先天性肌病是一種具有遺傳性和臨床異質性的肌肉疾病，其主要特徵為肌肉無力、肌張力減弱、運動發育遲緩。先天性肌病以肌肉切片所見的特徵進行命名。大部分的先天性肌病患者病情是進行性且罕見的，但目前的醫療對於先天性肌病沒有明確的治療方式。有名七歲的病童自嬰幼兒時期就患有先天性肌病，透過血清DNA定序發現病童原肌球蛋白3 (Tropomyosin 3, TPM3)基因在核苷酸第452位點單一點從A突變為G，導致第151個氨基酸從麩氨酸(Glutamic acid, E)改變為甘氨酸(Glycine, G)。先前文獻已報導過TPM3基因在核苷酸第452位點單一點從A突變為C，導致第151個氨基酸從麩氨酸(Glutamic acid, E)改變為丙氨酸(Alanine, A)會造成先天性肌病。本研究建立了轉基因斑馬魚動物模型，探討TPM3(E151G)和先天性肌病之間的病理關係，研究致病機轉和開發藥物篩選平台。我利用Tol2-Gateway轉基因技術，分別產生三種轉基因斑馬魚，將野生型TPM3、兩種突變型TPM3(E151A)、TPM3(E151G)表現在斑馬魚肌肉組織。我觀察了三種不同基因型斑馬魚的形態特徵，游泳速度和肌肉耐力，以及肌肉染色。首先，TPM3(WT)在外表呈現正常的外觀，一些TPM3(E151G)在F0及F1成魚具有沒有尾巴、彎曲的軀幹、異常的骨骼等外型上之變異。其次，我利用DanioVision測量幼魚游動能力，我發現TPM3(E151G)轉基因斑馬魚游動速度明顯比TPM3(WT)或TPM3(E151A)轉基因斑馬魚緩慢。我利用T-maze迷宮測量成魚游動能力之行為測試，與幼魚的結果相符，TPM3(E151G)轉基因斑馬魚游動速度明顯比TPM3(WT)或TPM3(E151A)轉基因斑馬魚緩慢。此外，透過游泳隧道測量肌耐力，TPM3(E151G)成魚明顯比TPM3(WT)或TPM3(E151A)表現出較弱的肌耐力。再者，我利用冷凍切片技術作為診斷肌肉疾病的工具，通過蘇木精 - 伊紅染色我觀察到TPM3(E151G)轉基因斑馬魚肌肉纖維排列不整、大小不成比例呈現先天性肌不均(congenital fiber type disproportion)的病變，TPM3(E151A)則呈現類似桿狀體肌病(Nemaline myopathy)的病變。 最後，我使用TPM3轉基因斑馬魚幼魚作為作為篩選某些天然物質的藥物篩選平台，我發現餵食TPM3(E151G)左旋肉鹼(L-carnitine）可以改善TPM3(E151G)幼魚的游動能力。左旋肉鹼可以貯存並調節能量ATP的供給，達到增強肌耐力。我還進行了肌肉標本的下一代測序以鑑定失調的基因/途徑。透過使用此斑馬魚模型作為臨床前模型，我不僅可以研究TPM3(E151G)突變引起之先天性肌病的致病機制，並為這種罕見的人類遺傳疾病開發個人化的藥物篩選。

Congenital myopathies (CM) are genetically and clinically heterogeneous muscle diseases, characterized by muscle weakness and hypotonia since birth, and pathologically defined by morphological features seen on muscle biopsy. Currently no definite treatment for CM. A 7-year-old boy has suffered from CM since infancy. His serum DNA sequencing revealed a de novo mutation in tropomyosin 3 (TPM3) at nucleotide 452 changed from A to G, resulting in amino acid 151 changing from glutamic acid (E) to glycine (G). The same position changing from A to C, and resulting TPM3 amino acid 151 changing from glutamic acid (E) to alanine (A) was previously reported causing CM. We have generated transgenic zebrafish for studying the pathological causality between TPM3(E151G) and CM, also as potential drug screening platform. Using Tol2-Gateway transgenesis technology, we established three transgenic zebrafish expressing TPM3(WT), TPM3(E151A) and TPM3(E151G) mutants in muscles, and observed the morphological features, swimming speed, and muscular endurance, as well as muscle staining. While TPM3(WT) showed normal appearance, some TPM3(E151G) fishes displayed either no tail, crooked body or abnormal skeletal in both F0 and F1 adults. TPM3(E151G) larvae exhibited dramatically slower swimming speed than TPM3(WT) and TPM3(E151A) mutant measured by DanioVision. TPM3(E151G) adults swam significantly slower than TPM3(WT) and TPM3(E151A) by T-maze. Furthermore, TPM3(E151G) transgenic fish exhibited weaker muscular endurance measured by swim tunnel. Moreover, we used cryosection technology as a tool for diagnosing muscle disease, TPM3(E151G) exhibited muscle fiber disproportion similar to congenital fiber type disproportion, and