| Summary: | 博士 === 國立中山大學 === 海洋生物科技暨資源學系研究所 === 102 === 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).
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