人類第一蛋白質複合體是目前所知最複雜精細的膜酵素。很多粒線體疾病的發生已被證實和此酵素複合體，特別是其組成中由粒線體基因所產生的次單體之機能性障礙有關，例如:ND4L會導致雷伯氏遺傳性視神經萎縮症(Leber Hereditary Optic Neuropathy, LHON)。然而不幸地，對於因為粒線體基因缺陷所誘發的疾病至今並沒有專一且有效率的治療方式。同素異位法(Allotopic expression)是一種最近被研發出來改善粒線體基因缺失的方法，並且已被認為在相關疾病治療上擁有克服這個具挑戰性任務的潛力。在這個方法裡，所選擇的標的粒線體基因將被重新設計並且表現在細胞質中，接著合成的基因產物藉由加入的粒線體標的序列(MTSs)而被運送回粒線體。在本篇的研究中，我們測試了應用這個方式於人類第一蛋白質複合體ND4L次單體蛋白質的可能性。我們成功地設計並合成了兩段轉殖基因COX8MTSND4L和 COX4MTSND4L，而且利用它們在一個可被四環黴素誘導系統裡藉由短暫性轉染同素異位地表現了ND4L。我們實驗證明可以藉由四環黴素的加入來誘導經改造的ND4L轉殖基因表現蛋白質，而且此可誘導的系統亦可以應用於由細胞核基因所產生人類第一蛋白質複合體的NDUFV2 (24k)。然而，我們所選擇兩種粒線體標的序列COX4MTS和COX8MTS卻無法攜帶ND4L到粒線體的正確位置。ND4L具高度疏水性的特質可能是阻礙此蛋白質粒線體運送的主因。我們認為使用其他的粒線體標的序列；在目標轉殖基因中加入3’未轉譯區域 (3'UTR)；尋找可以用來穩定產生粒線體蛋白質的mRNA之蛋白質，或者結合此三種方法之間之組合，也許可以改進ND4L同素異位表現之應用性。

Human complex I is the most intricate membrane-bound enzyme known to date, and the dysfunction of this enzyme complex, especially the mitochondrially encoded subunits such as ND4L, has linked to a wide variety of mitochondrial diseases, including Leber Hereditary Optic Neuropathy (LHON). Unfortunately, there is no specific and efficient therapy for treatment of diseases resulting from defects in the mitochondrial genome. Allotopic expression is a newly developed approach which has been considered to have the potential in conquering this challenging task. In this method, a mitochondrial gene is reengineered and expressed in the cytoplasm, and the resultant gene product is then imported back to mitochondria by the addition of mitochondrial targeting sequences (MTSs). In this study, we tested the possibility of applying this approach for human complex I ND4L subunit. We successfully designed and synthesized two transgenes, COX8MTSND4L and COX4MTSND4L, and used them in a tetracycline–inducible system for allotopic expression of ND4L by transient transfection. We demonstrated that the recoded ND4L transgene could be expressed by the addition of tetracycline, and this inducible system was also applicable to a nuclear-encoded mitochondrial gene for NDUFV2 (24k) subunit of human complex I. However, the two mitochondrial targeting sequences, COX8MTS and COX4MTS, we chose were unable to carry our targeted ND4L to the correct location in the mitochondria. The highly hydrophobic property of the ND4L may impede its mitochondrial import. We suggest that using other different MTSs, adopting 3’ untranslated region (3’ UTR) in the targeted transgene, looking for proteins which can stabilize the mRNAs encoding mitochondrial proteins or any combination of these three methods may improve the applicability of allotopic expression of ND4L.

摘要 1
Abstract 3
Table of contents 5
Introduction 6
Materials and Methods 16
Cell culture 16
Construction of vectors 16
1. For nuclear-encoded NDUFV2 subunit 16
2. For mitochondrial-encoded ND4L subunit 17
Transient transfection 20
Immunofluorescence staining 21
Results 24
Construction of recoded ND4L genes for allotopic expression 24
Analysis of immunohistochemistry in T-REx 293 cells 26
Discussion 29
Reference 33
Tables 36
Table 1: The wavelengths applied at the excitation spectrum and the emission spectrum for different fluorescent dyes. 36
Figures 37
Figure 1: The mitochondrial oxidative phosphorylation (OXPHOS) system in the lipid bi-layer of the inner mitochondrial membrane. 37
Figure 2: Schematic representation of the strategy for constructing the MTS-ND4L fusion genes and expressing the gene of interest in an inducible tetracycline-regulated expression system (T-REx system) 38
Figure 3: The thermodynamically balanced inside-out (TBIO) method of PCR-based gene synthesis of COX8MTS ND4L transgene. 40
Figure 4: The result of the first assembly PCR generated by TBIO method of PCR-based gene synthesis 41
Figure 5: The results of the second assembly PCR generated by PCR-based gene synthesis and 24k gene from MGC-15943 clone amplified by PCR. 42
Figure 6: Cloning transgenic gene into the pCRScript vector and replacing the mitochondrial targeting sequence (MTS) 43
Figure 7: Switching the targeted genes from the pCRScript vector to the tetracycline-regulated expression system plasmid pcDNA4TM/TO/myc-His A vector 44
Figure 8: Determining the optimal concentration of tetracycline for induction of protein expression under the tetracycline-regulated system 46
Figure 9: Subcellular localization of MTS-ND4L in T-REx 293 cells 48
Figure 10: Immunocytochemistry of MTS-ND4L in T-REx 293 cells using ATP synthase subunit α as the marker for mitochondria 50
Figure 11: Studies on the subcellular localization the allotopic-expressed ND4L and its association with cytoskeleton in the cytosol 51
Figure 12: Comparison of the original sequences with the recoded transgenes 53
Figure 13: The hydrophobicity profiles of MTS-ND4L and 24k 55

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