TPM3(E151A) seemed like nemaline myopathy by hematoxylin-eosin staining. Finally, using TPM3(E151G) transgenic zebrafish larvae as a drug screening platform to screen some natural substances, we identified L-carnitine specifically improved the swimming speed of TPM3(E151G) larvae. L-carnitine can stored and regulated the supply of energy ATP to enhance muscle endurance. Next generation sequencing for the muscle specimens were also performed to identify the dysregulated genes/pathways. By using the TPM3(E151G) transgenic zebrafish as a preclinical model, we not only can uncover the molecular mechanisms of TPM3(E151G) mutation mediated CM, and develop a personalized drug screening for this rare human genetic disease.

中文摘要 I
Abstract II
致謝 IV
Chapter 1 Introduction 1
1.1 Congenital myopathy as a rare disease 1
1.1.1 Congenital myopathy 1
1.1.2 Treatment for congenital myopathy 1
1.1.3 A patient with congenital myopathy 2
1.1.4 Tropomyosin family 2
1.2 Zebrafish model 3
1.2.1 Advantages of zebrafish model 3
1.2.2 Established transgenic zebrafish lines in this study 4
1.2.3 Skeletal muscle of zebrafish 4
Chapter 2 Materials and Methods 6
2.1 Zebrafish husbandry 6
2.2 Zebrafish lines 6
2.3 Amplification of TPM3 gene 6
2.4 Gateway cloning 7
2.5 Site-directed mutagenesis 8
2.6 Embryos collection 9
2.7 Microinjection 10
2.8 Selection and confirmation of transgenic zebrafish 10
2.9 Fish measurement 11
2.10 Morphological analysis 11
2.11 Tracking of larvae fish swimming behavior 11
2.12 Analysis of larvae fish swimming behavior 12
2.13 T-maze apparatus 12
2.14 Adult fish T-maze behavior test 12
2.15 Analysis of adult fish T-maze behavior test 13
2.16 Adult fish muscle endurance swimming performance 13
2.17 Analysis of adult fish muscle endurance swimming performance 14
2.18 Chemicals treatment for larvae fish 14
2.19 Chemicals treatment for adult fish 15
2.20 Tissue collection and frozen section 15
2.21 Hematoxylin and eosin stain (H&E stain) 16
2.22 Gömöri’s trichrome stain 17
2.23 Nicotinamide adenine dinucleotide tetrazolium reductase stain (NADH-TR stain) 18
2.24 ATPase stain 18
2.25 Total RNA isolation 20
2.26 RNA clean up 21
2.27 Reverse transcription polymerase chain reaction (RT-PCR) 22
2.28 Real time quantitative polymerase chain reaction (QPCR) 22
2.29 Statistical analysis 23
Chapter 3 Results 24
3.1 Establishment of TPM3 transgenic zebrafish 24
3.2 TPM3(E151G) mutant displayed abnormal appearance 25
3.3 TPM3 mutants’ adult displayed abnormal skeleton 26
3.4 TPM3 mutants’ larva exhibited lower survival rate 26
3.5 TPM3 mutants swimming slower compared to TPM3(WT) transgenic fish 27
3.6 TPM3 mutants exhibited weaker muscle endurance 28
3.7 Evaluation of histopathology on adult TPM3 transgenic zebrafish 29
3.8 Screening of the therapeutic drugs using TPM3(E151G) larvae fish 29
3.9 Evaluation of the therapeutic using TPM3(E151G) adult fish 30
Chapter 4 Discussion 32
4.1 Behavioral testing of swimming velocity and learning memory 32
