus cells are pro-tected, whereas in non-hematopoietic cancer cells, where miR-142 level is significant-ly lower, apoptosis will be triggered. We have successfully cloned and characterized all individual parts in the circuit and have observed the circuit’s capability to render death in HeLa cell. Moreover, we are currently optimizing the circuit by co-expressing an-other apoptotic gene and built the stable cell lines for increasing the transfection effi-cacy. However, when we construct a vector containing the two different types of pro-moter, i.e. constitutive and doxycycline-inducible, there appears to be interference between promoters. In resolving this, we tested an insulator core element and found that two tandem copies of this sequence can effectively suppress the interference. We envision our circuit will serve as a useful alterative tool for targeted cancer therapy in the near future. | en |
dc.description.provenance | Made available in DSpace on 2021-05-12T09:33:31Z (GMT). No. of bitstreams: 1 ntu-107-R05b43018-1.pdf: 2542161 bytes, checksum: 92fb20e00052ff90af6e5a61c1e8fc60 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 口試委員審定書 #
謝誌 i 摘要 iii Abstract v Contents vii Figure List x Table List xii CHAPTER 1 Introduction 1 1.1 Antimitotic chemotherapy 1 1.2 The design of cell treatment approached by Synthetic biology 1 1.3 Two sensors of the gene circuit 2 1.4 The choice of toxic gene 4 1.5 The insulator to prevent interference 5 CHAPTER 2 Materials and Methods 9 2.1 Bacterial Strain and Culture method 9 2.2.1 Strain 9 2.2.2 Culture 9 2.2 Cloning Methods of Recombinant Constructs 10 2.2.1 Vector 10 2.2.2 Plasmids preparation 11 2.2.3 PCR 12 2.2.4 Enzyme Digestion 13 2.2.5 Gel extraction and PCR Clean Up 14 2.2.6 Oligonucleotides annealing design 15 2.2.7 Ligation 16 2.2.8 Transformation & colony PCR 16 2.3 Cell lines and cell culture 17 2.4 Transfection 18 2.5 Drugs treatment and cell synchronization 19 2.6 Fluorescence microscopy and time-lapse imaging 19 2.7 Flow cytometry 20 2.8 Cell Proliferation Assay 20 2.9 Trypan Blue exclusion assay 21 2.10 primer list 22 CHAPTER 3 Results 23 3.1 Comparison between full-length and truncated cyclin B1 promoter 23 3.2 The degree of apoptosis triggered by hBax-β, cleaved-caspase-3 and cleaved-capsepase-7 25 3.3 Activation of the complete circuit in HeLa cells 26 3.4 Insulator between two promoters 28 CHAPTER 4 Discussion and Future Work 31 CHAPTER 5 Figures 33 CHAPTER 6 Reference 52 | |
dc.language.iso | en | |
dc.title | 利用細胞週期及微核糖核酸為基礎的邏輯匣專一性殺死上皮癌細胞 | zh_TW |
dc.title | A cell-cycle and microRNA-based AND logic gate
to specifically kill epithelial cancer cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 吳?承,朱雪萍 | |
dc.subject.keyword | 癌症,抗有絲分裂,基因治療,合成生物學,核糖酸干擾,細胞週期素,微型核糖核酸,四環素控制的表現系統,絕緣子, | zh_TW |
dc.subject.keyword | cancer,anti-mitotic,gene therapy,synthetic biology,RNA interference,cyclin B1,microRNA,insulator, | en |
dc.relation.page | 54 | |
dc.identifier.doi | 10.6342/NTU201804309 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2018-11-29 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
顯示於系所單位: | 分子與細胞生物學研究所 |
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