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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃楓婷 | |
| dc.contributor.author | Jia-Yu Lin | en |
| dc.contributor.author | 林家伃 | zh_TW |
| dc.date.accessioned | 2021-06-16T05:10:17Z | - |
| dc.date.available | 2014-08-25 | |
| dc.date.copyright | 2014-08-25 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-19 | |
| dc.identifier.citation | Bassing, C. H., Swat, W., & Alt, F. W. (2002). The mechanism and regulation of chromosomal V(D)J recombination. Cell, 109 Suppl, S45-55.
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Transcription-coupled events associating with immunoglobulin switch region chromatin. Science, 302(5653), 2137-2140. Okazaki, I. M., Hiai, H., Kakazu, N., Yamada, S., Muramatsu, M., Kinoshita, K., & Honjo, T. (2003). Constitutive expression of AID leads to tumorigenesis. J Exp Med, 197(9), 1173-1181. Okazaki, I. M., Kotani, A., & Honjo, T. (2007). Role of AID in tumorigenesis. Adv Immunol, 94, 245-273. Peled, J. U., Kuang, F. L., Iglesias-Ussel, M. D., Roa, S., Kalis, S. L., Goodman, M. F., & Scharff, M. D. (2008). The biochemistry of somatic hypermutation. Annu Rev Immunol, 26, 481-511. Roscigno, R. F., & Garcia-Blanco, M. A. (1995). SR proteins escort the U4/U6.U5 tri-snRNP to the spliceosome. RNA, 1(7), 692-706. Sanford, J. R., Gray, N. K., Beckmann, K., & Caceres, J. F. (2004). A novel role for shuttling SR proteins in mRNA translation. Genes Dev, 18(7), 755-768. Stavnezer, J., & Schrader, C. E. (2006). 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R., Jr., Ye, Z., Liu, F., Rosenfeld, M. G., Manley, J. L., Ross, J., Jr., Chen, J., Xiao, R. P., Cheng, H., Fu, X. D. (2005). ASF/SF2-regulated CaMKIIdelta alternative splicing temporally reprograms excitation-contraction coupling in cardiac muscle. Cell, 120(1), 59-72. Xu, Z., Zan, H., Pone, E. J., Mai, T., & Casali, P. (2012). Immunoglobulin class-switch DNA recombination: induction, targeting and beyond. Nat Rev Immunol, 12(7), 517-531. Yu, K., Chedin, F., Hsieh, C. L., Wilson, T. E., & Lieber, M. R. (2003). R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat Immunol, 4(5), 442-451. Zhang, Z., & Krainer, A. R. (2004). Involvement of SR proteins in mRNA surveillance. Mol Cell, 16(4), 597-607. Zhou, Z., & Fu, X. D. (2013). Regulation of splicing by SR proteins and SR protein-specific kinases. Chromosoma, 122(3), 191-207. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55893 | - |
| dc.description.abstract | 為能有效保護宿主不受到不同的外來抗原入侵，B細胞必須具備製造多樣化抗體的能力，而其中之一便是透過抗體類型轉換重組機制 (Class switch recombination, CSR) 使細胞表面從表現抗體IgM轉變為其他類型抗體。Srsf1能藉由阻止R-loop結構生成以穩定基因體，且其異構型Srsf1-3可能參與於SHM中。由於R-loop結構被認為存在於CSR中，且SHM和CSR兩機制有許多相似處，我們推測Srsf1亦有可能參與在CSR中。在論文中我們透過兩部分的實驗探討Srsf1於CSR中的可能角色：觀察CSR進行時的Srsf1特性變化包含srsf1基因表現及其於細胞中分佈情形，以及於小鼠B細胞株 (CH12F3) 中進行srsf1基因剔除。由實驗結果發現，細胞經刺激後，Srsf1-1的mRNA含量於刺激兩天組別無顯著變化，於刺激三天組別下降至九成左右，其蛋白質表現量則於刺激兩天及三天後下降至未受刺激組別的八成左右，此外，Srsf1-3的mRNA含量亦於刺激兩天及三天後下降至未受刺激組別的八成左右。透過核質分離觀察Srsf1-1於細胞中的分佈，發現於未受刺激及受刺激組別中，大部份Srsf1-1皆位於細胞核中，且細胞經刺激後，細胞質中Srsf1-1下降至未受刺激組別之六成左右，而細胞核部分則有待內部控制蛋白質 (internal control) 決定後才能進一步分析。接著第二部分實驗於CH12F3細胞中進行srsf1 基因剔除，欲確認其對CSR的重要性，目前針對srsf1+/-細胞株經刺激後初步檢測其CSR發生頻率，發現和wild type組別相比並無顯著變化，而srsf1 基因可能對於細胞生存具重要性，致使目前尚未得到srsf1-/-細胞株。綜合目前的實驗結果，Srsf1於CSR中的功能仍需更多實驗證據進行推論。 | zh_TW |
| dc.description.abstract | To effectively protect the host against different kinds of pathogens, B cells are capable to secret various isotypes of antibodies. Through class switch recombination (CSR), the immunoglobulin isotype of B cells switch from IgM to other isotypes. Srsf1 was known to maintain genome stability by preventing R-loops formation, and its isoform Srsf1-3 might be involved in somatic hypermutation (SHM). Since R-loop structures exist in the CSR process and SHM shares similar mechanisms with CSR, we speculated that Srsf1 might also participate in CSR.