4.2 How L-carnitine improve swimming velocity in larvae TPM3(E151G) zebrafish 33
4.3 Comparison of larvae fish and adult fish in a drug screening platform for congenital myopathy 34
4.4 Future perspective 36
Figures and Tables 37
Figure 1. Confirmation the construct of TPM3 transgenic zebrafish by colony PCR 37
Figure 2. Confirmation the sequence of TPM3 transgenic zebrafish 39
Figure 3. Appearance of adult TPM3 transgenic zebrafish 41
Figure 4. Statistical the proportion of abnormal appearance of larvae TPM3 transgenic zebrafish 43
Figure 5. The X-ray image of adult F1 TPM3 transgenic zebrafish 45
Figure 6. Statistical the survival rate of larvae TPM3 transgenic zebrafish 47
Figure 7. T-maze behavior test for F0 adult TPM3 transgenic zebrafish 49
Figure 8. T-maze behavior test for F1 adult TPM3 transgenic zebrafish 51
Figure 9. The swimming velocity of larvae F1 TPM3 transgenic zebrafish 53
Figure 10. Muscle endurance test for adult F1 TPM3 transgenic zebrafish 55
Figure 11. Principal component analysis of endurance test for adult F1 TPM3 transgenic zebrafish 57
Figure 12. 50X histopathological of F1 adult TPM3 transgenic zebrafish 59
Figure 13. 400X histopathology of F1 adult TPM3 transgenic zebrafish 61
Figure 14. Chemicals treatment on F3 larvae TPM3 transgenic zebrafish 63
Figure 15. Chemicals treatment on F2 adult TPM3 transgenic zebrafish 65
Figure 16. Principal component analysis of chemicals treatment on F2 adult TPM3 transgenic zebrafish 67
Table 1. The primer information for TPM3 gene amplification 69
Table 2. The primer information for generated pME-TPM3 70
Table 4. The primer information for site-directed mutagenesis 72
Table 5. The primer information for confirm TPM3 transgenic zebrafish 73
Table 6. The primer information for QPCR analysis in zebrafish 74
Supplementary Information 75
Supplementary Figure 1. A three-armed T-maze apparatus for adult fish behavior test 75
Supplementary Figure 2. Preparing the muscle tissue of zebrafish for frozen section 77
Supplementary Figure 3. Human TPM3 gene expression level of F1 adult TPM3 transgenic zebrafish 79
Supplementary Figure 4. The swimming velocity of larvae F1 TPM3 transgenic zebrafish at 3 dpf to 6 dpf 81
Supplementary Figure 5. ATPase 4.7 staining of F1 adult TPM3 transgenic zebrafish 83
Supplementary Figure 6. Pax7 gene expression level of F1 adult TPM3 transgenic zebrafish 85
Supplementary Figure 8. Heatmap of TPM3 transgenic zebrafish skeletal muscle by RNA-seq 89
Supplementary Figure 8. Heatmap of TPM3 transgenic zebrafish skeletal muscle by RNA-seq 90
Supplementary Data 1. Cloning of Tg(MLC2:TPM3(WT);myl7:EGFP) transgenic zebrafish 91
Supplementary Data 2. Cloning of Tg(MLC2:TPM3(E151A);myl7:EGFP) transgenic zebrafish 94
Supplementary Data 3. Cloning of Tg(MLC2:TPM3(E151G);myl7:EGFP) transgenic zebrafish 97
References 100
Honors & Awards 108

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