首先利用斑馬魚2.5 kb mylz2啟動子來構築EGFP及DsRed螢光蛋白的表現
質體，並在模式魚種斑馬魚中測試使用該質體建立螢光轉殖魚的可行性。吾人接
續利用該質體，研究神仙魚（Pterophyllum scalare）生殖腺之體內電穿孔基因轉殖
的適用性。先將質體注射入麻醉之神仙魚生殖腺內，接者利用正負電極針於生殖
腺兩側施予電脈衝。待實驗魚隻穩定後進行自然交配並產卵，再利用PCR擴增法
對魚苗檢測外源基因的存在。儘管基因轉殖率偏低（約1.33%），但活體內電穿孔
對於不易取得精子或受精卵之觀賞魚而言，為建立基因轉殖初代（transgenic
founder）的一種相對簡單之基因轉殖技術。另一方面，由於僅有少量的轉基因魚
表現出肉眼可見之綠螢光，本研究亦選殖並解析吳郭魚(Oreochromis niloticus)之
肌球蛋白輕鏈3啟動子區域（~4.3 kb）。序列分析顯示該選殖片段與吳郭魚mlc3
啟動子、外顯子 1 及內含子 1 之基因具有高度相似性。它包含幾個預測之轉錄因
子結合序列，如MEF-2、MYOG、MyoD、PKNOX1及AREB6。直接將質體DNA
注射入白獅頭（Amatitlania nigrofasciatusvar.；convict cichlid）之骨骼肌，並以冷
光素酶活性檢測肌肉專一性報導基因之活性，結果證實吳郭魚mlc3啟動子在刪除
-801至-3881 bp的基因體片段（-4314-3882/-800-1/i1）之後，具有高度的啟動子活
性，其表現高於斑馬魚mylz2啟動子達32倍之多。吾人亦於觀賞魚種--白獅頭中
建立帶有由該mlc3啟動子驅動之台灣珊瑚紅螢光蛋白（TcRFP）的穩定轉基因種
系。F1白獅頭成魚在日光下於骨骼肌中展現出真實亮眼之粉紅螢光，更具有觀賞
的特性。綜上所述，本研究證實活體內電穿孔基因轉殖技術及吳郭魚mlc3啟動子
於生產螢光觀賞魚種（例如慈鯛科魚類）的可行性。

In this study, we first examined the feasibility of establishing various lines of
transgenic fluorescent zebrafish by combining the Tol2 transposon system with our
newly-constructed fluorescent protein expression plasmids, in which EGPF or DsRed
are expressed under the control of a 2.5 kb mylz2promoter. Of these plasmids, the
construct containing the fluorescent EGFP gene was subsequently used to investigate
the suitability of in vivoelectroporation for gene transfer directly into the gonads of
Pterophyllum scalare. We injected gonads with plasmid DNA expressing zebrafish
mylz2 promoter-driven EGFP, and electric pulses were thenapplied to both sides of the
gonad with a pair of electrode needles. The fish were allowed to mate and spawn, and
PCR was used to confirm the presence of the transferred gene in the next generation fry.
Despite a low gene transfer rate (about 1.33%), the presence of the GFP gene in certain
hatched offspring indicates that in vivo electroporation is an alternative and relatively
simple transgenic tool with which to establish transgenic founders for ornamental fish
with a low availability of sperm or fertilized eggs. However, only a small proportion of
the transgenic fish exhibited green fluorescencevisible to the naked eye. In an attempt
to increase the proportion of fluorescent transgenic fish, we first isolated and
characterized a tilapia (Oreochromis niloticus) myosin light chain 3promoter region
(~4.3 kb). Sequence analysis of the clone revealed high similarity with a tilapia gene
encoding the promoter region, exon 1, and intron 1 of mlc3. This clone contained
several putative binding sequences for transcription factors, including MEF-2, MYOG,
MyoD, PKNOX1, and AREB6. A genomicfragment (-4314-3882/-800-1/i1)
encompassing the tilapia mlc3promoter region but with a deletion from -801 to -3881
bp exhibited promoter activity, as evidencedby luciferase reporter activity following
direct intramuscular injection of plasmid DNA into the skeletal muscle of Amatitlania
nigrofasciatus var. (convict cichlid); luciferase activity was 32-fold greater than that
observed using constructs containing the zebrafish mylz2promoter. Stable transgenic
germlines carrying the gene encoding Taiwan coral red fluorescent protein (TcRFP)
under the control of the mlc3promoter were established in the ornamental fish species,
A. nigrofasciatusvar. F1 adult transgenic A. nigrofasciatusvar. exhibited brilliant-pink
fluorescence in skeletal muscles under visible light, and as such, may be suitable for
ornamental exhibition. Overall,these findings demonstrate the feasibility of employing
in vivoelectroporation and the tilapia mlc3promoter to produce novel, medium-sized,
ornamental fish species (such as Cichlidae).

論文審定書.................................................................................................................................... i
誌 謝........................................................................................................................................... ii
中文摘要...................................................................................................................................... iii
Abstract ....................................................................................................................................... iv
1. Introduction ............................................................................................................................. 1
1.1. General overview of the ornamental fish industry ........................................................ 1
1.2. Current status of the fluorescent fish industry .............................................................. 3
1.2.1. Development and marketing .................................................................................... 3
1.2.2. Regulations ................................................................................................................. 4
1.3. Transgenesis ...................................................................................................................... 5
1.3.1. Background ................................................................................................................ 5
1.3.2. Techniques ................................................................................................................. 8
1.4. The Tol2transgenesis system ........................................................................................ 14
1.5. Regulation of muscle growth and development ........................................................... 16
2. Specific Aims .......................................................................................................................... 20
3. Materials and Methods ......................................................................................................... 22
3.1. Materials ......................................................................................................................... 22
3.2 Equipment and apparatus .............................................................................................. 26
3.3. Methods ........................................................................................................................... 27
3.3.1. Establishment of fluorescent zebrafish using the mylz2promoter...................... 27
3.3.2. Establishment of fluorescent angelfish using the mylz2promoter ...................... 34
3.3.3 Establishment of fluorescent convict cichlid using the mlc3promoter ...... 37
4.1. Establishment of fluorescent zebrafish using the mylz2promoter ............................ 43
4.2. Establishment of fluorescent angelfish by electroporation ......................................... 44
4.3. Establishment of fluorescent convict cichlid using the mlc3promoter ..................... 45
5. Discussion ............................................................................................................................... 48
5.1. Establishment of fluorescent cichlids by electroporation ........................................... 48
5.2. Tol2transposon system .................................................................................................. 49
5.3. Establishment of novel fluorescent lines of convict cichlids using the Nile tilapia
mlc3promoter ........................................................................................................................ 50
6. Future perspectives ............................................................................................................... 53
7. Conclusion .............................................................................................................................. 54
8. Figures .................................................................................................................................... 55
9. Tables ..................................................................................................................................... 72
References .................................................................................................................................. 79
Appendix .................................................................................................................................... 88

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