The aim of the thesis is to investigate the role of Srsf1 in CSR from two aspects. First, the characteristics changes of Srsf1 during CSR were addressed, including the expression level and subcellular location of Srsf1. Second, the srsf1 gene was knocked out in CH12F3 cells, a murine B cell line as the CSR model. After CSR stimulation, the mRNA amounts of srsf1-1 remained unchanged in two-day-stimulated group, and decrease by 10% in three-day-stimulated group, and the protein amount of Srsf1-1 decreased slightly by about 20% after stimulation for two days and three days. The mRNA amounts of srsf1-3 decreased slightly by about 20% after stimulation for two days and three days. Next, Srsf1-1 was found mostly located in the nuclear fraction in both unstimulated and stimulated cells. Moreover, the Srsf1-1 amount in the cytoplasm fraction decreased apparently by about 40%. However, the change of the Srsf1 amount in the nuclear fraction was not determined yet due to the uncertainty of the suitable internal control. Finally, srsf1+/- cell clones were generated by knocking out the srsf1 gene in CH12F3 cells. Preliminary results of srsf1+/- cells showed no significant influence on CSR. Nevertheless, srsf1-/- cells failed to be obtained possibly due to the important role of Srsf1 in cell survival. In conclusion, the role of Srsf1 in CSR needs more experiments to be confirmed. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T05:10:17Z (GMT). No. of bitstreams: 1 ntu-103-R01b22015-1.pdf: 15294518 bytes, checksum: 12670f346ec62121a4b9afe47ebbcd58 (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 謝辭 i
中文摘要 ii Abstract iii Abbreviations v Table of contents vi Chapter 1 Introduction 1 1.1 Immunoglobulin and immunoglobulin diversity 1 1.2 Class switch recombination (CSR) 2 1.3 Serine arginine rich splicing factor 1 (Srsf1) 5 1.3.1 SR protein family 5 1.3.2 Functions of Srsf1 6 1.3.3 Localization of Srsf1 7 1.4 Research purpose 9 Chapter 2 Materials and Methods 10 2.1 Cell culture and CSR rate determination 10 2.1.1 Cell culture 10 2.1.2 Induction of CSR 10 2.1.3 Cell staining and flow cytometry analysis for CSR rate 10 2.2 Srsf1 gene knockout 11 2.2.1 Srsf1 knockout plasmid construction 11 2.2.2 Srsf1 knockout plasmid transfection 11 2.2.3 Genomic DNA extraction (Rough method) 12 2.2.4 Genomic DNA extraction (Phenol-chloroform extraction) 13 2.3 mRNA quantification 13 2.3.1 Total RNA extraction 13 2.3.2 DNase treatment 14 2.3.3 cDNA synthesis 15 2.3.4 Reverse-transcription quantitative PCR (RT-qPCR) 15 2.3.5 Relative quantification 15 2.4 Protein analysis 16 2.4.1 Whole cell lysate preparation 16 2.4.2 Subcellular fractionation 16 2.4.3 Gel electrophoresis 17 2.4.4 Transfer 17 2.4.5 Immunoblotting 18 2.5 Southern blot 18 2.5.1 Genomic DNA digestion 18 2.5.2 Electrophoresis and gel denaturation 18 2.5.3 Transfer and DNA-crosslink 19 2.5.4 Probe labeling 19 2.5.5 Pre-hybridization and probe hybridization 19 2.5.6 Immunological detection 20 Chapter 3 Results 21 3.1 Characteristics of Srsf1 