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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 江運金 | zh_TW |
| dc.contributor.advisor | Yun-Jin Jiang | en |
| dc.contributor.author | 王辰瑜 | zh_TW |
| dc.contributor.author | Chen-Yu Wang | en |
| dc.date.accessioned | 2024-09-10T16:17:46Z | - |
| dc.date.available | 2024-09-11 | - |
| dc.date.copyright | 2024-09-10 | - |
| dc.date.issued | 2024 | - |
| dc.date.submitted | 2024-08-05 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/95481 | - |
| dc.description.abstract | 為了研究基因功能以及與疾病的關聯性，我們會使用許多方法來使基因喪失功能，但生物體常常能夠演化出需多不同的機制，來調適基因功能的缺失並達成所謂的遺傳穩健性。
在我們實驗室先前所做過的研究中，我們使用斑馬魚作為模式動物來模擬偏頭痛(migraine)並且具有不寧腿症候群(Restless legs syndrome)共病的病人。我們實驗室使用不同方法降低/去除ccdc141與vstm2l之基因表現，有利用嗎啉基(morpholino)影響蛋白質生成或產物改變、利用失活的常間回文重複序列叢集關聯蛋白(CRISPR/dCas9)系統進行轉錄阻礙，並且利用注射CRISPR/Cas9的4天大幼魚三種方式進行分析，均觀察到類似的性狀表現，包含有表現酪胺酸羥化酶(tyrosine hydroxylase)之視網膜無長突細胞(amacrine cell)的數目減少，以及胸鰭(pectoral fin)擺動頻率上升。然而，在使用CRISPR/Cas9創造的單一位點基因敲除突變種中，卻沒有發現上述性狀表現。回顧文獻後發現先前已經有許多機制被發表，包含遺傳補償效應(genetic compensation)/轉錄適應(transcriptional adaptation)、外顯子跳躍(exon skipping)、替代性轉譯起始(alternative translation initiation)等等。 首先，經由雙基因突變種(double mutant line)的創造，我們排除了ccdc141和vstm2l間可能具有遺傳冗餘性(genetic redundancy)。在vstm2lnn2608-/- 中，在3’端的基因表現/擴增子(amplicon)量遠低於5’端，顯示可能在此同型合子突變種中可能發生由3’端往5’端的信使核糖核酸降解; 此外，在注射upf3a嗎啉基進入突變種後，表現酪胺酸羥化酶之視網膜無長突細胞的數目下降。綜合以上結果，我們認為轉錄適應為最有可能的基因功能補償機轉，在使用核糖核酸定序(RNA-seq)進行基因表現量分析後，發現三個在突變種中表現量上升但在嗎啉基注射之幼魚中表現量無改變的基因，而這些基因表現的上升是否可以彌補vstm2l的失能還需要後續研究再證實。在ccdc141nn2704-/-中，透過將互補去氧核醣核酸克隆至載體中並進行定序，確認了此突變種未經歷外顯子跳躍，並且透過利用人類胚腎細胞株HEK293T，確認表現ccdc141nn2704-/-序列的蛋白是一種缺少N端約104個胺基酸的胜肽，並且可能是透過在提前終止密碼子(pre-mature stop codon)下游之替代性轉譯起始點開始轉譯出蛋白。為了確認真正基因被失活後的突變株之表現性狀，我創造了以無信使核糖核酸表現為目標的幾個基因敲除突變種，但發現這些突變種仍有用一些未知機制表現信使核糖核酸，並且也未看到相似於先前研究中的性狀，而這些原因仍待之後的研究來進行釐清。 總結上述內容，此研究的結果支持ccdc141與vstm2l分別以不同的方法來減低基因被突變所造成的影響。並且未來仍有些項目可繼續進行，如研究出真正彌補vstm2l功能的基因與機轉，以及測試在細胞株中所證實的機轉是否可以運用在ccdc141斑馬魚突變種中，還有解決大片段基因敲除魚的問題並創造真正具有基因失活特性的突變種等。 | zh_TW |
| dc.description.abstract | Lots of loss-of-function strategies were used to study the function of genes, whereas biological systems may adapt the genomic defect through different mechanisms. In previous study, we found similar results in our zebrafish model for migraine patients with RLS (morphants, transcriptional knockdown larvae, and crispants of ccdc141 and vstm2l), which displayed a lower number of tyrosine hydroxylase-expressing amacrine cells, and some of them also displayed hyperkinetic pectoral fin movements. However, in the CRISPR/KO mutant lines of these two genes, showed neither decreased amacrine cell number nor hyperkinetic pectoral fin movements. Literatures had shown some genetic compensation mechanisms in different model organisms, such as transcriptional adaptation, exon skipping, alternative translation initiation, and so on. We would like to find out whether these mechanisms could happen in our zebrafish larvae.
