心血管疾病在大多數國家都是主要死因之一。其中，心肌梗塞每年累計有數百萬病例。心肌梗塞發生後，人體心臟會累積纖維蛋白，並發生心室擴大的現象，使病患心臟的血液供給功能下降。相比於人類，斑馬魚可以在心臟受損後完整修復組織，並且恢復心臟功能。因此斑馬魚這種模式生物近年被廣泛用於研究心臟再生。在人類個案研究和小鼠模式中，syndecan-4 (SDC4) 此一蛋白被指出可調控多種細胞和分子的功能。為了研究syndecan-4在斑馬魚心臟修復過程中的功能，我們建立了一個帶有sdc4基因突變的斑馬魚品系。使用的基因編輯工具為clustered regularly interspaced short palindromic repeats and the CRISPR-associated protein 9 (CRISPR/Cas9) 系統。將Cas9 mRNA和針對sdc4基因編輯的guide RNA以顯微注射技術注射到野生斑馬魚胚胎後，在sdc4目標基因片段會產生DNA雙股斷裂，並在DNA雙股斷裂修復過程中產生序列突變。對十五隻注射過的sdc4CRISPR斑馬魚進行基因定序後，計算此次基因編輯效率為百分之六十。將帶有基因突變的sdc4CRISPR斑馬魚和野生種斑馬魚雜交後，產生了帶有異型對偶基因的第一子代。對野生種斑馬魚和sdc4CRISPR斑馬魚的初步測試結果，顯示兩種魚在幼魚生長速率、尾鰭再生、心臟受損後膠原蛋白表現量、心臟受損後心電圖圖形，都沒有顯著差異。未來的斑馬魚配種和基因定序，可篩選出同型對偶基因帶有sdc4缺失的斑馬魚，並藉此研究sdc4基因在斑馬魚心臟修復過程中的功能。

The cardiovascular disease is a class of diseases that has been ranked as the leading cause of death worldwide. Among the cardiovascular diseases, myocardial infarction (MI, or commonly known as heart attack) occurs in millions of patients each year. After MI occurrence, human heart would accumulate fibrotic molecules and undergo ventricle dilation. As a result, the heart's pumping ability will be compromised. In contrast to human, zebrafish has the capacity to fully regenerate the damaged heart tissue without significant loss of cardiac function. Hence zebrafish has been widely used in heart regeneration studies. During healing, a protein called syndecan-4 (SDC4) has been reported to regulate multiple cellular and molecular functions in human case study and mice model. To investigate the function of Sdc4 in zebrafish heart repair, we established a zebrafish line containing mutation on sdc4 gene with clustered regularly interspaced short palindromic repeats and the CRISPR-associated protein 9 (CRISPR/Cas9) system. Wildtype zebrafish embryos were injected with Cas9 mRNA and guide RNA targeting sdc4 gene to induce DNA double strand break (DSB) and mutagenized after DNA repair. The genotypic analysis showed a 60% efficacy of gene-editing. The mating between gene-edited sdc4CRISPR zebrafish and wildtype zebrafish generated heterozygous mutants in the first filial (F1) generation. Preliminary analysis comparing wildtype and F1 sdc4CRISPR zebrafish showed no significant difference in larval growth and adult fin regeneration. After ventricular cryoinjury, collagen deposition and electrophysiology properties were also similar. We expected to generate homozygous zebrafish mutants containing specified sdc4 mutation on both alleles in the ongoing work, and this mutant line shall serve as a useful tool to reveal the role of sdc4 during heart repair and regeneration.