during CSR 21 3.1.1 Induction of CSR in CH12F3 cells 21 3.1.2 The mRNA level of srsf1-1 and srsf1-3 during CSR 21 3.1.3 The protein level of Srsf1-1 during CSR 21 3.1.4 Subcellular localization of Srsf1-1 during CSR 22 3.1.5 Phosphorylation status of Srsf1-1 23 3.1.6 Sorting of IgA+- and IgA--expressing CH12F3 cells 23 3.2 Srsf1 gene knockout in the CH12F3 cell line 24 3.2.1 First allele knockout of the srsf1 gene 24 3.2.2 Southern blot for srsf1+/- genotyping 25 3.2.3 Second allele knockout of the srsf1 gene 26 3.2.4 Expression of srsf1 gene in srsf1 knockout CH12F3 26 3.2.5 Effect of srsf1 gene knockout in CH12F3 cells on CSR 27 Chapter 4 Discussion 28 4.1 Regulation of srsf1 gene in CSR 28 4.2 Internal controls for the nuclear fraction 29 4.3 Importance of Srsf1 in cell survival of CH12F3 29 4.4 Antibody for Srsf1-3 30 Chapter 5 Future work 31 Chapter 6 Figures and tables 32 Figure 1. CSR rate detection in stimulated CH12F3 cells by flow cytometry 32 Figure 2. mRNA level of srsf1-1 and srsf-3 in stimulated CH12F3 cells 33 Figure 3. Protein level of Srsf1-1 in stimulated CH12F3 cells 34 Figure 4. Subcellular fraction of Srsf1 in CH12F3 cells by Western blotting 36 Figure 5. The phosphorylation status of Srsf1-1 in different subcellular fractions in stimulated CH12F3 cells 37 Figure 6. Srsf1 gene expression level in IgA-- and IgA+-expressing cells by RT-qPCR and Western blotting 38 Figure 7. Southern blot for srsf1+/- genotype check 39 Figure 8. Southern blot for genotyping of different CH12F3 cell clones 40 Figure 9. Srsf1 gene expression in srsf1+/- cells 41 Figure 10. The CSR rate of stimulated srsf1+/- cells by flow cytometry 42 Reference list 43 Appendixes 48 A. Details of primers 48 B. PCR conditions 49 C. Antibodies for western blotting 50 D. Candidate α-Srsf1-3 antibodies and the immunoblotting results 51 E. Diagram of pKY-TV-Srsf1 and PCR primers for genotyping 52 F. EcoRV sites and probe-detecting fragments for each allele type 53 G. Diagram of RT-qPCR primers for quantification of srsf1-1 and srsf1-3 54 口試委員之提問與建議 55 | |
| dc.language.iso | en | |
| dc.subject | 抗體類型轉換重組 | zh_TW |
| dc.subject | Srsf1 | zh_TW |
| dc.subject | Srsf1異構型 | zh_TW |
| dc.subject | 核質分離 | zh_TW |
| dc.subject | 基因剔除 | zh_TW |
| dc.subject | gene knockout | en |
| dc.subject | Srsf1 | en |
| dc.subject | Srsf1 isoform | en |
| dc.subject | subcellular fractionation | en |
| dc.subject | class switch recombination (CSR) | en |
| dc.title | Srsf1於抗體類型轉換重組機制中扮演之角色探討 | zh_TW |
| dc.title | Investigation of the Role of Srsf1 in Class Switch Recombination | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 常怡雍,冀宏源,黃兆祺 | |
| dc.subject.keyword | 抗體類型轉換重組,Srsf1,Srsf1異構型,核質分離,基因剔除, | zh_TW |
| dc.subject.keyword | class switch recombination (CSR),Srsf1,Srsf1 isoform,subcellular fractionation,gene knockout, | en |
| dc.relation.page | 57 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-08-19 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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