Through creating the double mutant line, we demonstrated that these two genes do not play a redundant role of each other. In vstm2lnn2608-/-, the mRNA amplicon level at 3’ end is much lower than that at 5’ end, implicating a degradation event. Moreover, upf3a MO injection caused decreased amacrine cell number. Combing these two results, transcriptional adaptation was concluded as the possible mechanism to compensate genomic defect. RNA-sequencing results found that some genes were up-regulated in mutants but not morphants, and the compensatory effect needs to be verified further. In ccdc141nn2704-/-, we excluded exon skipping mechanism through cDNA cloning, and demonstrated an alternative peptide would be translated from downstream AUG using HEK293T cell lines. Some large fragment deletion lines were created to validate the phenotypes in null allele mutants. However, we failed to create mRNA-less mutants, and the reason should be discussed further. In conclusion, the results suggested that these two mutant lines in our lab may exploit different mechanisms to reduce the impact of mutations. We should verify the compensatory role in vstm2l mutants, and test the alternative translation initiation mechanism in ccdc141 larvae. Moreover, solve the problems in large fragment deletion lines in the future. | en |
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| dc.description.tableofcontents | 中文摘要……………………………………………………………..………………..... i
Abstract...……………………………………………………………………………... iii Table of Contents ...…………………………………………………………...………...v Index of Figures …………………………………………………………….……….... vi Index of Tables ...…………………………………………………………………....... vii Index of Supplementary Figures ...………………………………………………….. vii Index of Supplementary Table ......………………………………………………….. vii Chapter 1. Introductions ...…………………………………………………………… 1 1.1 Introduction to genetic robustness and gene compensation ………...………… 1 1.2 Introduction to migraine and restless legs syndrome and their risky genes identified in our lab ……………………………...…………………………... 2 1.3 Introduction to loss-of-function methods and their molecular effects in this study ………………...………………………………………….……………... 3 1.4 Previous works, hypotheses and aims …………………………….…………... 4 Chapter 2. Materials and Methods ...………………………………….……………... 8 2.1 Maintenance of zebrafish …………………….……...………………………... 8 2.2 Information and identification of mutant lines …………...……….…………... 8 2.3 Whole mount in situ hybridization (WISH) .…………………….…………... 12 2.4 Analysis of pectoral fin flapping frequency …………………….………….... 13 2.5 Quantitative reverse transcription PCR (qRT-PCR) .……………..……..…... 13 2.6 Morpholino knockdown …………………….……...……………….……….. 15 2.7 Large fragment deletion knockout .……………….……...……………….….. 16 2.8 cDNA cloning (transcript screening) …………………….……....….……….. 17 2.9 myc fusion clone expression in HEK293T cell lines ………….…….……….. 18 2.10 Mass spectrometry ……………..………….……....……………….……….. 20 2.11 RNA sequencing (RNA-seq) ……………..………….….…....…………….. 21 Chapter 3. Results ...……………………………………………………….…………. 23 3.1 Tyrosine hydroxylase expressing cell number and pectoral fin flapping frequency in mutant lines ..…………………………………………..………. 23 3.2 ccdc141 and vstm2l may not be the redundant genes in RLS pathogenesis 23 3.3 Relative mRNA expression level in mutant lines (ccdc141 & vstm2l) …....… 24 3.4 Transcripts clones exclude the possibility that exon skipping events happened in mutant lines ..….………………………………………………..……………. 25 3.5 upf3a MO injection partially restores the phenotype in vstm2lnn2608 homozygous larvae ……………….…………………………………………..……………. 26 3.6 Screening up-regulating genes in vstm2lnn2608 mutants through RNA-Seq ….. 27 3.7 ccdc141 mutant clone can translate in-frame peptide in HEK293T cells ….. 27 3.8 ccdc141 mutant clone can translate in-frame peptide from downstream alternative translation initiation site ……………..…..….….….…....……….. 28 3.9 Phenotypes and molecular effects of large fragment deletion ….………….. 29 3.10 5’-RACE of ccdc141 promoter-less knockout line (ccdc141nn2700) …...…… 30 Chapter 4. Discussions ....………………….……………………………………….... 46 4.1 How to choose proper crRNA target site for loss-of-function model ……...… 46 4.2 Alternative translation initiation usage, and efficiency …..……….……….. 47 4.3 Implications of RNA-seq data from vstm2l morphant and mutant …..