中文摘要 I
Abstract II
致謝 III
Table of contents IV
List of Abbreviations VII
1. Introduction 1
1.1 Cardiovascular disease 1
1.1.1 Cardiovascular disease is a global health problem 1
1.1.2 Cardiac fibrosis leads to cardiac function loss in human 2
1.2 Zebrafish model for heart regeneration research 2
1.2.1 Model organisms for heart regeneration research 2
1.2.2 Biological advantages of zebrafish model 3
1.2.3 Technical advantages of zebrafish model 4
1.2.4 Review of key findings in zebrafish heart regeneration 5
1.3 Syndecan-4 plays a critical role during heart healing 5
1.3.1 Syndecan-4 interacts with key modulators in the injured heart 5
1.3.2 Syndecan-4 is a multifunctional membrane protein 6
1.3.3 Syndecan-4 regulates signaling pathways for heart healing 7
1.4 CRISPR/Cas9 system conducts gene knockout in animals 7
1.4.1 Introduction of CRISPR/Cas system 7
1.4.2 CRISPR/Cas9 applications in research and therapy 8
1.5 The specific aim of this study 9
2. Material and methods 10
2.1 Zebrafish husbandry 10
2.2 Ventricular cryoinjury to induce heart damage 10
2.3 Establish the syndecan-4 knockout zebrafish line with CRISPR/Cas9 system 10
2.3.1 Design of guide RNA 11
2.3.2 Preparation of Cas9 nuclease and guide RNA 11
2.3.3 Microinjection of zebrafish embryos 11
2.4 Gene-edited fish Identification: Genomic DNA isolation, PCR and sequencing 12
2.5 Transcription profiling: RNA isolation and real-time PCR 12
2.5.1 mRNA isolation 12
2.5.2 Real-time qPCR 12
2.6 Histological analysis: AFOG staining of heart cryosection 13
2.7 Zebrafish ECG 13
3. Results: 15
3.1 Establishment of syndecan-4 knockout zebrafish 15
3.2 Identification of edited gene sequence 15
3.3 sdc4CRISPR zebrafish characterization 17
3.4 Preliminary analysis on cryo-injured heart in sdc4CRISPR zebrafish 18
4. Discussion 19
4.1 Summary 19
4.2 Different breeding methods of zebrafish after CRISPR/Cas9 system gene editing 19
4.3 Different methods to genotype the F0 zebrafish after CRISPR/Cas9 system gene editing 20
4.4 DNA repair mechanisms activated after CRISPR/Cas9 system gene editing 22
4.5 Brief review of Sdc4 function in zebrafish 22
4.6 Brief review on SDC4 functional domains 23
4.7 Future experimental plans on verifying sdc4 loss in homozygous mutant zebrafish 24
5. Perspectives 25
6. References 26
List of Tables 29
Table. 1 Sequences of guide RNA in CRISPR/Cas9 system 29
Table. 2 List of primer sequences 29
List of Figures 30
Fig. 1 Gene editing with CRISPR/Cas9 system was designed to target exon 2 of sdc4. 30
Fig. 2 Schematic depiction of zebrafish breeding strategy after CRISPR/Cas9 sdc4 knockout. 31
Fig. 3 Mating scheme of sdc4CRISPR zebrafish. 32
Fig. 4 Sample sequence of a non-edited zebrafish. (F0 female #4 forward product) 33
Fig. 5 Sample of coding sequence deletion in CRISPR/Cas9 Gene Knockout zebrafish. (F0 male #3 forward product) 35
Fig. 6 Sample sequence of substitution mutation in CRISPR/Cas9 Gene Knockout zebrafish. (F1 male #11 forward product) 37
Fig. 7 Summary of mutation identification in sdc4CRISPR zebrafish 39
Fig. 8 Predicted amino acid sequence of Sdc4 precursor transcribed from the altered coding region. 40
Fig. 9 Preliminary phenotypic characterization of sdc4CRISPR zebrafish 41
Fig. 10 Preliminary AFOG staining analysis of heart from wildtype and sdc4CRISPR zebrafish showing collagen and fibrin deposition after cryoinjury 43
Fig. 11 Preliminary analysis on wildtype and sdc4CRISPR zebrafish ECG before and after heart cryoinjury 45
Supporting information 46
Fig. S1 Photograph recording of the cryoinjury procedure. 46
Fig. S2 DNA microarray showing sdc4 and tgfb1a expression in zebrafish heart post cryoinjury and post amputation revealed similarities in expression profile 47
Fig. S3 Protein-protein interaction among Sdc4, Tgf-β1a and Fgf2 predicted with STRING v11.0 48
Fig. S4 Comparison of DNA sequence on sdc4 coding region revealed unexpected variations among eight F1 mutants. (F1 male #11, #12, #13, #14, female#11, #12, #13, #14 forward product) 49

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