| Summary: | 碩士 === 國立臺灣大學 === 動物學系 === 85 === In order to study the gene regulation in cell lines and
embryos, several expression vectors containing different
heterologous promoters as well as lacZ reporter gene were
constructed. The promoter regions were obtained from a
variety of fish genes including common carp (Cyprinus carpio)
JAK1 and cdc2 genes as well as the round-spotted pufferfish
(Tetraodon fluviatilis ) JAK1, RET and c-fos genes. The results
of promoter assay were different in cell lines and in
embryos. When transfected into carp CF cell line, pf-fos
promoter displayed strong lacZ activity, about 15% of that of
pCMVβ, While pf-JAK1 promoter had only 7% of that of
pCMVβ. When these constructs were injected into
zebrafish (Danio rerio) embryos at one-cell- stage, the pf-
JAK1 promoter displayed stronger expression activity than
that of pf-fos promoter. Analyzing the expression pattern of 24
-h (hours after fertilization at 28℃) embryos, the β-gal-
expressed cells of pf-JAK1 and c-JAK1 promoters were
predominantly located in notochord and muscle in the trunk
segments of embryos, whereas those of c-cdc2 promoter
were located ubiquitously. The majority of expressing cells of
pf-RET promoter was located in the head and skin region.
We also performed deletion analysis of c-cdc2 promoter
and a positive regulation region between -211 and -146
was defined. A sequence in the region (5'-AAATT
AACAAA-3') has high similarity with rat cdc2 enhancer element
(-276AA GTTACAAA-267).
With the goal of detecting gene expression in the embryo at
different developmental stages, the green fluorescent
protein (GFP) from the jellyfish (Aequorea victoria) was chosen
as reporter gene. GFP fluorescence can be observed non-
invasively in living cells without any substrate or cofactor. A
GFP expression vector driven by c-JAK1 promoter was constructed
and microinjected into zebrafish embryos. At 44-h and
72-h after injected, the green fluorescence was visible in
muscle and notochord. Our data
indicated that the transient expression system, by means of
transgenic zebrafish, could be used as rapid analysis of
promoter activity and gene regulation in vivo. Utilizing this
system, we found that some promoters were expressed
ubiquitously whereas other promoters were expressed
at restricted area. Thus, analysis of promoter activity would be
useful for further studies.
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