…….. 49 4.4 Loss-of-function or gain-of-function effects from pathogenic association (SNP) of patients …...….………………………………………………………….. 49 4.5 Prospection of drug selection model using morphants, or crispants ……….… 50 References ....…………………………………………………………………………. 56 List of Abbreviations ....………….…………………………………………………... 65 Index of Figures Figure A. Information of ccdc141 mutant lines ……..…….…………………………... 10 Figure B. Information of vstm2l mutant lines ……..…….…...………………………... 10 Figure 1. Tyrosine hydroxylase-expressing amacrine cell number in 5 single site mutant lines ……………………………………………………………………………… 31 Figure 2. Pectoral fin flapping frequency in single site mutant lines ……….………… 32 Figure 3. Double knockout line does not display phenotypes like morphants ……… 33 Figure 4. Extremely lower amplicons level at 3’ end of mRNA implicates the degradation of transcript in vtsm2lnn2608-/-.………….………………………………………….. 34 Figure 5. Transcripts cloning exclude the possibility that exon skipping events happened in ccdc141nn2704-/- andvstm2lnn2608-/- ……………………………………………… 35 Figure 6. th+ amacrine cell number were decreased after upf3a MO injection in vstm2lnn2608-/- mutants …………………………………………………………... 36 Figure 7. RNA sequencing results reveals differential expression level in vstm2lnn2608-/-compared with vstm2lnn2608+/+ ………………………...…………………………. 37 Figure 8. ccdc141nn2704 mutant clone could translate in-frame peptide with shorter length than wild type clone ……………………….………...…………...………………. 39 Figure 9. Mass spectrometry confirm the sequence identity of mutant protein is similar with wild type protein .…………………………………………………………… 40 Figure 10. Homozygote clone could not translate proper peptide after mutating the alternative translation start site at 105th amino acid …………………………… 41 Figure 11. Large deletion of genomic region of ccdc141 and amacrine cell phenotype 42 Figure 12. Large Deletion of genomic region of vstm2l and amacrine cell phenotype .. 44 Index of Tables Table 1. Genotyping methods and primer sequences of mutant lines ……….………... 11 Table 2. qRT-PCR primers ……….……………………………….…………………... 14 Table 3. Information and phenotype observation of morpholinos …………………… 15 Index of Supplementary Figures Supplementary Figure 1. ccdc141 crRNA activity test and efficiency of genomic deletionin injected embryos …………………………….…….….……………………….. 52 Supplementary Figure 2. vstm2l crRNA activity test and efficiency of genomic deletion in injected embryos ……………………………….….………………………….. 53 Supplementary Figure 3. Translation initiation in ccdc141 mutant was affected when injecting AUG morpholino …………………………………………………...….. 54 Index of Supplementary Tables Supplementary Table 1. Top 20 annotated GO terms (categorized by molecular function) …………………….…………………………………..............................….. 55 Supplementary Table 2. Top 20 annotated GO terms (categorized by cellular component) …….…………………………………………………..........................….. 55 | - |
| dc.language.iso | en | - |
| dc.subject | 替代性轉譯起始 | zh_TW |
| dc.subject | 遺傳穩健性 | zh_TW |
| dc.subject | 斑馬魚 | zh_TW |
| dc.subject | vstm2l | zh_TW |
| dc.subject | 遺傳補償效應 | zh_TW |
| dc.subject | ccdc141 | zh_TW |
| dc.subject | alternative translation initiation | en |
| dc.subject | zebrafish | en |
| dc.subject | genetic robustness | en |
| dc.subject | ccdc141 | en |
| dc.subject | vstm2l | en |
| dc.subject | transcriptional adaptation | en |
| dc.title | 斑馬魚變異種以不同的方式達成遺傳穩健性 | zh_TW |
| dc.title | Genetic Robustness of Zebrafish ccdc141 and vstm2l Mutants in Different Ways | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 112-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 陳俊豪;賴時磊 | zh_TW |
| dc.contributor.oralexamcommittee | Chun-Hao Chen;Shih-Lei Lai | en |
| dc.subject.keyword | 斑馬魚,遺傳穩健性,替代性轉譯起始,遺傳補償效應,ccdc141,vstm2l, | zh_TW |
| dc.subject.keyword | zebrafish,genetic robustness,ccdc141,vstm2l,transcriptional adaptation,alternative translation initiation, | en |
| dc.relation.page | 66 | - |
| dc.identifier.doi | 10.6342/NTU202401763 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2024-08-09 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | - |
| dc.date.embargo-lift | 2027-08-31 | - |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-112-2.pdf 未授權公開取用 | 3.38 MB | Adobe PDF | 檢視/開啟 |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。