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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭永銘(Yung-Ming Jeng) | |
| dc.contributor.author | Shih-Yao Peng | en |
| dc.contributor.author | 彭詩窈 | zh_TW |
| dc.date.accessioned | 2021-06-15T12:48:41Z | - |
| dc.date.available | 2021-08-26 | |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-07-22 | |
| dc.identifier.citation | 1. Schafer, D.F. and M.F. Sorrell, Hepatocellular carcinoma. Lancet, 1999. 353(9160): p. 1253-7.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50611 | - |
| dc.description.abstract | Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system (CRISPR/Cas9 system) is a highly efficient genome editing tool. It consists of two components: a strand of ribonucleic acid called small guide RNA (sgRNA) and an enzyme called CRISPR-associated endonuclease 9 (Cas9). In this system, sgRNA directs Cas9 to a targeted DNA sequence for site-specific cleavage. Recently, the mutagenic function of CRISPR/Cas9 system is widely used in genome and cancer researches. In this study, we exploited CRISPR/Cas9 system in two topics: CRISPR/Cas9 library screening in live cancer cell line and CRISPR/Cas9-mediated chromosomal translocation between Fibroblast growth factor receptor 2 (FGFR2) gene and BicC Family RNA Binding Protein 1 (BICC1) gene. In CRISPR/Cas9 library screen, we used HepG2 cell line to produce a mixture of cells with loss-of-function mutations of genes and then made tumor formation in NOD/SCID mice by subcutaneous injection of those cells. According to the consequences of next-generation sequencing, we found that some populations of CRISPR/Cas9 knockout cells dominated in quantity in late-phase tumors, so the possible explanation is that those genes knockout by the CRISPR/Cas9 system are suppressor of tumor progression. Further experiment revealed that one of the genes identified, DEDD2, can inhibit cell death caused by oxidative stress. In CRISPR/Cas9-mediated chromosomal translocation study, we used BICC1 and FGFR2-targeting CRISPR/Cas9 plasmids to produce the predicted FGFR2-BICC1 fusion DNA products. Although this method worked, the output was low. As a result, we developed the two-target CRISPR/Cas9 plasmids to improve the efficiency by increasing the possibility of cleavage of two target genes in one single cell. However, the experiment results showed that those plasmids were unable to produce the predicted chromosomal translocation products. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T12:48:41Z (GMT). No. of bitstreams: 1 ntu-105-R03444005-1.pdf: 2723109 bytes, checksum: f50209d655da314284c22155cfd0b482 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 口試委員會審定書 I
謝辭 II 中文摘要 III ABSTRACT IV CONTENTS VI 1. INTRODUCTION 1 1.1 Hepatocellular carcinoma 1 1.2 Functional screening of oncogenic and tumor suppressor proteins 3 1.3 CRISPR/Cas9 system 4 1.4 CRISPR/Cas9 knockout library screen 5 1.5 CRISPR/Cas9-mediated chromosomal translocation 6 1.6 FGFR2-BICC1 fusion 7 2. MATERIALS AND METHODS 9 2.1 Cell culture 9 2.2 Viral production and transduction 9 2.3 In vivo xenograft tumor formation in mice 10 2.4 Genomic DNA extraction and sgRNA analysis 11 2.5 Soft agar assay 12 2.6 MTT assay 13 2.7 Apoptosis assay 14 2.8 ELISA assay for 8-OHdG 14 2.9 Plasmids 15 2.10 RNA isolation 16 2.11 PCR and RT-PCR for FGFR2-BICC1 fusion 16 2.12 Surveyor assay 17 3. RESULTS 18 3.1 The preliminary testing of CRISPR library screening 18 3.1.1 CRISPR/Cas9 library-mediated mutagenesis promotes tumor growth in vivo 18 3.1.2 Enriched sgRNAs from the CRISPR screen in 6-week tumors 19 3.1.3 Knockout of DEDD2 showed enhanced anchorage-independence growth abil-ity in vitro and tumorigenic ability in vivo 20 3.1.4 DEDD2 knockout HepG2 cell line showed higher survival rate after H2O2 treatment 21 3.2 The second CRISPR screen 23 3.2.1 Dynamic evolution of sgRNA representation during tumor growth 24 3.2.2 Enriched sgRNAs from the CRISPR screen in 6-week tumors 25 3.3 CRISPR-mediated chromosomal rearrangement 26 3.3.1 Generation of FGFR2-BICC1 rearrangements and FGFR2-BICC1 fusion mRNA by the CRISPR/Cas9 System in vitro 27 3.3.2 The two-target New plasmids did not produce the FGFR2-BICC1 fusion 28 4. DISCUSSION 30 4.1 The common problems between the preliminary testing and the second CRISPR screen 30 4.2 The inconsistency between our CRISPR experiments and previous studies 31 4.3 The efficiency of generation of CRISPR-mediated FGFR2-BICC1 translocations was low 32 4.4 The FGFR2-BICC1 fusion mRNA was not detected 34 4.5 The two-target plasmid method needs improvement 34 4.6 Future improvements of our translocation experiment 35 5. FIGURES AND TABLES 36 5.1 Tables 36 5.2 Figures 42 REFERENCES 57 | |
| dc.language.iso | en | |
| dc.subject | 染色體轉位 | zh_TW |
| dc.subject | BICC1 | zh_TW |
| dc.subject | FGFR2 | zh_TW |
| dc.subject | 染色體轉位 | zh_TW |
| dc.subject | library screen | zh_TW |
| dc.subject | CRISPR/Cas9 | zh_TW |
| dc.subject | BICC1 | zh_TW |
| dc.subject | CRISPR/Cas9 | zh_TW |
| dc.subject | library screen | zh_TW |
| dc.subject | FGFR2 | zh_TW |
| dc.subject | CRISPR/Cas9 | en |
| dc.subject | CRISPR/Cas9 | en |
| dc.subject | BICC1 | en |
| dc.subject | library screen | en |
| dc.subject | chromosomal translocation | en |
| dc.subject | FGFR2 | en |
| dc.subject | BICC1 | en |
| dc.subject | library screen | en |
| dc.subject | chromosomal translocation | en |
| dc.subject | FGFR2 | en |
| dc.title | CRISPR/Cas9系統在肝癌研究之應用 | zh_TW |
| dc.title | Applications of CRISPR/Cas9 System in Liver Cancer Research | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張亨?,陳彥榮,袁瑞晃 | |
| dc.subject.keyword | CRISPR/Cas9,library screen,染色體轉位,FGFR2,BICC1, | zh_TW |
| dc.subject.keyword | CRISPR/Cas9,library screen,chromosomal translocation,FGFR2,BICC1, | en |
| dc.relation.page | 60 | |
| dc.identifier.doi | 10.6342/NTU201601097 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-07-22 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 病理學研究所 | zh_TW |
| 顯示於系所單位： | 病理學科所 | |
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